Growth Factor Receptor Family Research Articles

GraphEGFR: Multi-task and transfer learning based on molecular graph attention mechanism and fingerprints improving inhibitor bioactivity prediction for EGFR family proteins on data scarcity.

The proteins within the human epidermal growth factor receptor (EGFR) family, members of the tyrosine kinase receptor family, play a pivotal role in the molecular mechanisms driving the development of various tumors. Tyrosine kinase inhibitors, key compounds in targeted therapy, encounter challenges in cancer treatment due to emerging drug resistance mutations. Consequently, machine learning has undergone significant evolution to address the challenges of cancer drug discovery related to EGFR family proteins. However, the application of deep learning in this area is hindered by inherent difficulties associated with small-scale data, particularly the risk of overfitting. Moreover, the design of a model architecture that facilitates learning through multi-task and transfer learning, coupled with appropriate molecular representation, poses substantial challenges. In this study, we introduce GraphEGFR, a deep learning regression model designed to enhance molecular representation and model architecture for predicting the bioactivity of inhibitors against both wild-type and mutant EGFR family proteins. GraphEGFR integrates a graph attention mechanism for molecular graphs with deep and convolutional neural networks for molecular fingerprints. We observed that GraphEGFR models employing multi-task and transfer learning strategies generally achieve predictive performance comparable to existing competitive methods. The integration of molecular graphs and fingerprints adeptly captures relationships between atoms and enables both global and local pattern recognition. We further validated potential multi-targeted inhibitors for wild-type and mutant HER1 kinases, exploring key amino acid residues through molecular dynamics simulations to understand molecular interactions. This predictive model offers a robust strategy that could significantly contribute to overcoming the challenges of developing deep learning models for drug discovery with limited data and exploring new frontiers in multi-targeted kinase drug discovery for EGFR family proteins.

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.

Targeting Cardiomyocytes with Adeno Associated Viruses to Protect Hearts from Trastuzumab-induced Cardiotoxicity

Amplification of HER2 occurs in 20% of breast cancers. HER2 (known as ErbB2 in mouse) belongs to the Epidermal Growth Factor Receptor (EGFR) family that is responsible for cell growth and survival. While HER2 does not bind ligands, it forms heterodimers with HER1 (also known as EGFR) in the presence of its ligand EGF, and HER4 following stimulation with Neuregulin. In HER2-positive breast cancer where HER2 expression is high, HER2 homodimerizes leading to excessive activation of downstream signalling and cell proliferation. Current HER2-positive breast cancer treatment involves chemotherapy with drugs called anthracyclines, such as Doxorubicin, and an antibody targeting HER2 called Trastuzumab. This combination is effective in killing HER2-positive breast tumours, however, Trastuzumab alone or in combination with anthracyclines leads to severe cardiotoxicity and heart failure in 2.6-11% of cases, making it a dose-limiting side effect. We hypothesise that cardiac toxicity can be mitigated by targeting cardiomyocytes exclusively to express a mutated (but functional) version of HER2 that is not recognised by trastuzumab. We designed 3 HER2 variants wherein the trastuzumab binding domain was mutated to disrupt binding. These HER2 mutants were transfected into HEK293T cells, and we confirmed that all 3 successfully localised to the cell membrane, and did not bind trastuzumab. We have used bioluminescence resonance energy transfer to examine the capacity of all 3 mutants to activate recruitment of Grb2 to the HER2 as part of HER1/HER2 and HER4/HER2 heterodimers; Western blotting also confirming mutants were capable of stimulating ERK and Akt phosphorylation. Future studies will evaluate the effcacy of using AAVs to instruct cardiomyocytes to express these HER2 mutants in a murine model of breast cancer. We predict that this new therapy would not only mitigate cardiac toxicity, but would also permit higher doses of trastuzumab to be used to treat breast cancer. This work is supported by Australia's National Health and Medical Research Council and the Australian Research Council. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DB-1310, an ADC comprised of a novel anti-HER3 antibody conjugated to a DNA topoisomerase I inhibitor, is highly effective for the treatment of HER3-positive solid tumors

BackgroundHER3 (ErbB3), a member of the human epidermal growth factor receptor family, is frequently overexpressed in various cancers. Multiple HER3-targeting antibodies and antibody–drug conjugates (ADCs) were developed for the solid tumor treatment, however none of HER3-targeting agent has been approved for tumor therapy yet. We developed DB-1310, a HER3 ADC composed of a novel humanized anti-HER3 monoclonal antibody covalently linked to a proprietary DNA topoisomerase I inhibitor payload (P1021), and evaluate the efficacy and safety of DB-1310 in preclinical models.MethodsThe binding of DB-1310 to Her3 and other HER families were measured by ELISA and SPR. The competition of binding epitope for DB-1310 and patritumab was tested by FACS. The sensitivity of breast, lung, prostate and colon cancer cell lines to DB-1310 was evaluated by in vitro cell killing assay. In vivo growth inhibition study evaluated the sensitivity of DB-1310 to Her3 + breast, lung, colon and prostate cancer xenograft models. The safety profile was also measured in cynomolgus monkey.ResultsDB-1310 binds HER3 via a novel epitope with high affinity and internalization capacity. In vitro, DB-1310 exhibited cytotoxicity in numerous HER3 + breast, lung, prostate and colon cancer cell lines. In vivo studies in HER3 + HCC1569 breast cancer, NCI-H441 lung cancer and Colo205 colon cancer xenograft models showed DB-1310 to have dose-dependent tumoricidal activity. Tumor suppression was also observed in HER3 + non-small cell lung cancer (NSCLC) and prostate cancer patient-derived xenograft (PDX) models. Moreover, DB-1310 showed stronger tumor growth-inhibitory activity than patritumab deruxtecan (HER3-DXd), which is another HER3 ADC in clinical development at the same dose. The tumor-suppressive activity of DB-1310 synergized with that of EGFR tyrosine kinase inhibitor, osimertinib, and exerted efficacy also in osimertinib-resistant PDX model. The preclinical assessment of safety in cynomolgus monkeys further revealed DB-1310 to have a good safety profile with a highest non severely toxic dose (HNSTD) of 45 mg/kg.ConclusionsThese finding demonstrated that DB-1310 exerted potent antitumor activities against HER3 + tumors in in vitro and in vivo models, and showed acceptable safety profiles in nonclinical species. Therefore, DB-1310 may be effective for the clinical treatment of HER3 + solid tumors.

Abstract CT215: Frontline vaccination with the B-cell peptide compound HER-Vaxx (IMU-131), combined with standard-of-care chemotherapy induces high levels of HER2-specific antibodies mediating ADCC and intracellular phosphorylation inhibition resulting in overall survival benefit in patients with HER2+ metastatic or advanced gastric/GEJ adenocarcinoma - Final results from Phase II/HERIZON study

Abstract Background: HER2, a member of the epidermal growth factor receptor (EGFR) family, is overexpressed in approximately 30% of gastric cancers. HER-Vaxx is a B cell peptide-based anti-HER2 vaccine (IMU-131) comprising trastuzumab’s binding site. Here, we present the results of the randomized open-label, multicenter phase 2 HERIZON trial (NCT02795988), which compared HER-Vaxx plus chemotherapy to chemotherapy alone. The study aimed to evaluate clinical (primary endpoint) and antibody (secondary endpoint) responses in patients with HER2-overexpressing gastric/gastro-esophageal junction (GC) cancer naïve to anti HER2 therapy. Methods: Patients were randomized to chemotherapy alone (n=17) or HER-Vaxx (50µg, recommended phase 2 dose) plus chemotherapy (n=19). In the HER-Vaxx group, patients received the vaccine at days 0, 14, 35, and 77, then every 63 days until disease progression. Chemotherapy consisted of oxaliplatin plus capecitabine and was started at day 0, repeated every 21 days for a maximum of 6 cycles. The clinical responses were assessed by RECIST 1.1 criteria. FACS analysis of PBMCs for immunophenotyping was carried out, as well as in vitro phosphorylation inhibition assays of HER2 and the signaling pathway kinases Akt and MAPK, and mediation of ADCC to characterize the vaccine-induced IgG antibodies. Results: A 42% survival benefit for patients treated with HER-Vaxx plus chemotherapy [median overall survival: 13.9 months (7.5, 14.3)] compared to chemotherapy alone [mOS: 8.3 months (6.0, 9.6)] was shown, which translated into an OS HR of 0.580 (80% 2-sided CI: 0.362, 0.927). A highly significant HER2-specific IgG and IgG1 antibody response at all time points, particularly after 3 or more doses of HER-Vaxx were induced (P<0.001). The HER-Vaxx antibody response correlated well with mediating ADCC (IgG, P=0.003; IgG1, P=0.05) and the anti-tumor effect (IgG, P=0.001; IgG1, P=0.016). Moreover, the HER-Vaxx-induced IgG antibodies exhibited binding to the gastric cancer cell line N87, with capacity to inhibit HER2 signaling pathway kinases Akt and MAPK phosphorylation. It also demonstrated decreased levels of Foxp3+ Tregs (P=0.0013). Conclusions: The previously shown safety profile of HER-Vaxx, along with the improved clinical and antibody responses in HER2-overexpressing GC patients shown here, validate the proof of concept for a first-in-class B-cell immunotherapy based on HER2 B-cell peptides. Clinical trials evaluating further treatment with HER-Vaxx in HER2+ GC are ongoing. Citation Format: Joshua Tobias, Michael Kundi, Erika Garner-Spitzer, Christoph Zielinski, Marina Maglakelidze, Zoran Andric, Zoran Petrovic, Rajnish Nagarkar, Tanuj Chawla, Leslie Mi Ok Chong, Bonnie Nixon, Sharon Yavrom, Nicholas J. Ede, Ursula Wiedermann. Frontline vaccination with the B-cell peptide compound HER-Vaxx (IMU-131), combined with standard-of-care chemotherapy induces high levels of HER2-specific antibodies mediating ADCC and intracellular phosphorylation inhibition resulting in overall survival benefit in patients with HER2+ metastatic or advanced gastric/GEJ adenocarcinoma - Final results from Phase II/HERIZON study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT215.

Immunotherapy for gastrointestinal cancer

Cancers of gastrointestinal tract make up the largest group of solid tumour diseases in Germany. The prognosis at diagnosis is often critical. Drug therapies reduce the risk of relapse after resection and can halt the progression of metastatic disease. Immunotherapies contribute increasingly to the treatment of gastrointestinal tumours. Monoclonal antibodies (mAB) against surface receptors from the epidermal growth factor receptor family (EGFR, Her2) are well established. The effect is partly based on the interruption of the oncogenic downstream signalling cascades and partly on immune effector mechanisms such as antibody-dependent cellular cytotoxicity. In clinical practice mAB directed against programmed cell death protein 1 (PD-1), its ligand (PD-L1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) - so-called immune checkpoint inhibitors - play an increasing role and change the natural history of some subgroups of gastrointestinal cancers, especially those with deficient DNA mismatch repair which leads to genomic microsatellite instability.

Genomic Profiling and Molecular Characterisation of Metastatic Urothelial Carcinoma.

The clinical management of metastatic urothelial carcinoma (mUC) is undergoing a major paradigm shift; the integration of immune checkpoint inhibitors (ICIs) and antibody-drug conjugates (ADCs) into the mUC therapeutic strategy has succeeded in improving platinum-based chemotherapy outcomes. Given the expanding therapeutic armamentarium, it is crucial to identify efficacy-predictive biomarkers that can guide an individual patient's therapeutic strategy. We reviewed the literature data on mUC genomic alterations of clinical interest, discussing their prognostic and predictive role. In particular, we explored the role of the fibroblast growth factor receptor (FGFR) family, epidermal growth factor receptor 2 (HER2), mechanistic target of rapamycin (mTOR) axis, DNA repair genes, and microsatellite instability. Currently, based on the available clinical data, FGFR inhibitors and HER2-directed ADCs are effective therapeutic options for later lines of biomarker-driven mUC. However, emerging genomic data highlight the opportunity for earlier use and/or combination with other drugs of both FGFR inhibitors and HER2-directed ADCs and also reveal additional potential drug targets that could change mUC management.

The impact of nitric oxide on HER family post-translational modification and downstream signaling in cancer.

The human epidermal growth factor receptor (HER) family consists of four members, activated by two families of ligands. They are known for mediating cell-cell interactions in organogenesis, and their deregulation has been associated with various cancers, including breast and esophageal cancers. In particular, aberrant epidermal growth factor receptor (EGFR) and HER2 signaling drive disease progression and result in poorer patient outcomes. Nitric oxide (NO) has been proposed as an alternative activator of the HER family and may play a role in this aberrant activation due to its ability to induce s-nitrosation and phosphorylation of the EGFR. This review discusses the potential impact of NO on HER family activation and downstream signaling, along with its role in the efficacy of therapeutics targeting the family.

Abstract 5975: Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family

Abstract Antibodies targeting HER receptors have led to successful therapeutics for multiple human malignancies. Targeting the family of HER ligands provides an alternative strategy to inhibit signaling and subsequent proliferation of cancer cells. We have engineered ligand traps that bind the growth factors activating the HER family and have characterized a series of molecules (EN2000-Fc) using Surface Plasmon Resonance (SPR). Five distinct molecules were further evaluated for improved in vivo pharmacokinetics in mice that express human neonatal Fc receptor (FcRn). Mutations in the IgG Fc known to extend the in-vivo half-life of monoclonal antibodies were incorporated into the parental EN2000-Fc to generate 3 more distinct pan HER ligand traps. In addition, an EN2000-Fc variant with a full-length hinge was engineered, which is 5-amino-acid longer N-terminally than the parental molecule. These five molecules were synthesized (> 300 mg of each), purified (>98%), and characterized for in vitro and in vivo testing. Two variants were compared by evaluating the ligand binding affinity to 11 HER ligands using SPR. Both molecules showed comparable affinity against all ligands tested, confirming reproducibility of the pan HER ligand trap engineering technology. Among the ligands, HB-EGF had the highest affinity. High binding affinities to EGF, NRG1, NRG2, betacellulin and TGFα were also measured. The serum pharmacokinetics of the different ligand traps were compared in 6- to 8-week-old female C57BL/6 mice engineered to express human FcRn. Ligand traps were administered by single tail vein injection, and serum samples collected over a 28-day interval were analyzed by ELISA. Ligand traps with engineered Fc-mutations had 3- to 4-fold greater systemic exposure (area-under-the-curve) compared to ligand traps with the parental sequence, demonstrating improved in vivo characteristics of selected engineered fusion proteins. These results provide useful information for product candidate selection and for further preclinical development of a ligand trap targeting HER family receptors. Citation Format: Daniel C. Maneval, Cindy Yang, Cynthia Trevisiol, H. Michael Shepard, Pei Jin. Characterization and pharmacokinetics of ligand traps targeting the human epidermal growth factor receptor (HER) family [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5975.

Abstract 6264: Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate cancer

Abstract Objective: Prostate cancer (PCa)-specific nuclear expression of ErbB3, a receptor tyrosine kinase of the epidermal growth factor receptor (EGFR) family, has long been recognized. While an 80kDa nuclear variant of ErbB3 has been identified, full-length 185 kDa ErbB3 also translocates to the nucleus in PCa in a bone microenvironment and androgen status-dependent manner. Here, we studied potential mechanisms by which nuclear ErbB3 may regulate PCa progression. Methods: ErbB3 localization was investigated in vitro (human prostate cancer cell lines, LNCaP, C4-2, 22Rv1) using amiloride or heregulin-1β (ErbB3-specific ligand). ErbB3 expression and subcellular localization were analyzed using immunofluorescent microscopy and subcellular fractionation followed by immunoblot. Physiological readouts included viability, migration and reporter gene assays. Results: Here we show that ErbB3 can occupy a nuclear/perinuclear location in prostate cancer cells. The hormone sensitive PCa line LNCaP expressed higher levels of nuclear ErbB3 compared to castration resistant lines C4-2 and 22Rv1. The diuretic amiloride was previously shown to inhibit ErbB3 internalization by macropinocytosis thus we investigated whether it could also modulate ErbB3 nuclear localization in PCa cells. In LNCaP cells, amiloride dose dependently prevented ErbB3 nuclear transport, whereas in C4-2 and 22Rv1, no such effect was observed. Further analysis revealed that amiloride promoted ErbB3 expression in the membrane, where ErbB3 underwent ligand-dependent phosphorylation, formed ErbB2/ErbB3 dimers, and in turn phosphorylated the downstream targets AKT and ERK1/2 (p42/p44MAPK). On the other hand, EGFR phosphorylation and dimerization were unaffected by amiloride. Our results suggest that ErbB3 is stored in the nuclear region in an inactive form, whereas upon stress induction, including androgen deprivation, ErbB3 is translocated to the plasma membrane, where it is activated by ligand binding, and causes downstream signaling. Conclusion: We previously showed that the EGFR/ErbB2 dual inhibitor lapatinib failed to affect PCa - we now show that this may be due to modulation of nuclear and cytoplasmic ErbB3 expression. Amiloride prevented translocation (and thus expression of) ErbB3 to the nucleus and retained this RTK in the membrane, resulting in the formation of active ErbB2/ErbB3 dimers. Lapatinib was then able to inhibit and inactivate its specific target, ErbB2, thereby inducing significant apoptosis. The above indicates a novel role for nuclear ErbB3 in PCa that can be regulated by the combination of amiloride and lapatinib. Citation Format: Maitreyee K. Jathal, Maria M. Mudryj, Paramita M. Ghosh. Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6264.

Abstract 4553: DXC004A, a novel EGFR antibody-tubulysin analog conjugate demonstrated potential to broaden therapeutic opportunities for non-small cell lung cancer

Abstract EGFR is a member of the epidermal growth factor receptor (HER) family, and the over-expression of EGFR plays an important role in the growth and progression of various tumors, including non-small cell lung cancer. Monoclonal EGFR antibodies, such as cetuximab and panitumumab, have proven efficacy in various types of cancer. However, treatment with the anti-EGFR agents can be associated with toxicities of the skin, nails, hair, and eyes. Nimotuzumab (Nimo) is the first humanized monoclonal antibody targeting EGFR and has been granted approval for use in squamous cell carcinoma of head and neck (SCCHN), glioma and nasopharyngeal cancer in different countries. In contrast to cetuximab, Nimotuzumab is distinguished by achieving higher or similar complete remission rate (CRR) or overall remission rate (ORR) of the primary tumor in clinical applications but much less toxicity, resulting in a better safety profile, which has been attributed to its about 10-fold lower affinity. Paclitaxel combined with cisplatin is the first-line chemotherapy regimen for non-small cell lung cancer. A phase 2 clinical trial has confirmed that Nimotuzumab combined with concurrent chemoradiation therapy (radiation concurrent with docetaxel and cisplatin, CCRT) was well tolerated for locally advanced squamous cell lung cancer. However, the combination strategy of Nimo-CCRT has only demonstrated similar OS and PFS to those in the CCRT group. DXC004A is an ADC drug targeting EGFR, in which a Nimotuzumab derivative and a Tubulysin B analog is conjugated by a functional linker. DXC004A is in phase I clinical trial and has demonstrated clinical activity in advanced non-small cell lung cancers and good tolerability. In vitro and in vivo results had confirmed that DXC004A monotherapy had better anti-tumor activity than the combination of Nimotuzumab and Tubulysin analog in HCC827 cell lines with high EGFR expression. Meanwhile, a single injection of low-dose DXC004 (2.5 mg/kg) in HCC827 xenograft model showed very good durable anti-tumor effect. Moreover, the combination of DXC004A with Cisplatin exhibited significantly better activities than that of Nimo-CCRT combination, DXC004A or cisplatin alone, in vitro and in vivo, which might solve the predicament of poor efficacy of Nimo-CCRT combination therapy. This synergy results suggested that DXC004A plus cisplatin would possibly be a new adjuvant therapy for non-small cell lung cancer in further clinical studies. Citation Format: Xiaobo Cai, Min Cao, Huihui Guo, Qingliang Yang, Yongxiang Chen, Xiangfei Kong, Yong Du, Zhicang Ye, Zhixiang Guo, Lingli Zhang, Lu Bai, Junxiang Jia, Yunxia Zheng, Wei Zheng, Jun Zheng, Wenjun Li, Yuanyuan Huang, Zhongliang Fan, Binbin Chen, Meng Dai, Robert Y. Zhao. DXC004A, a novel EGFR antibody-tubulysin analog conjugate demonstrated potential to broaden therapeutic opportunities for non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4553.

Abstract 5083: Discovery of an asymmetric IgG-like bispecific ADC targeting EGFR/HER3

Abstract Background: The epidermal growth factor receptor (EGFR) family, consisting of EGFR, HER2, HER3, and HER4, plays a critical role in tumorigenesis and tumor progression. Inhibition of ERBBs using either monoclonal antibodies (mAb) or small-molecule tyrosine kinase inhibitors have been approved for use for the treatment of various types of cancers. Despite relative success, many patients acquire resistance to long-term benefit due to alternative compensatory signaling pathways. Accordingly, activating mutations or overexpression of HER3 has been reported in many cancers, such as breast, ovarian, lung, colorectal, melanoma, head and neck, cervical and prostate cancers. Here, we report the discovery of a humanized anti-EGFR x HER3 IgG-like bispecific antibody-drug conjugate (ADC). The ADC takes a asymmetric 1+1 form through HC-HC and HC-LC charge-based heterodimerization, which is then conjugated to a topoisomerase 1 inhibitor (TOP1i) payload via a cleavable linker. The affinity of EGFR and HER3 was optimized to improve the therapeutical window, and has shown potent anti-tumor efficacy in various tumor models with limited toxicity supporting further development in clinic. Methods: An anti-EGFR x HER3 bispecific ADC (PM1300) was generated by introducing unique mutations to the CH1-CL domains of each binding arm to avoid HC-LC mispairing. KIH mutations were also introduced to each CH3 domain to support HC/HC heterodimerization and generate an IgG-like bispecific with one arm binding to EGFR and the other to HER3. TOP1i payload was conjugated to the IgG-like bispecific by the endogenous cysteine residues at DAR 8 via a clearable linker. Results: Through affinity optimization, PM300 was found to preferentially bind to EGFR/HER3 double-positive cells rather than EGFR single-positive cells. PM1300 showed significantly increased internalization and anti-proliferation activity against EGFR/HER3 double-positive cells, with limited activity towards single positive-cells. PM1300 used an optimized linker for better payload release and serum stability. PM1300 induced a potent anti-tumor efficacy in various CDX models representing different EGFR and HER3 expressing levels and mutations. Importantly, PM1300 exhibited a good safety profile in NHP consistent with the optimal affinity for EGFR and HER3. These results demonstrate that PM1300 has a promising efficacy/safety therapeutical window in preclinical models supporting further development in the clinic. Conclusion: A bispecific ADC targeting EGFR x HER3 was discovered, named PM1300, which has an asymmetric 1+1 IgG-like structure and optimized affinity. This allows for preferential binding to EGFR/HER3 double-positive cancer cells, rather than EGFR single-positive cells. This may significantly contribute to minimizing safety risk that is common for EGFR-targeting agents. Citation Format: Liandi Chen, Shaogang Peng, Chao Wang, Xiaoniu Miao, Yao Yan, Jun Xing, Weifeng Huang, Andy Tsun, Yingda Xu, Xiaolin Liu, Yi Luo. Discovery of an asymmetric IgG-like bispecific ADC targeting EGFR/HER3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5083.

Abstract 5152: Co-expression and prognostic significance of HER family members and EGFRvIII in patients with hepatocellular carcinoma

Abstract Hepatocellular carcinoma is one of the most common type of liver cancer. Despite advances in its treatment including the approval of several targeted agents in the past few decades, many patients do not respond or have a response of short duration to such therapeutic interventions. As a result, liver cancer is the third leading cause of cancer-related deaths worldwide. Therefore, it is essential to identify additional therapeutic targets and to develop more effective therapeutic interventions. In the past few decades, increased expression, and activation of the human epidermal growth factor receptor (HER) family members have been detected in various cancer types and of these, EGFR and HER2 are important therapeutic targets for the treatment of patients with a wide range of cancers, but currently not in liver cancer. In some studies, cross-talk between different members of the HER family has been associated with tumor progression and resistance to therapy with EGFR inhibitors but there has been no comprehensive study of the expression of all members of the HER family in patients with hepatocellular carcinoma. In this study, we examined the relative expression of all members of the HER family, type-III deletion mutant of EGFR (i.e.EGFRvIII), which is a tumor specific antigen, and two putative liver cancer cell biomarkers (i.e. CD44, EpCAM) in 43 patients with hepatocellular carcinoma by immunohistochemistry. The prognostic significance of such antigens was also investigated. Of these, the expression of EGFR, HER2, HER3, HER4 and EGFRvIII was found in tumor specimens from 35%, 58%, 0%, 19%, and 26% of the cases, respectively. Interestingly, at a cut off value of >5% of tumor cells with staining, 23%, 14% and 9% of the patients had co-expression of EGFR with HER2, EGFRvIII, and HER2/EGFRvIII. In addition, 40% and 33% of the cases were CD44 and EpCAM positive. Of these, the expression of EGFRvIII positivity at >5% ( HR=3.35, p=0.002) and 20% (HR=7.22, p=0.003) of tumour cells and membranous CD44 ( HR=2.12, p=0.04) were associated with a poorer survival in patients with hepatocellular carcinoma. In contrast, the expression of cytoplasmic CD44 was associated with a better overall survival (HR=0.30, p=0.02). These were found to be independent prognostic markers in multivariate analysis. Interestingly, the co-expression of EGFR with EGFRvIII was associated with a poorer overall survival (59.5 vs 21.0 months, p=0.004). Taken together, our results suggest that the expression of EGFRvIII occurs in patients with hepatocellular carcinoma and highlight its importance as biomarker for tumor progression and as a potential target for therapeutic intervention with drugs targeting EGFRvIII and other members of HER family. Citation Format: Ozlem Sherif, Said Khelwatty, Izhar Bagwan, Alan Seddon, Angus Dalgleish, Satvinder Mudan, Helmout Modjtahedi. Co-expression and prognostic significance of HER family members and EGFRvIII in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5152.

Unraveling Proto-Oncogene (ErbB2) Expression in Patients With Carcinoma Head of Pancreas and Chronic Pancreatitis Patients: A Case-Control Study.

Background The pre-malignant tendency of the normal, non-affected portion of the pancreas is not as well explored as the multicentricity documented in pancreatic cancer cases. In order to ascertain the expression of inflammatory markers and Erythroblastic Oncogene B (ErbB2) in the non-affected pancreas in patients with pancreatic cancer, a case-control study was carried out. Materials and methods In patients who underwent pancreatoduodenectomy for pancreatic cancer (PC), pro-inflammatory genes and a tumor marker, erythroblastic oncogene 2 (ErbB2) in the epidermal growth factor receptor family were analyzed in the pancreatic tissue at the cut surface of the normal pancreas using qRT-PCR. Twenty patients diagnosed with Chronic pancreatitis (CP) after Frey's surgical procedure were selected, and their pancreatic tissues were analyzed as controls. The HPLC-purified primers were designed using National Center for Biotechnology Information (NCBI) software. The primer's specificity was verified for gene expression analysis using the Basic Local Alignment Search Tool (BLAST). The genes under study were normalized using β-actin as the housekeeping gene, and the 2-ddct method was used to compute the fold change compared to the control sample. Results Patients with margin-positive were not included. Pro-inflammatory genes (TNF-α, NF-kβ,andCOX-2) had significantly lower foldchange in PC patients compared to the CP group. The CP control group had higher levels ofIL-6gene expression than the PC group. Patients with pancreatic cancer had a considerably higher expression of theErbB2gene than patients with CP. Conclusion The upregulatedErbB2gene in the unaffected pancreatic tissue of pancreatic cancer patients, when compared to controls, indicates that the remaining pancreas may have the capacity to cause cancer. Proto-oncogene may play a role in the pathophysiologic process in patients with pancreatic cancer.

Molecular docking and molecular dynamics approach to identify EGFR and HER2 receptors inhibitory effect of compounds in Zanthoxylum simulans Hance

AbstractEpidermal growth factor receptor (EGFR) and human epithelium receptor‐2 (HER2) are members of the epidermal growth factor receptor (EGFR/ErbB) family implicated in the development of many types of carcinoma. Zanthoxylum simulans Hance has the effect of inhibiting growth and causing the death of many types of cancer cells. In this study, the molecular docking method and molecular dynamics simulations were used to evaluate the inhibition abilities of EGFR and HER2 receptors of 120 compounds in Zanthoxylum simulans Hance. The results showed two compounds that inhibit both EGFR and HER2 targets including simulanoquinoline and 6‐acetonyldihydroavicine. Therefore, in vivo and in vitro studies of these potential compounds should be carried out to become anti‐cancer drugs in the future.

Romidepsin and Afatinib Abrogate Jak–Signal Transducer and Activator of Transcription Signaling and Elicit Synergistic Antitumor Effects in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphomas (CTCL) are mature lymphoid neoplasias resulting from the malignant transformation of skin-resident T cells. A distinctive clinical feature of CTCL is their sensitivity to treatment with histone deacetylase (HDAC) inhibitors. However, responses to HDAC inhibitor therapy are universally transient and non-curative, highlighting the need for effective and durable drug combinations. Here we demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an epidermal growth factor receptor (EGFR) family inhibitor, induces strongly synergistic antitumor effects in CTCL models in vitro and in vivo via abrogation of JAK-STAT signalling. These results support a previously unrecognized potential role for HDAC inhibitor plus afatinib combination in the treatment of CTCL.

A phase I dose-escalation study of pulsatile afatinib in patients with recurrent or progressive brain cancer.

Afatinib (BIBW2992; Gilotrif®) is a selective and irreversible inhibitor of the epidermal growth factor receptor (ErbB; EGFR) family. It inhibits EGFR, HER2, and HER4 phosphorylation, resulting in tumor growth inhibition and regression. This phase I dose-escalation trial of pulsatile afatinib examined the safety, drug penetration into the central nervous system, preliminary antitumor activity, and recommended phase II dose in patients with progressive or recurrent brain cancers. Afatinib was taken orally once every 4 days or once every 7 days depending on dose cohort, until disease progression or unacceptable toxicity. A total of 24 patients received the investigational agent and were evaluable for safety analyses, and 21 patients were evaluable for efficacy. Dosing was administered at 80mg every 4 days, 120mg every 4 days, 180mg every 4 days, or 280mg every 7 days. A recommended phase II dose of pulsatile afatinib was established at 280mg every 7 days as there were no dose-limiting toxicities in any of the dosing cohorts and all toxicities were deemed manageable. The most common drug-related toxicities were diarrhea, rash, nausea, vomiting, fatigue, stomatitis, pruritus, and limb edema. Out of the 21 patients evaluable for efficacy, 2 patients (9.5%) exhibited partial response based on Response Assessment in Neuro-Oncology criteria and disease stabilization was seen in 3 patients (14.3%). Afatinib taken orally was safe and well-tolerated up to 280mg every 7 days in brain cancer patients.

Expert Consensus on the Diagnosis and Treatment of NRG1/2 Gene Fusion Solid Tumors.

The fusion genes NRG1 and NRG2 , members of the epidermal growth factor (EGF) receptor family, have emerged as key drivers in cancer. Upon fusion, NRG1 retains its EGF-like active domain, binds to the ERBB ligand family, and triggers intracellular signaling cascades, promoting uncontrolled cell proliferation. The incidence of NRG1 gene fusion varies across cancer types, with lung cancer being the most prevalent at 0.19 to 0.27%. CD74 and SLC3A2 are the most frequently observed fusion partners. RNA-based next-generation sequencing is the primary method for detecting NRG1 and NRG2 gene fusions, whereas pERBB3 immunohistochemistry can serve as a rapid prescreening tool for identifying NRG1 -positive patients. Currently, there are no approved targeted drugs for NRG1 and NRG2 . Common treatment approaches involve pan-ERBB inhibitors, small molecule inhibitors targeting ERBB2 or ERBB3, and monoclonal antibodies. Given the current landscape of NRG1 and NRG2 in solid tumors, a consensus among diagnostic and treatment experts is proposed, and clinical trials hold promise for benefiting more patients with NRG1 and NRG2 gene fusion solid tumors.

Identification and engineering of potent cyclic peptides with selective or promiscuous binding through biochemical profiling and bioinformatic data analysis.

As our understanding of biological systems grows, so does the need to selectively target individual or multiple members of specific protein families in order to probe their function. Many targets of current biological and pharmaceutical interest are part of a large family of closely related proteins and achieving ligand selectivity often remains either an elusive or time-consuming endeavour. Cyclic peptides (CPs) occupy a key niche in ligand space, able to achieve high affinity and selectivity while retaining synthetic accessibility. De novo cyclic peptide ligands can be rapidly generated against a given target using mRNA display. In this study we harness mRNA display technology and the wealth of next generation sequencing (NGS) data generated to explore both experimental approaches and bioinformatic, statistical data analysis of peptide enrichment in cross-screen selections to rapidly generate high affinity CPs with differing intra-family protein selectivity profiles against fibroblast growth factor receptor (FGF-R) family proteins. Using these methods, CPs with distinct selectivity profiles can be generated which can serve as valuable tool compounds to decipher biological questions.

Abstract A165: FGFR alterations in pediatric cancers: Opportunity for targeted therapy

Abstract Introduction: The Fibroblast Growth Factor Receptor (FGFR) family consists of four members, FGFR1–4, and is involved in regulating cell proliferation, survival, and differentiation. FGFR alterations, such as fusions, mutations and amplification, have been found in multiple adult cancers, for which several FGFR inhibitors are in clinical development. Although cancer is a leading cause of death in children, the understanding of genetic causes of pediatric cancers is urgently lacking and the progress in developing targeted therapy in children significantly lags that in adults. This study aimed to uncover the FGFR alterations in pediatric cancers and provide preclinical rationale for FGFR inhibitors as potential therapy in children with cancer. Methods: The prevalence of FGFR alterations in pediatric cancers such as neuroblastoma (NB) , gliomas and sarcomas was calculated based on systematic queries of multiple cancer databases that house real-world molecular profiling data. A pan-FGFR inhibitor, BGJ398, was used as a tool compound for FGFR inhibition. Effect of BGJ398 on cell proliferation was tested by Incucyte in a panel of 12 NB cell lines. Cell migration, survival and signaling pathway modulation were carried out in a subset of NB lines. Results: The overall rates of genomic alterations (fusions, mutations and amplification) of FGFR1, 2, 3 and 4 in pediatric cancers are 0.5%, 0.1%, 1.1% and 0.8%, respectively, with pediatric gliomas exhibiting higher rates in all subtypes. Although FGFR genomic alterations are rare (<0.1%) in neuroblastoma and rhabdoid sarcoma, overexpression of FGFR1-4 is observed in subsets of these tumors. In pediatric glioma, FGFR1, 2, 3 and 4 alterations are found in 1%, 1.6%, 2.1% and 0.9% tumor samples, respectively, and the mutation profile substantially differs from that of adult glioma. Dose-dependent inhibition of cell proliferation and migration, as well as induction of cell death, were achieved with BGJ398 treatment in a panel of NB cells, accompanied by inhibition of MAPK pathway and induction of apoptosis. Conclusions: Adult and pediatric cancers share common mechanisms of FGFR activation, but differ in overall alteration rates and in relative abundance of specific aberrations. Preliminary preclinical data indicates the therapeutic potential of FGFR inhibitors in the treatment of certain pediatric cancers. Citation Format: Ivan Li, Yuchen Huo, Gary Li, Peter Zage. FGFR alterations in pediatric cancers: Opportunity for targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A165.