HER2-expressing Cells Research Articles

Evaluation of Potency and Specificity of Cryptophycin-Loaded Antibody-Drug Conjugates.

An enhanced variant of the antimitotic toxin cryptophycin was conjugated to the anti-Her2 monoclonal antibody (mAb) Trastuzumab upon Michael addition. Either antibodies with freed hinge-region cysteines or THIOMAB formats with engineered cysteines in the mAbs light chain were added to a maleimide derivative of cryptophycin. These Antibody-Drug Conjugates (ADCs) showed retained binding to Her2 positive tumor cells and highly efficient cell killing in double-digit pM range on high Her2-expressing SK-BR-3 cells. Two ADCs (DAR6, DAR3) showed superior cell killing of the cell lines JIMT-1 and RT112 with medium receptor expression level in comparison with a DAR6 MMAE ADC serving as reference. The observed cell cytotoxicity is target-dependent since no impact on cell viability was observed for low Her2-expressing MDA-MB468 cells. Particularly the DAR3 ADC in THIOMAB format exhibiting desirable biophysical properties and high potency emerged as a promising candidate for further in vivo investigations.

HER2 Nanobody-Poly(rhamnose) Conjugates Efficiently Recruit Anti-Rhamnose Antibodies from Serum to the Surface of HER2-Expressing Cells

HER2 Nanobody-Poly(rhamnose) Conjugates Efficiently Recruit Anti-Rhamnose Antibodies from Serum to the Surface of HER2-Expressing Cells

Influence of PEGylation on HER2-targeting retro A9 peptide analogue

Elevated levels of HER2 receptor in breast cancer can be targeted through receptor-specific peptides for precise detection and therapy by nuclear medicine approach. Previously reported retro analogue of A9 peptide had shown HER2-specificity with promising pharmacokinetic features. Hence, with an aim of further improving the circulation time of rL-A9 radiopeptide, long polyethylene glycol chain (PEG12) was introduced at the N-terminus of the peptide during solid phase synthesis and influence of PEGylation on biological profile was studied. [177Lu]Lu-DOTA-PEG12-rL-A9 demonstrated high specific cellular uptake (5.94 ± 0.09 %) in HER2-expressing human breast carcinoma SKBR3 cells and low nanomolar binding affinity (Kd = 34.58 ± 12.78 nM). Uptake in SKBR3 tumors induced in female SCID mice was higher at all the time points investigated (3, 24, 48 h) in comparison to the non-PEGylated radiopeptide, [177Lu]Lu-DOTA-rL-A9. Blocking studies led to 51 % reduction in accumulation of radioactivity in the tumor indicating specificity of the radiopeptide. Improved tumor-to-stomach and tumor-to-intestine ratios for [177Lu]Lu-DOTA-PEG12-rL-A9 compared to [177Lu]Lu-DOTA-rL-A9 at 48 h shall pave the way for better contrast and delineation of metastatic sites.

P2.10A.01 Phase 2 Study of Disitamab Vedotin in Patients with HER2-Expressing Non-Small Cell Lung Cancer (DV-005; Trial in Progress)

P2.10A.01 Phase 2 Study of Disitamab Vedotin in Patients with HER2-Expressing Non-Small Cell Lung Cancer (DV-005; Trial in Progress)

177Lu-labeling of nuclear localization sequence (NLS)-grafted HER2-receptor affine peptide

Tagging of cell permeable nuclear localization sequence (NLS) with receptor targeting peptide vectors is an attractive strategy for selectively targeted translocation of therapeutic cargoes. The present study aimed at grafting nuclear localization sequence (NLS) onto breast cancer targeting rL-A9 peptide. Molecular docking analysis revealed higher binding affinity of the peptide, DOTA-NLS-rL-A9 (−26.1 kJ/mol) towards HER2 receptor in comparison to DOTA-rL-A9 peptide (−22.2 kJ/mol). Confocal microscopy data suggested significantly enhanced cellular internalization of NLS-tagged peptide. The engineered HER2-selective, DOTA-NLS-rL-A9 peptide scaffold was radiolabeled with Lu-177 for intracellular delivery of the theranostic radionuclide into tumor cells. [177Lu]Lu-DOTA-NLS-rL-A9 exhibited significantly enhanced binding affinity (4.58 ± 1.77 nM) towards human breast carcinoma SKBR3 cells and cellular internalization (85 % at 24 h) compared to its original analog, [177Lu]Lu-DOTA-rL-A9. In vivo biodistribution studies showed consistent retention of [177Lu]Lu-DOTA-NLS-rL-A9 in the tumor with negligible washout of radioactivity (∼4.1 % ID/g at 48 h). Prolonged tumor activity with rapid off-target tissue clearance resulted in significantly high tumor-to-background ratios. The radiopeptide, [177Lu]Lu-DOTA-NLS-rL-A9 thus, being precisely confined into HER2-expressing tumor cells and exhibiting favourable pharmacokinetic features is an efficient candidate for further screening.

Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model.NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells.Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients.

Abstract PO4-25-12: Improving HER2 Low Scoring Consistency and Accuracy: Insights from the Australian HER2 Low Concordance Study for Invasive Breast Cancer

Abstract Introduction: The Destiny Breast-04 Trial (DB-04) demonstrated the survival benefits of Trastuzumab Deruxtecan (T-DXd) for women with metastatic HER2 Low breast cancer, characterised by 1+ or 2+ IHC staining without amplification. While the DB-04 study applied the standard 2018 ASCO CAP IHC scoring criteria, in clinical practice, distinguishing HER2 0 from 1+ cancers is challenging as i) HER2 Low is not a biologically distinct subset of breast cancer, ii) there are no reference standards for HER2 Low cancers, iii) second-tier test, like ISH, are not applicable, and iv) there are no known controls for cases that have 0 or 1+ HER2 scores. For two decades this distinction was clinically immaterial, but now differentiating between HER2 0 and 1+ has now become crucial for determining patient eligibility for T-DXd therapy. Concerns regarding the subjectivity, imprecision and poor concordance between pathologists in scoring IHC in HER2 Low cancers raise the potential for misalignments in patient treatment. Ensuring pathologists have access to focused training for interpreting IHC scores at the low end of the HER2 expression spectrum, quality assurance procedures and reference sets are essential steps to help pathologists assess HER2 Low breast cancers more accurately and consistently. Design: In this study, a group of 9 experienced breast pathologists compiled a deidentified set of 60 breast cancer core biopsies from 3 laboratories. The Ventana 4B5 HER2 assay had been used for evaluation and the local laboratories had scored the samples as HER2 0 or 1+. We teased out the ASCO CAP 2018 criteria and used our collective expertise of reporting HER2 IHC for many years to specify HER2 Low-focused scoring conventions, including some potential pitfalls. Subsequently, using these conventions, each pathologist reviewed digitized whole slide images of the IHC slides and scored HER2 expression for each case. At a consensus workshop, the cases were jointly reviewed to establish consensus scores and determine the percentage of HER2-expressing tumor cells in each case. We then evaluated the concordance between individual pathologists' HER2 scores and the consensus opinion and ascertained reasons for discordance. Results: Among the cases discussed during the consensus conference, 43 out of 60 (71.7%) were classified as HER2 Low, with 40 cases designated as 1+ and three as 2+ (known to be not amplified). The consensus score matched the majority opinion of the pathologists' independent scores in 93.3% (56 out of 60) of the cases. Utilizing the HER2 Low-focused IHC scoring conventions, 7 out of 17 (41.2%) cases locally reported as HER2 0 were reclassified as HER2 Low. Conversely, among the 32 cases with local scores of 1+, 7 (21.8%) were reclassified as ultralow or null. When compared to the consensus score, individual pathologists' scores demonstrated concordance levels ranging from 71.7% to 91.7%, with a mean concordance rate of 81.3%. Cases with less than 20% of tumor cells expressing HER2 had lower inter observer concordance. This reference set of cases with expert consensus HER2 scores obtained through our study will be invaluable for peer training and the development of external quality assurance programs for HER2 Low cancers, including the quality assurance program of the Royal College of Pathologists of Australasia. Conclusion: This study revealed that when breast pathologists were provided explicit instructions on scoring pitfalls and HER2 Low-focused scoring conventions, their HER2 scores were concordant with expert consensus scores in 71.7% to 91.7% of cases. Discordant cases primarily involved cases with less than 20% of tumor cells expressing HER2. Utilising such an approach, peer training and quality assurance procedures will improve the accuracy and consistency of HER2 IHC assessment for better patient care. Reassessing older cases using HER2 Low focused scoring conventions may result in revisions of HER2 scores from HER2 Low to zero, and vice versa. Table. Individual pathologists' concordance with the consensus HER2 IHC score Applying our HER2 Low-focused IHC scoring conventions in a set of 60 core biopsies of invasive breast cancer with low or 0 HER2 protein expression. Citation Format: Gelareh Farshid, Beena Kumar, Nirmala Pathmanathan, Hema Mahajan, Ben Dessauvagie, Jane Armes, Cameron Snell, Amardeep Gilhotra. Improving HER2 Low Scoring Consistency and Accuracy: Insights from the Australian HER2 Low Concordance Study for Invasive Breast Cancer [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 PO4-25-12.

Abstract PO1-04-07: Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical breast cancer models

Abstract Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that targets cancers expressing HER2. DV consists of an anti-HER2 monoclonal antibody, disitamab, conjugated with the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a cleavable vedotin linker. DV has multimodal antitumor mechanisms of action that include direct cytotoxicity of HER2-expressing cancer cells and bystander effect-based cytotoxicity of neighboring cells, both of which are mediated by the intracellular release of MMAE. Released MMAE can also induce immunogenic cell death, which promotes immune cell recruitment to the tumor. In addition, DV stimulates Fc-gamma receptor mediated antibody-dependent cellular cytotoxicity, which can lead to target cell death. DV also inhibits HER2-activated downstream signaling pathways, further blocking cell growth, survival, and proliferation. Previously, we showed the cytotoxic activity of DV against a panel of breast cancer cell lines with varying levels of HER2 expression, including the HER2-low range. Here, we sought to further characterize the preclinical antitumor activity of DV in patient-derived 3D models and patient-derived xenographs (PDX). In cultured breast cancer cells, DV was more potent and internalized to a greater magnitude than the HER2-directed ADCs trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). In patient-derived 3D models, DV had superior activity compared to T-DXd. We further explored whether dual HER2 targeting with DV in combination with the HER2-selective oral TKI tucatinib improved the antitumor outcomes. In PDX models with varying HER2 IHC levels and models refractory to T-DXd, DV as a monotherapy and in combination with tucatinib showed significant tumor growth inhibition. This result is consistent with previous in vitro results that showed increased cytotoxicity of the combination of DV and tucatinib in cell lines with a wide range of HER2 expression, which may be mechanistically attributed to elevated HER2 cell surface levels upon treatment with tucatinib. Overall, these findings provide scientific rationale to explore DV in HER2-positive and HER2-low breast cancer patients as a monotherapy or in combination with tucatinib. Citation Format: Kelsi Willis, Renee Hein, Katie Snead, Robert Thurman, Anita Kulukian. Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical breast cancer models [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 PO1-04-07.

Construction of Switch Modules for CAR-T Cell Treatment Using a Site-Specific Conjugation System.

Chimeric antigen receptor T-cell (CAR-T cell) therapy has become a promising treatment option for B-cell hematological tumors. However, few optional target antigens and disease relapse due to loss of target antigens limit the broad clinical applicability of CAR-T cells. Here, we conjugated an antibody (Ab) fusion protein, consisting of an Ab domain and a SpyCatcher domain, with the FITC-SpyTag (FITC-ST) peptide to form a bispecific safety switch module using a site-specific conjugation system. We applied the safety switch module to target CD19, PDL1, or Her2-expressing tumor cells by constructing FMC63 (anti-CD19), antiPDL1, or ZHER (anti-Her2)-FITC-ST, respectively. Those switch modules significantly improved the cytotoxic effects of anti-FITC CAR-T cells on tumor cells. Additionally, we obtained the purified CD8+ T cells by optimizing a shorter version of the CD8-binding aptamer to generate anti-FITC CD8-CAR-T cells, which combined with the CD4-FITC-ST switch module (anti-CD4) to eliminate the CD4-positive tumor cells in vitro and in vivo. Overall, we established a novel safety switch module by site-specific conjugation to enhance the antitumor function of universal CAR-T cells, thereby expanding the application scope of CAR-T therapy and improving its safety and efficacy.

Peptide-drug conjugate designated for targeted delivery to HER2-expressing cancer cells.

Targeted therapy of the highest globally incident breast cancer shall resolve the issue of off-target toxicity concurring with augmented killing of specific diseased cells. Thus, the goal of this study was to prepare a peptide-drug conjugate targeting elevated expression of HER2 receptors in breast cancer. Towards this, the rL-A9 peptide was conjugated with the chemotherapeutic drug doxorubicin (DOX) through a N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker. The synthesized peptide-drug conjugate, rL-A9-DOX, was characterized by mass spectrometry. Molecular docking studies, based on binding energy data, suggested a stronger interaction of rL-A9-DOX with the HER2 receptor in comparison to the unconjugated peptide, rL-A9. The cytotoxic effect of the rL-A9-DOX conjugate was observed to be higher in HER2-positive SKOV3 cells compared to HER2-negative MDA-MB-231 cells, indicating selective cell killing. Cellular internalization of the rL-A9-DOX conjugate was evident from the flow cytometry analysis, where a noticeable shift in mean fluorescent intensity (MFI) was observed for the conjugate compared to the control group. This data was further validated by confocal microscopy, where the fluorescent signal ascertained nuclear accumulation of rL-A9-DOX. The present studies highlight the promising potential of rL-A9-DOX for targeted delivery of the drug into a defined group of cancer cells.

Abstract LB058: N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models

Abstract Background: Current clinical application of trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC) has been severely limited by interstitial lung disease (ILD). Recent studies suggest a correlation between ILD and the uptake of T-DXd by alveolar macrophages (AMs), which are major innate immune cells in lung tissues. This underlying mechanism of AMs phagocytosing T-DXd was predicted to be the binding interaction between the crystallizable fragment (Fc) of trastuzumab and the Fcγ receptors on the cell surface of AMs, exerting effector functions. Since monoclonal antibodies typically undergo N-glycosylation in the Fc fragment, here we investigate the impacts of antibody N-glycan deglycosylation on T-DXd-induced ILD in preclinical models. Methods: N-glycan removal was performed on trastuzumab and T-DXd using PNGase F. Using immunofluorescence imaging combined with flow cytometry, we conducted a comparative analysis of the binding affinity and internalization efficiency of wild type and fully N-glycan deglycosylated HER2 antibodies. Next, we determined the in vitro cytotoxicity of WT and N-glycan deglycosylated T-DXd against both macrophages and HER2-positive cancer cells using CCK-8 assay. Furthermore, the deposition of N-glycan deglycosylated T-DXd in healthy lungs and their effects in ILD incidence and severity was determined in immunocompetent BALB/c mice carrying HER2-positive tumors. Results: First, we obtained complete N-glycan deglycosylated HER2 antibodies confirmed by SDS-PAGE and mass spectrometry. Next, we showed that N-glycan removal by PNGase F did not affect the Fab fragments of HER2 antibodies binding to the HER2 antigens on HER2-expressing cancer cells (OE19, SK-OV-3 and CT26-hHER2). Importantly, we did observe an approximately 30-50% reduction in the N-glycan deglycosylated HER2 antibodies binding with a panel of three macrophages (MH-S, RAW264.7, and PMA-induced THP-1) in comparison with WT antibodies, indicating a significant impact of N-glycan removal on innate immune cell-mediated effector functions. Moreover, in vitro cytotoxicity studies further support these observations that N-glycan deglycosylated T-DXd has significantly lower cytotoxicities against MH-S (p=0.046) and RAW264.7(p=0.020) in comparison with WT T-DXd. Correlating with our in vitro findings, in vivo biodistribution data showed a reduced lung deposition of HER2 antibodies and T-DXd after N-glycan removal. Immunofluorescence staining confirms the co-localization of HER2 antibodies with CD68-positive macrophages, indicating that AMs are the major contributor to the lung deposition of HER2 antibodies and T-DXd and N-glycan removal could effectively avoid ADC being phagocytosed by AMs. We also demonstrated chronic administration of N-glycan deglycosylated T-DXd could reduce ADC-associated ILD incidence and ameliorate ILD severity. Conclusions: Collectively, our study suggests that antibody N-glycan deglycosylation has important roles in regulating interactions between ADC and innate immune cells. This finding provides valuable clues for further optimizing ADC design and treatment strategies. Citation Format: Yu-Xuan Yang, Le-Tao Ma, Peng Guo. N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models [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 LB058.

Abstract CT098: Phase I study of GQ1005, a new generation HER2-ADC, in patients with HER2-expressing and HER2 mutated advanced solid tumors

Abstract Background: GQ1005 is a new generation antibody-drug conjugate, targeting HER2-expressing tumor cells with Topoisomerase I inhibitors (DXd) as its payload via a stable and cleavable linker. In preclinical studies, GQ1005 demonstrated comparable antitumor activity compared to Enhertu in multiple tumor cell line xenograft models with no noticeable toxicity, indicating higher therapeutic index. GLP toxicity studies demonstrated that GQ1005 was well tolerated, with the highest non-severe toxic dose determined to be 60mg/kg. This report presents the preliminary findings of an ongoing phase I study, which aims to evaluate the safety, tolerability, pharmacokinetics, and antitumor activity of GQ1005 in patients with advanced solid tumors expressing HER2 or harboring HER2 mutations. Methods: GQ1005 was administrated intravenously every 3 weeks. An accelerated titration design was employed for the initial 2mg/kg dose, followed by Bayesian Optimal Interval Design for subsequent dose levels of 4, 6, 7.2, and 8.4 mg/kg. The primary objective of this study was to evaluate the safety, tolerability, and to determine the MTD or RP2D. Results: As of Oct. 13th, 2023, 46 (30 in dose-escalation and 16 in dose-expansion phase) subjects with HER2-expressing/mutated advanced solid tumors, predominantly in breast cancer (18), and lung cancer (18) , were enrolled into the study. Patients received a median of 3 (range, 1-11) prior lines of treatment, and 86.4% of pts previously received anti-HER2 therapies before. During dose escalation, No DLT was observed in all doses, MTD was not reached up to 8.4 mg/kg. Treatment-related adverse events (TRAEs) occurred in 44 (95.7%) subjects. The most common TRAEs were grade 1 or 2, including nausea (52.2%), vomiting (39.2%), aspartate aminotransferase (AST) increased (39.1%), alanine aminotransferase (ALT) increased (23.9%), anemia (23.9%), anorexia (21.7%), fatigue (19.6%), leukopenia (15.2%), infusion-related reactions (15.2%) , decreased lymphocyte count (15.2%) and dizziness (15.2%). Grade ≥3 TRAEs only occurred in 4 subjects (8.7%), including anemia, decreased lymphocyte count and γ-glutamyltransferase (GGT) increased. Interstitial lung disease was observed in 6 pts received highest dose level and most of them have recovered. No drug-related deaths. The pharmacokinetics analysis showed the concentration of GQ1005 and TAb generally peaked rapidly and declined in a roughly biphasic manner. Among 38 evaluable subjects, 1 pt achieved complete response, 18 cases achieved confirmed partial response, and 11 had stable disease, the objective response rate was 50% (19/38), the disease control rate was 78.9% (30/38) , and the 6-month PFS rate was 43.0% in all patients. Conclusions: GQ1005 demonstrated a promising antitumor activity in HER2-expressing/mutated advanced solid tumors patients previously heavily treated, and showed a good tolerance and manageable safety profile. The dose expansion study is on-going at 7.2 mg/kg to establish the RP2D in four cohorts. (NCT06154343; sponsored by GeneQuantum Healthcare (Suzhou) Co., Ltd.) Citation Format: Caicun Zhou, Bo Yang, Ting Deng, Biyun Wang, Jian Zhang, Yuping Sun, Linlin Wang, Hongwei Yang, Jingfen Wang, Wei Li, Qi Song, Yuchong Yang. Phase I study of GQ1005, a new generation HER2-ADC, in patients with HER2-expressing and HER2 mutated advanced solid tumors [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 CT098.

Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors.

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three 99mTc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G3C), (Gly-Gly-Gly-Ser)3-Cys ((G3S)3C), or Glu-Glu-Glu-Cys (E3C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with 99mTc(CO)3 using the (HE)3-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50-80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5-3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of 99mTc-labeled DARPin G3 variants to HER2 in vitro; however, [99mTc]Tc-G3-(G3S)3C-HYNIC had the highest affinity. Comparative biodistribution of [99mTc]Tc-G3-G3C-HYNIC, [99mTc]Tc-G3-(G3S)3C-HYNIC, [99mTc]Tc-G3-E3C-HYNIC, and [99mTc]Tc-(HE)3-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [99mTc]Tc-G3-E3C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [99mTc]Tc-G3-(G3S)3C-HYNIC and [99mTc]Tc-(HE)3-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [99mTc]Tc-G3-(G3S)3C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [99mTc]Tc-(HE)3-G3. Radiolabeling of DARPin G3-HYNIC conjugates with 99mTc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the 99mTc-tricarbonyl-labeled DARPin G3. At this stage, [99mTc]Tc-(HE)3-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.

Abstract 3190: Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement

Abstract Immunoglobulin G (IgG)-based mAbs have become a dominant class of cancer immunotherapy due to their excellent drug-like properties, long half-life, and generally safe profiles. Typical IgG-based therapeutic mAbs have strong binding to target antigens owing to the valency effect of two antigen-binding fragments (Fabs). However, the single crystallizable fragment (Fc) lacks the valency effect to engage immune effector elements. As Fcγ receptors (FcγR) such as FcγRIIa and FcγRIIIa and C1q complement component have low affinity for Fc, IgG-based mAbs require high antibody opsonization density to exhibit avidity for these low affinity Fcγ receptors and C1q. This may also account for the suboptimal efficacy of IgG-based mAbs against tumors with less abundant antigen. To enhance effector functions of IgG1 by overcoming such limitation, we devised a novel platform, Twin Fc-ICE, in which a single Fab region is attached by two Fc regions which allow for high avidity binding to Fcγ receptors and C1q. The two Fc regions of Twin Fc-ICE are arranged in parallel, not in tandem, to adopt natural topologies of multiple Fc regions presented by IgG1-opsonized targets. Supporting the avidity effect of Twin Fc-ICE for FcγR binding, we showed that CTN001, a Twin Fc-ICE with trastuzumab Fab, have markedly increased binding to FcγRs including FcγRI, FcγRIIa and FcγRIIIa compared with trastuzumab. The avid FcγR binding was further demonstrated by flow cytometry analysis of human blood which revealed significantly increased binding of CTN001 relative to trastuzumab for immune cells expressing activating FcγRs such as natural killer (NK) cells, monocytes, and dendritic cells (DCs). The monovalent antigen binding of Twin Fc-ICE requires Fab with high affinity. However, provided that high affinity is achieved, the monovalent Fab adds to the two Fc regions to maximize the Fc load on cancer cells expressing low levels of antigen. Indeed, flow cytometry analysis revealed that treatment of CTN001 alone results in higher Fc load on HER2-expressing cancer cells than combination of two anti-HER2 IgG1 mAbs, trastuzumab and pertuzumab. In consistent with increased Fc load on target cells and avid FcγR binding, CTN001 induces enhanced effector functions such as CDC, ADCC and ADCP against HER2-expressing cancer cells compared with trastuzumab, which will be presented in a separate poster. CTN001 exhibits comparable thermal stability with trastuzumab and can be readily purified to >95% purity with typical purification platforms. The serum half-life of CTN001 at 10 mg/kg intravenous dose in rats is found non-inferior to that of trastuzumab (~12 days versus ~7 days). Taken together, Twin Fc-ICE provides an optimized platform with drug-like properties, which leverages avidity effects of Fc to potentiate therapeutic efficacy of antibody. Citation Format: Eun Shik Choi, Hyunkyu Park, Jiseon Bae, Ki-June Lee, Eunmi Kim, Sujin Yoon, Deokjae Lee, Gi-Hyeok Yang. Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement [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 3190.

Abstract 1901: Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy in preclinical urothelial cancer models

Abstract Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that selectively delivers cytotoxic drug to HER2-expressing cancer cells. DV is composed of disitamab, a HER2-directed monoclonal antibody, and a vedotin linker-payload system, which enables conjugation of the microtubule-disrupting agent monomethyl auristatin E (MMAE) to the antibody via a protease-cleavable linker. The proposed mechanism of action (MOA) of DV occurs through direct cytotoxicity of HER2-expressing tumor cells following internalization of DV and intracellular release of MMAE. DV may also induce antitumor activity through bystander-mediated cytotoxicity of neighboring cells and via the inhibition of HER2 signaling pathways. Moreover, cellular response to MMAE includes the induction of immunogenic cell death and recruitment of immune cells to the tumor site. DV is currently being evaluated globally in subjects with HER2-expressing locally advanced or metastatic urothelial carcinoma (la/mUC) and has gained conditional approval in China following an overall response rate of 50.5% (pooled analysis from studies NCT03507166 and NCT03809013). RNA sequencing data from a large cohort of urothelial cancer patient tumors revealed expression of HER2 in many urothelial tumors; however, the relationship between HER2 expression and efficacy of DV is not well understood. Here, we sought to evaluate the antitumor activity of DV monotherapy in preclinical urothelial cancer models with varying levels of HER2 expression. Multiple patient-derived xenografts (PDX) and patient-derived 3D in vitro urothelial cancer models showed potent antitumor response to DV monotherapy in tumors with varying HER2 expression levels including HER2-low tumors. Overall, these findings support ongoing clinical trials in subjects with HER2-expressing la/mUC and provide scientific rationale to further explore DV monotherapy in HER2-positive and HER2-low urothelial cancer clinical settings. Citation Format: Renee Hein, Kelsi Willis, Gina LoMastro, Suhas Vasaikar, Vinita Gupta, Katie Snead, Sharsti Sandall, Anita Kulukian. Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy in preclinical urothelial cancer models [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 1901.

Abstract 6354: The HER2/4-1BB bispecific antibody, YH32367 (ABL105) demonstrates optimal efficacy and safety for HER2-expressing tumors and exhibits synergistic efficacy when combined with anti-PD-L1

Abstract Overexpression/amplification of the human epidermal growth factor receptor 2 (HER2) is associated with clinically aggressive subtypes in various cancers. Currently, new HER2-targeted therapy like trastuzumab deruxtecan have improved outcomes not only for HER2-positive tumors but also for HER2-low tumors. However, curing cancer patients who develop resistance to current HER2-targeted therapies or experience unwanted side effects remains a significant challenge. Therefore, there is a pressing need for more advanced treatments, and one promising approach involves combination with immunotherapy. 4-1BB (CD137) is a key costimulatory receptor expressed on activated T-cells and natural killer cells, making it a promising therapeutic target in cancer. This study presents the anti-tumor efficacy of the HER2/4-1BB bispecific antibody, YH32367 (ABL105), against both HER2-positive and HER2-low tumors. Additionally, the study suggests that YH32367 could be a viable treatment option for combination with anti-PD-1 antibody (Ab).​ YH32367 has been designed to overcome the challenges with HER2 resistance via tumor-directed 4-1BB agonism. YH32367 induced IFN-γ secretion and thereby eliciting tumor cell death in co-culture condition with hPBMC and HER2-expressing tumor cells. YH32367 showed superior efficacy in eradicating tumors and triggered long-term anti-tumor immunity in MC38/hHER2-bearing h4-1BB KI mouse models, compared to benchmark 4-1BB/HER2-bispecific molecule and anti-HER2 Ab. Even in HER2-low tumor, YH32367 was more effective than benchmark 4-1BB/HER2-bispecific molecule. Additionally, when YH32367 was administered in combination with anti-PD-1 Ab to h4-1BB KI mice bearing HER2-low tumors, a synergistic effect was observed, surpassing the effects of anti-PD-1 Ab alone or anti-HER2 Ab-drug conjugate. The potent anti-tumor efficacy of YH32367 was also verified in HER2 expressing tumor-bearing hPBMC humanized mice. In terms of toxicity, YH32367 demonstrates a favorable safety profile, which was confirmed through a 4-week GLP toxicity study using cynomolgus monkeys at doses of 10, 30, and 100 mg/kg. In summary, YH32367 has demonstrated significant anti-tumor effects, impacting tumor immunity in h4-1BB KI mice with HER2-low and HER2-positive tumors. Furthermore, its combination with anti-PD-1 Ab has shown synergistic efficacy, indicating that YH32367 may offer treatment options not only as a monotherapy but also in combination therapy for HER2 expressing tumors. The phase 1 dose escalation study is currently underway (NCT05523947), and a dose optimization study to determine the recommended phase 2 dose (RP2D) is scheduled for the second half of 2024. Citation Format: Eunjung Lee, Young Bong Park, Minji Choi, Jangwoo Shin, Kyong Bae Kim, Ju Young Park, Junhwan Kim, Kyoung Kyu Ahn, Yangsoon Lee, Kyeongsu Park, Wonjun Son, Donghoon Yeom, Jaehyun Eon, Hanbyul Lee, Hanbyul Jung, Jonghwa Won, Se-Woong Oh, Yeul Hong Kim. The HER2/4-1BB bispecific antibody, YH32367 (ABL105) demonstrates optimal efficacy and safety for HER2-expressing tumors and exhibits synergistic efficacy when combined with anti-PD-L1 [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 6354.

Abstract 4692: A novel ceramide synthase 2 agonist and its implication in triple-negative breast cancer therapy

Abstract Based on the biological/molecular markers, breast cancer can be classified into five subgroups: Luminal A (ER+/PR+/HER2-), Luminal B (ER+/PR-/HER2- or ER+/PR+/HER2+, triple-positive), ER-/PR-/HER2+, and ER-/PR-/HER2- (triple-negative). Significant progress has been made in treating hormone-responsive and HER2-expressing breast cancer. However, the targeted therapies are limited for triple-negative breast cancer (TNBC) due to lack of well-defined druggable targets. Additionally, all breast cancer subtypes can quickly develop resistance to current treatments. Thus, there is an urgent need for therapeutic strategies targeting pathways common to all subtypes, irrespective of receptor status. In this effort, we have synthesized a novel biisoquinoline-derivative, DH20931 (US Patent ID: US9914733B2) that induces cytotoxicity in breast cancer cells, including TNBC cells. The spatial molecular modeling and enzymatic assays show that DH20931 interacts with and stimulates ceramide synthase 2 (CerS2) activity, reduces the growth of hormone-responsive, HER2-expressing, and TNBC cells in monolayer and 3D spheroid culture models. DH20931 treatment increases the level of very long chain fatty acid (VLCFA) containing ceramides, induces lipotoxic endoplasmic reticulum (ER) stress, and ATF4/CHOP/PUMA pathway of apoptosis. Based on Swiss-ADME analysis, DH20931 possess all the properties of a druggable molecule and obeys the Lipinski-rule-of-five. Thus, DH20931 can be developed as a novel targeted therapeutic agent independent of ER, PR and HER2 status that potentially may reduce the morbidity and mortality for women with breast cancer. Citation Format: Hissah Alatawi, Nayeong Koo, Iqbal Mahmud, Haritha Nair, Amandeep Singh, Timothy J. Garrett, Arun K. Sharma, Sukwon Hong, Satya Narayan. A novel ceramide synthase 2 agonist and its implication in triple-negative breast cancer therapy [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 4692.

Abstract 4005: HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC

Abstract Approximately 3% of non-small cell lung cancer (NSCLC) cases harbor ERBB2/HER2 mutations. Notably, the majority of actionable HER2 mutations in NSCLC are in-frame insertions in exon 20. Current HER2-targeting approaches, including antibody-drug conjugates (ADCs) such as trastuzumab deruxtecan, have shown promising response rates. To explore novel strategies for targeting HER2-positive NSCLC, we investigated chimeric antigen receptor (CAR)-based therapies to enhance the anti-tumor activity of immune effector cells. We engineered a series of HER2 CAR constructs utilizing scFvs derived from trastuzumab, pertuzumab, and FRP5, recognizing distinct domains of HER2. These scFvs were integrated into second-generation BB3z (with 4-1BB and CD3z intracellular domains) and third-generation 28BB3z (with CD28, 4-1BB, and CD3z intracellular domains) CAR backbones, which were subsequently transduced into activated T and natural killer (NK) cells to generate CAR-T and CAR-NK cells, respectively. High transduction efficiencies were achieved, with over 80% in CAR-T cells and over 50% in CAR-NK cells. We next evaluated their activity against a panel of NSCLC cell lines expressing varying levels of HER2 in vitro using firefly luciferase assays. All HER2 CAR-T cells demonstrated more than 90% killing against H2170 (HER2 high) and H322 (HER2 medium) at an effector:target (E:T) ratio of 10:1, whereas HER2 CAR-T cells yielded a killing effect of 35% against A549 (HER2 low) cells. HER2-targeting CAR-NK cells also displayed potent cytotoxicity against HER2-expressing NSCLC cells. Moreover, we observed that treatment of HER2 mutant and HER2 amplified NSCLC cells with low doses of HER2 tyrosine kinase inhibitors (TKIs) such as poziotinib enhanced the expression of HER2 on the cell surface. This upregulation resulted in enhanced activation of HER2 CAR-T and CAR-NK cells in coculture experiments as measured by CD107a expression on effector cells. Additionally, treatment with the HER2 TKI poziotinib potentiated the killing activity mediated by HER2 CAR-T and CAR-NK cells. Using a HER2 YVMA duplication patient-derived xenograft (PDX) NSCLC model, we observed that the in vivo combination of poziotinib with HER2 CAR-NK cells exhibited superior anti-tumor activity as compared to poziotinib treatment alone. In conclusion, HER2 CAR-T and CAR-NK cells effectively target HER2-mutant and HER2-positive NSCLC, and their combination with HER2 TKIs enhances vulnerability to HER2-targeting strategies. Citation Format: Yan Yang, Monique B. Nilsson, Alissa Poteete, Junqin He, Qian Huang, John V. Heymach. HER2 TKIs enhance the anti-tumor activity of HER2-targeting CAR-T and CAR-NK cells against NSCLC [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 4005.

Abstract 6352: Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types

Abstract Traditional antibody-drug conjugates leverage the specificity of antibodies to deliver cytotoxic payloads to tumor microenvironments (TMEs), thereby reducing systemic toxicities associated with non-targeted chemotherapeutics. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to selectively deliver synthetic immune agonists to the TME without systemic immune activation. The first generation of ISACs and the majority of approved and investigational ADCs contain payloads linked to native lysine or cysteine residues, which can lead to heterogenous drug-antibody ratios (DARs), reduced antibody stability, and labile conjugation, all of which limit therapeutic index. Here we describe the generation and preclinical characterization of ARX622, a site-specific TLR7-selective agonist ISAC targeting HER2. To avoid the instability issues of previous generation ADCs and ISACs, ARX622 has a homogenous and stable DAR via oxime bond-mediated payload conjugation to a synthetic amino acid, para-acetyl phenylalanine (pAF), which is recombinantly incorporated at defined heavy chain positions. Similar to cytotoxic ADCs built on this site-specific conjugation platform, ARX622 ISAC and total mAb pharmacokinetic (PK) profiles in non-human primates (NHPs) are indistinguishable, demonstrating in vivo stability and minimal payload deconjugation. Accordingly, ARX622 displays an extended PK profile in mice compared to a DAR-matched random cysteine ISAC containing the same antibody and payload. In the presence of HER2-expressing cells, ARX622 selectively induces proinflammatory cytokine production, type I and III IFN secretion, and APC maturation. In a syngeneic tumor model, ARX622 promotes complete tumor regressions, immunologic memory, and epitope spreading. In xenograft systems, single doses of ARX622 induce complete regression of large, established tumors (>1000mm3), and robustly inhibit tumor growth in mice that have progressed through prior treatment with HER2-targeted mAbs or ADCs. In HER2-low xenografts, ARX622 monotherapy inhibits tumor growth, and promotes complete regression of late-stage (800mm3) tumors when sequentially combined with a HER2-ADC. Based on PK data at efficacious doses in mice and tolerated exposures in NHP toxicity studies, ARX622 has an estimated therapeutic index of ~60x. Taken together, ARX622’s mAb-like properties, high stability, broad immune mechanism induction, strong anti-tumor activity alone or in combination with ADCs, and wide therapeutic index make it a strong candidate for further development toward treating patients with a variety of HER2-expressing solid tumors. Citation Format: David Mills, Ji Young Kim, Erica Wood, Keith Tatsukawa, Nick Knudsen, Jay Nelson, Lillian Skidmore, Kedar GC, Hon Tran, Manoj Pal, Hamidreza Hashemi, Ying Buechler, Shawn Zhang, Dan O'Connor. Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types [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 6352.

Preparation of trastuzumab-DM1 conjugate with a high drug-to-antibody ratio for breast cancer therapy

Antibody-drug conjugates (ADCs) are important in active tumor-targeting therapy. However, improving the efficiency of payload delivery to tumors without affecting the tumor-targeting ability of the antigen-binding fragment (Fab) structure remains a challenge. In this study, a Fab-nondestructive ADC loaded with maytansine (DM1) (T-NPLG-DM135) was prepared with drug-to-antibody ratio (DAR) of up to 35 by utilizing the carboxyl groups on the side-chain of poly(L-glutamic acid) (PLG) as the payload binding sites. The use of the Fc-III-4C peptide and the preparation condition of aqueous environment were conducive to avoiding the impact of the Fab structure of monoclonal antibodies on tumor-targeting capability. T-NPLG-DM135 was recognized and rapidly internalized by HER2-expressing SKBR-3 tumor cells, with a 3.8-fold increase in accumulation compared to that of the non-tumor-targeting IgG-NPLG-DM135. T-NPLG-DM135 also showed excellent tumor inhibition and its therapeutic effect improved as the DAR increased. The successful development of T-NPLG-DM135 demonstrates the potential of this approach for future ADC design and development.