HER2-expressing Breast Cancer Research Articles

Preliminary safety and efficacy of TQB2930, a HER2-targeted bispecific antibody in patients with advanced breast cancer: Results from a phase 1b study.

1026 Background: TQB2930 is a HER2-targeted bispecific antibody, binding to two distinct HER2 epitopes, the same domain as trastuzumab (ECD4) and pertuzumab (ECD2). This study aims to evaluate the safety and efficacy of TQB2930 for patients with advanced breast cancer after failure of standard therapy. Methods: This phase 1b, dose-escalation and expansion trial included patients preferred HER2-expressing or HER2-amplified breast cancer who had received standard therapy. In the dose-escalation phase, TQB2930 was given weekly (10mg/kg, QW), biweekly (20mg/kg, Q2W), or triweekly (30mg/kg, Q3W). After the dose-escalation phase, eligible patients were enrolled and dosed biweekly or triweekly in the expansion cohort. The primary endpoints were safety, maximum tolerated or maximum administered dose, and the secondary endpoints were immunogenicity, pharmacokinetics, and tumor response per RECIST v1.1. Results: As of Dec 20, 2023, 34 patients(pts) have received TQB2930 weekly (n=3), biweekly (n=16), and triweekly (n=15). All pts were female, the median age was 59. 22 pts had HER2 expression of ICH3+ or ICH2+ with FISH+, and the others had ICH2+ with FISH- or ICH1+. The median prior lines of therapies were 3 (range: 0-8), with one pt intolerance to first-line treatment enrolled, and the median prior lines of HER2-targeted therapies were 2 (range: 0-8). No dose-limited toxicity was observed. The main treatment-related adverse events (TRAEs) were infusion reaction, occult blood of urine, leukopenia, and neutropenia. 3 pts were reported grade≥3 TRAEs, including neutropenia, anemia, and hypokalemia. Of 31 response-evaluable pts, the objective response rate (ORR) was 25.8% with 8 partial responses (PRs) were observed (including 5 confirmed PRs and 3 PRs awaiting confirmation). The disease control rate was 80.6%. Moreover, 5 pts achieved PR at 30mg/kg (ORR 41.7%), including one with prior exposure to trastuzumab, pyrotinib, tucatinib, ARX788, and the other one prior to trastuzumab, pertuzumab, lapatinib, MRG002. The median progression-free survival for all pts was 5.5 months (95%CI 3.58-NE). Additionally, Pharmacokinetic analysis showed exposure (Cmax and AUC0-t) of TQB2930 increased linearly with doses within 10~30mg/kg. After dosing, the mean peak concentration reached at 2.5-4.3 h, and the elimination half-life of TQB2930 was 109-161 h. Conclusions: TQB2930 is well tolerated and has demonstrated promising single-agent anti-tumor activity in HER2-positive breast cancer who have failed standard anti-HER2 therapies, including multiple lines of prior HER2 targeted agents. These early signs of activity support the further exploration of combination therapy of TQB2930 and the study is ongoing. Clinical trial information: NCT06202261 .

Abstract PO5-04-06: Enhancing Antitumor Activity of Her2 CAR T cells through TR2BB Co-Expression and Cytokine Signal 3 Incorporation

Abstract Chimeric antigen receptor T cells (CARTs) targeting Her2 represent a promising approach for the treatment of patients with Her2 expressing breast cancers (BC). Her2 CARTs confront major challenges within the immune-suppressive tumor microenvironment (TME) of breast cancer, which inhibits sustained T cell proliferation and persistent antitumor activity. To bolster Her2 CARTs' expansion and persistence, we engineered a TNF-related apoptosis-inducing ligand receptor 2 (TR2) specific scFv connected to the endodomain of 4-1BB (TR2BB). TR2BB co-expressing Her2 CARTs exhibited enhanced antitumor potential against BC tumors by triggering myelod derived suppressor cell (MDSC) apoptosis, thereby remodeling the TME and promoting T cell proliferation. Despite better tumor control in mice receiving CAR.TR2BB T cells, recent trials have demonstrated the importance of incorporating a cytokine signal for optimal tumor control in patients with solid tumors. Thus, we hypothesized that we could further augment anti-tumor activity of Her2 CAR.TR2BB T cells against BC by incorporating a cytokine signal 3 through IL-15 or C7R. We engineered the CARTs to (1) ectopically produce IL-15, a crucial T-cell homeostasis and survival cytokine, and (2) express a constitutively active IL-7 receptor (C7R) that triggers IL-7 receptor signaling independently of ligand or the common receptor gamma chain (γc). Our repeat weekly tumor challenge assay revealed that CAR.TR2BB.IL15 T cells outperformed CAR.TR2BB.C7R and CAR.TR2BB in T cell proliferation. Both CAR.TR2BB.IL15 and CAR.TR2BB.C7R exhibited improved cytotoxic potential after three weeks of antigen stimulation compared to CAR.TR2BB alone in comparison with CAR.TR2BB alone. Furthermore, in our in vivo xenograft NSG mouse models implanted with Her2 expressing breast cancer cell line and MDSCs, CAR.TR2BB.IL15 T cells showed superior tumor control and improved survival rates. In summary, the addition of IL15 transgene to our TME targeting Her2 CARTs provides an advantage in terms of T cell persistence, proliferation, and tumor control in in vitro and in vivo models. These promising preclinical results propel us to apply this optimized Her2 targeting CART therapy in a phase I clinical trial for patients with metastatic Her2-expressing breast cancers. Citation Format: Diego Chamorro, Lauren Somes, Emily Madaras, Saisha Nalawade, Paul Shafer, Andres Mosquera, Mary Kathryn McKenna, Valentina Hoyos. Enhancing Antitumor Activity of Her2 CAR T cells through TR2BB Co-Expression and Cytokine Signal 3 Incorporation [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 PO5-04-06.

Abstract PO3-20-06: TRUDI: A phase II study of neoadjuvant TRastuzumab derUxtecan and Durvalumab for stage III HER2-expressing Inflammatory breast cancer

Abstract Background: Despite being a rare presentation of breast cancer, inflammatory breast cancer (IBC) is responsible for up to 10% of all breast cancer-related deaths. Most IBCs express HER2, with up to 40% of all IBCs being HER2-positive and 40% being HER2-low. Additionally, IBC more often exhibits PD-L1 expression compared to non-IBC, suggesting a preeminent role for immune evasion in the development and progression of IBC. No systemic treatment approaches have been yet developed specifically for IBC, and standard treatments commonly lead to poor outcomes. Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate currently approved for patients with pretreated HER2-positive and HER2-low metastatic breast cancer. Early-phase data highlighted the safety and potential synergy of combining T-DXd with the immune checkpoint inhibitor durvalumab. The aim of TRUDI is to evaluate the clinical activity and safety of neoadjuvant T-DXd with durvalumab among patients with HER2-expressing IBC. Trial Design: TRUDI is an ongoing, open label, multicenter, two cohort investigator-initiated phase II neoadjuvant trial for patients with stage III HER2-expressing IBC. The trial was activated in May 2023, with enrollment ongoing. Eligible participants are women and men with previously untreated, stage III (T4d, any N) breast cancer, clinically determined to be IBC. Patients will be included in two cohorts, depending on the locally determined HER2 status (with any hormone receptor [HR] status): Cohort 1 (n=36) for patients with HER2-positive disease (HER2 IHC 3+ or 2+/ISH amplified); Cohort 2 (n=27) for patients with HER2-low disease (HER2 IHC 1+ or 2+/ISH not amplified). Patients will receive eight cycles of T-DXd (5.4 mg/kg IV every 21 days) combined with durvalumab (1125 mg IV every 21 days), followed by modified radical mastectomy and post-mastectomy radiation. Post-surgical systemic treatment will follow local standards. Tumor tissue will be collected at baseline, cycle 1 day 8, and at surgery; blood will be collected at baseline, cycle 4 day 1, cycle 7 day 1 and at surgery; stool will be collected at baseline, after 3-6 weeks of treatment and surgery. The primary endpoint is pathologic complete response (pCR; ypT0/Tis ypN0). Patients in each cohort will be enrolled based upon Simon two-stage designs: in Cohort 1, if ≥8/20 patients with HER2-positive IBC experience pCR then a total of 36 patients will be enrolled. In Cohort 2, if ≥1/18 patients with HER2-low IBC experience pCR then a total of 27 patients will be enrolled. Secondary endpoints include the residual cancer burden at surgery, event free survival, distant progression or distant disease-free survival, as well as safety of the regimen. Exploratory objectives will investigate biomarker analyses on tissue, blood, and microbiome to explore the association of HER2 expression, immune variables, and stool composition on the efficacy of T-DXd with durvalumab among patient with HER2-expressing IBC. To our knowledge, this is the first and only ongoing study testing the combination of an anti-HER2 antibody-drug conjugate with immunotherapy for the neoadjuvant treatment of patients with IBC. Clinical trial information: NCT05795101. Citation Format: Paolo Tarantino, Samuel Niman, Antonio Giordano, Faina Nakhlis, Jennifer Bellon, Wendy Woodward, Azadeh Nasrazadani, Sadia Saleem, Anthony Lucci, Michelle DeMeo, Naoto Ueno, Sara Tolaney, Vicente Valero, Meredith Regan, Rachel Layman, Filipa Lynce. TRUDI: A phase II study of neoadjuvant TRastuzumab derUxtecan and Durvalumab for stage III HER2-expressing Inflammatory 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 PO3-20-06.

Abstract PO3-19-10: Phase 1b study of EZH1/2 inhibitor valemetostat in combination with trastuzumab deruxtecan in subjects with HER2 low/ultra-low/null metastatic breast cancer

Abstract Background Low HER2-expressing breast cancers have traditionally been classified as HER2-negative and treated as TNBC or hormone receptor (HR)-positive. Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate, composed of an anti-HER2 antibody conjugated to topoisomerase I payload, is approved for clinical use in HER2-low (IHC 1+ or IHC 2+/ISH negative) metastatic breast cancer (MBC). In the DAISY study (NCT04132960), meaningful clinical response was observed with T-DXd in HER2 IHC 0 MBC. Valemetostat is an oral, selective dual inhibitor of enhancer of zeste homolog 1 and 2 (EZH1/2, methyltransferases that specifically methylate histone H3 lysine 27. It is currently approved for patients with relapsed or refractory adult T-cell leukemia/lymphoma in Japan. EZH2-mediated PP2A inactivation has been shown to confer resistance to HER2-targeted therapy. Additionally, valemetostat has been shown to upregulate Schlafen11 (SLFN11), a putative DNA/RNA helicase, that regulates the sensitivity to DNA damaging agents such as topoisomerase I inhibitors. Consequently, this study examines the safety and anti-tumor activity of valemetostat in combination with T-DXd in subjects with HER2 low/ultra-low/null MBC. Trial Design This is a single-arm, phase-1b study to evaluate the safety and clinical activity of T-DXd in combination with valemetostat in patients with HER2 low/ultra-low/null MBC. The dosing for T-DXd is 5.4 mg/kg Q3W administered intravenously as indicated for current clinical use. Valemetostat will be evaluated at three dose levels (100 mg, 150 mg, and 200 mg orally), with starting dose (level 1) at 100 mg QD. The dose-limiting toxicity (DLT) evaluation period will be the first 2 treatment cycles (42 days). Eligibility criteria Specific aims Statistical methods Approximately 12 evaluable patients will be enrolled for the dose-escalation portion based on the Bayesian optimal interval design with a target DLT rate of 25%. Patients enrolled in cohorts of 3. The expansion will be performed at the RDE using the 2-stage Bayesian optimal dose-expansion design. In the first stage, 13 evaluable patients (including those treated at the RDE in the dose-escalation part) will be enrolled. If < 5 patients respond in the first stage, the study will be stopped for futility. If ≥ 5 responses are observed, 13 additional evaluable patients will be enrolled. If 11 or more responses are observed among the total of 26 patients the treatment will be regarded as promising. This two-stage design yields 78% power under the alternative hypothesis of ORR=50% (null ORR = 30%) while controlling the one-sided type I error at 10%. Contact information for people with a specific interest in the trial sdamodaran@mdanderson.org (NCT05633979) Citation Format: Senthil Damodaran, Toshiaki Iwase, Angela Marx, Jie Willey, Funda Meric-Bernstam, Debu Tripathy, Carlos Barcenas, Jangsoon lee, Naoto Ueno. Phase 1b study of EZH1/2 inhibitor valemetostat in combination with trastuzumab deruxtecan in subjects with HER2 low/ultra-low/null metastatic 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 PO3-19-10.

Targeting Breast Cancer Using 177 Lu-Labeled Trastuzumab and Trastuzumab Fragment : First-in-Human Clinical Experience.

A monoclonal antibody, trastuzumab, is used for immunotherapy for HER2-expressing breast cancers. Large-sized antibodies demonstrate hepatobiliary clearance and slower pharmacokinetics. A trastuzumab fragment (Fab; 45 kDa) has been generated for theranostic use. Fab was generated by papain digestion. Trastuzumab and Fab have been radiolabelled with 177 Lu after being conjugated with a bifunctional chelating. The affinity and target specificity were studied in vitro. The first-in-human study was performed. The bifunctional chelating agent conjugation of 1-2 molecules with trastuzumab and Fab was detected at the molar ratio 1:10 in bicarbonate buffer (0.5 M, pH 8) at 37°-40°C. However, 2-3 molecules of bifunctional chelating agent were conjugated when DMSO in PBS (0.1 M, pH 7) was used as a conjugation buffer at a molar ratio of 1:10. The radiolabelling yield of DOTA-conjugated Fab and trastuzumab at pH 5, 45°C to 50°C, with incubation time 2.5-3 hours was 80% and 41.67%, respectively. However, with DOTAGA-conjugated trastuzumab and Fab, the maximum radiolabelling yield at pH 5.5, 37°C, and at 2.5-3 hours was 80.83% and 83%, respectively. The calculated K d of DOTAGA Fab and trastuzumab with HER2-positive SKBR3 cells was 6.85 ± 0.24 × 10 -8 M and 1.71 ± 0.10 × 10 -8 M, respectively. DOTAGA-Fab and trastuzumab showed better radiolabelling yield at mild reaction conditions.177 Lu-DOTAGA-Fab demonstrated higher lesion uptake and lower liver retention as compared with 177 Lu-DOTAGA-trastuzumab. However, 177 Lu-DOTAGA-Fab as compared with 177 Lu-DOTAGA-trastuzumab showed a relatively early washout (5 days) from the lesion. 177 Lu-DOTAGA-Fab and trastuzumab are suitable for targeting the HER2 receptors.

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 2600: Novel dual TOP1i ADC inducing superior tumour growth inhibition at low-drug load vs. trastuzumab deruxtecan

Abstract The Araris’ site-specific and one-step conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using two different Topoisomerase-1 inhibitors (TOP1i) as payloads that shows superior anti-tumor efficacy compared to trastuzumab deruxtecan (T-DXd) in head-to-head in vivo studies. For proof-of concept and comparison to T-DXd, trastuzumab was used as the targeting antibody. The Araris ADC was designed to combine two features in one ADC to maximize tumor-specific activity by using two different exatecan-based payloads: one that is able to exert bystander activity to address tumor heterogeneity and low-target expression and one that accumulates in cancer cells (no bystander activity) to achieve greater potency. We found that the ADC was well-defined with a drug to antibody ratio (DAR) of 4 (2+2), as expected and no signs of aggregation under stressed conditions (40°C) during a period of 14 days. In in-vitro assays on target positive cell-lines, the ADC demonstrated taget-specific cell-cytotoxicity in the low nM-range similarly to T-DXd. Importantly, the non-bystander exatecan showed an increased intracellular concentration (up to 4x) and the bystander capable exatecan demonstrated high bystander activity in co-cultured, target-negative cell lines. In mouse pharmacokinetic studies, the ADC showed excellent stability in circulation with no signs of payload loss or linker-cleavage with a PK profile comparable to the unconjugated trastuzumab antibody which is key for a maximal and tumor-specific payload delivery. Finally, a head-to-head study vs T-DXd was done in a challenging, medium HER2-expressing breast cancer model (JIMT-1), known to be resistant against the FDA-approved ADC T-DM1 (trastuzumab emtansine) and for T-DXd only showing limited activity (Ogitani et al., 2016). Impressively, it was found that the dual-TOP1i DAR4 Araris ADC showed superior anti-tumor efficacy compared to the payload-dose-adjusted T-DXd (DAR8). Using published doses for T-DXd, the Araris ADC was administered at ADC doses of 2 × 10mg/kg on days 1 and 8, resulting in superior anti-tumor activity and tumor eradication compared to T-DXd lasting for more than 47 days. T-DXd dosed at 2 × 5mg/kg (same payload dose) on days 1 and 8, only showed a very limited tumor growth inhibition with tumor regrowth occurring in all animals already at around 21 days. We here show first encouraging results on a novel concept of combining TOP1i payloads that have two different features in one ADC to maximize efficacy. We believe that this concept in combination with a stable payload attachment at low DAR and an excellent exposure may help to develop ADCs with an improved therapeutic index for various solid tumor indications. Citation Format: Philipp Spycher, Rachael Fay, Romain Bertrand, Philipp Probst, Ramona Stark, Roger Santimaria, Patrick Maurhofer, Lia Kallenberger, Emma Renard, Bernd Schlereth, Dragan Grabulovski, Isabella Attinger-Toller. Novel dual TOP1i ADC inducing superior tumour growth inhibition at low-drug load vs. trastuzumab deruxtecan [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 2600.

Abstract B043: Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer

Abstract Breast cancer is the most common cause of mortality among women worldwide. Among all subtypes, HER2-positive breast cancers are highly aggressive and have poor prognosis. Surgical resection is choice of treatment. However, surgery may not be possible if disease is widespread. Trastuzumab, a monoclonal antibody, is globally used for immunotherapy. It is a large sized molecule (141kDa) and show slow pharmacokinetics and high liver uptake. We have explored trastuzumab fragment as a radio-theranostic agent. Trastuzumab Fab was generated using Papain enzyme tagged beads, incubation time 18 to 20 h at 850 rpm in phosphate buffer. Fab were collected with ultrafiltration device, cut off 30 kDa, in phosphate buffer pH 8. Quantification of Fab was done at 280 nm. Fab characterization was done using MALDI-TOF and SDS-PAGE. To enable radiolabelling of Fab with Ga-68 and Lu-177, Fab was conjugated with bifunctional chelator, DTPA. Fab-DTPA was separated by micro bio-spin P-6 chromatography columns and the average number of DTPA/Fab were calculated. Affinity of Fab conjugated Fab-DTPA for HER2 receptors was studied using the biolayer interferometry technique (BLI). Radiolabelling of Fab with Ga-68 for PET imaging and Lu-177 for therapy was optimized. Quality control included radiochemical purity using TLC in sodium citrate (pH 5) mobile phase, pyrogenicity using PTS, and sterility by tryptic soy broth culture media. Receptor affinity and apoptosis were studied with HER2 positive SKBR-3 cells using flow cytometry assay. The SKBR-3 cell (106) were cultured in 96 well plates and incubated with a known amount of Lu-177-DTPA-Fab (0- 18.5 MBq). First in human Ga-68 Fab PET/CT and Lu-177-Fab gamma camera images were acquired after obtaining clearance from Institutional Ethics Committee and inform signed consent. Patients were recruited with histopathological proven HER2 positive breast cancer and uptake on F-18 FDG PET/CT. Ga-68-DTPA-Fab (3.5-5 mCi) and 10-15 mCi Lu-177 DTPA-Fab was injected and images were acquired at 2/3 h and up to 7 days respectively. Dominant Fab peak at 45kDa on MALD-TOF, single band on SDS-PAGE (Non-reduced or reduced) have been obtained. Two DTPA/Fab were conjugated and confirmed by MALDI-TOF. BLI data showed Kd value of trastuzumab, Fab, and conjugated DTPA-Fab as 0.2 nM, 0.57 nM, and 20 nM for HER2. High labelling efficiency (≥98%) of Lu-177-DTPA-Fab and Ga-68-DTPA-Fab was achieved. Apoptosis (%) was 1.5%- 42.5% in SKBR-3 cells treated with Lu-177-DTPA-Fab. Both formulations were found sterile and pyrogen-free. Kidney and bladder activity of Ga-68/Lu-177-DTPA-Fab demonstrate renal clearance. Blood pool activity of both tracers were high. However, it was decreased, and lesion uptake was increased with time. All the lesions on Ga-68-DTPA-Fab, same as in F-18 FDG PET/CT, were picked up accept those near heart. Good lesion retention of Lu-177-DTPA-Fab was observed showing suitability for therapy. Fab-DTPA has potential to target HER2 receptor. It could be radiolabelled with Ga-68 for PET imaging and Lu-177 for targeted therapy. Citation Format: Jaya Shukla, Yogesh Rathore, Komalpreet Kaur, Santosh Irrinky, Rajender Kumar, Amanjit Bal, Bhagwant R Mittal. Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B043.

Antibody-mimetic drug conjugate with efficient internalization activity using anti-HER2 VHH and duocarmycin

Antibody-mimetic drug conjugate (AMDC) is a cancer cell-targeted drug delivery system based on the non-covalent binding of mutated streptavidin and modified biotin, namely Cupid and Psyche. However, the development of AMDCs is hampered by difficulties in post-translational modification or poor internalization activity. Here, we report an expression, refolding, and purification method for AMDC using a variable heavy chain of heavy chain-only antibodies (VHHs). Monomeric anti-HER2 VHH fused to Cupid was expressed in Escherichia coli inclusion bodies. Solubilization and refolding at optimized reducing conditions and pH levels were selected to form a functional, tetrameric protein (anti-HER2 VHH-Cupid) that can be easily purified based on molecular weight. Anti-HER2 VHH-Cupid non-covalently creates a tight complex with Psyche linked to a potent DNA-alkylating agent, duocarmycin. This complex can be absorbed by the HER2-expressing human breast cancer cell line, KPL-4, and kills KPL-4 cells in vitro and in vivo. The production of a targeting protein with internalizing activity, combined with the non-covalent conjugation of a highly potent payload, renders AMDC a promising platform for developing cancer-targeted therapy.

685P BL-M07D1, a HER2 antibody-drug conjugate in subjects with locally advanced or metastatic HER2 expressing breast cancer and other solid tumors

685P BL-M07D1, a HER2 antibody-drug conjugate in subjects with locally advanced or metastatic HER2 expressing breast cancer and other solid tumors

Comprehensive Analysis of Human Epidermal Growth Factor Receptor 2 Through DNA, mRNA, and Protein in Diverse Malignancies.

Human epidermal growth factor receptor 2 (HER2) expression (protein immunohistochemistry [IHC] or gene amplification [copy-number variation, CNV]) predicts anti-HER2 therapy responsiveness, although recently it was shown that even low HER2-expressing breast cancers benefit from trastuzumab-deruxtecan. Little is known about HER2 transcriptomic (mRNA) expression, and comparisons between genomic, mRNA, and protein HER2 assays. HER2 status was evaluated using clinical-grade IHC (protein), quantitative reverse transcription polymerase chain reaction (mRNA), and next-generation sequencing (NGS; amplifications). Multi-institutional HER2 testing was performed on 5,305 diverse cancers including non-small-cell lung (n = 1,175), breast (n = 1,040), and colon cancers (n = 566; N = 3,926 tested for CNV; N = 1,848, mRNA; N = 2,533, IHC). Overall, 4.1% (161/3,926) had NGS HER2 amplification; 33.3% (615/1,848) had mRNA overexpression; and 9.3% (236/2,533) were IHC-positive. In 723 patients with all three tests (CNV/mRNA/IHC), various amplification/expression patterns occurred: 7.5% (54/723) had all three HER2 tests positive; 62.8% (454/723) had all three tests negative. Discrepant patterns between amplification and overexpression emerged. For instance, 20% (144/723) of patients had mRNA overexpression alone with negative CNV and IHC. A range in values for only mRNA+ cases occurred in different tumor types (eg, 16.9%, breast; 5%, hepatobiliary). There were 53 patients with various tumors from our institution who had all three assays attempted; 22 tested positive for HER2, and seven received anti-HER2 therapy: two patients achieved response: one (esophageal cancer), complete response (≥42 months); one (cholangiocarcinoma), who only had HER2 mRNA positivity (tissue was inadequate for IHC and CNV assessment), partial response (≥24 months) on HER2-based regimens. We demonstrate variability of HER2 (protein and mRNA) expression and amplification using comprehensive assays (CNV, mRNA, and IHC) among diverse cancers. As HER2-targeted therapy indications expand, the relative importance of these modalities merits further evaluation.

Efficacy and safety of trastuzumab deruxtecan (T-DXd) in patients (pts) with HER2-expressing solid tumors: DESTINY-PanTumor02 (DP-02) interim results.

LBA3000 Background: T-DXd is an antibody drug conjugate targeting HER2 and is approved in HER2-expressing breast (BC) and gastric (GC) cancers. HER2 expression is prevalent in other solid tumors. The efficacy of current treatments (Tx) in these populations, including studies with HER2-directed Tx, is modest, revealing a significant unmet medical need. Clinically meaningful activity of T-DXd was seen in HER2-expressing tumors in a phase 1 study (NCT02564900). Methods: DP-02 (NCT04482309) is an open-label phase 2 study of T-DXd 5.4 mg/kg q3w in pts with HER2-expressing (immunohistochemistry [IHC] 3+ or IHC 2+ by local or central testing) locally advanced or metastatic disease that progressed after ≥1 systemic Tx or that has no Tx options. Cohorts with biliary tract (BTC), bladder (URO), cervical (CC), endometrial (EC), ovarian (OC), pancreatic (PC), or other tumors (excluding BC, GC, colorectal cancer, and non-small cell lung cancer) were enrolled. Efficacy and safety were analyzed in all pts who received ≥1 dose of T-DXd. The primary endpoint was investigator-assessed confirmed objective response rate (ORR). Secondary endpoints included duration of response (DOR), disease control rate, progression-free and overall survival, and safety. Results: At data cutoff (16 Nov 2022; median follow-up, 9.7 mo), 267 pts had been treated (median, 2 prior lines of Tx [range, 0-13]); 75 pts were IHC 3+ and 125 were IHC 2+ by central testing. In all 267 pts, the ORR was 37.1% and median DOR (mDOR) was 11.8 mo; in pts with IHC 3+ expression, the ORR was 61.3% and mDOR was 22.1 mo. ORR per cohort is shown in all pts and those with centrally confirmed HER2 IHC 3+ or IHC 2+ expression. Grade (G) ≥3 adverse events (AEs) occurred in 58.4% of pts; 11.6% discontinued Tx due to AEs. Adjudicated drug-related interstitial lung disease/pneumonitis occurred in 18 pts (6.7% [G1, n=6; G2, n=11; G5, n=1]). Conclusions: This is the first tumor-agnostic global study of T-DXd in a broad range of HER2-expressing solid tumors. T-DXd showed encouraging ORR, particularly in pts with IHC 3+ expression, durable clinical benefit, and a manageable safety profile in this heavily pretreated population. These interim results show T-DXd to be a potential new Tx option for pts with HER2-expressing solid tumors. Clinical trial information: NCT04482309 . [Table: see text]

Cancer therapy by antibody-targeted Cerenkov light and metabolism-selective photosensitization.

Photodynamic therapy (PDT) is an effective cancer treatment option, but it suffers from penetration limit of light, making it available only for superficial and endoscopically accessible cancers. Recently, there have been reports that Cerenkov luminescence originated from radioisotopes can be utilized as an excitation source for PDT without external light illumination. Here, cancer-selective agents, i.e., (1) clinically available 5-aminolevulinic acid (5-ALA), which promotes cancer metabolism-specific accumulation of protoporphyrin IX (PpIX), and (2) 64Cu-DOTA-trastuzumab, which has HER2-expressing cancer selective uptake, are separately applied as a photosensitizer and an in situ radiator, respectively, to potentiate tumor-specific Cerenkov luminescence energy transfer (CLET) from 64Cu to PpIX for high-precision PDT of cancer. It is shown that the combinational administration and tumor colocalization of 5-ALA and 64Cu-DOTA-trastuzumab exert significant in vitro cytotoxicity (cell viability <9%) as well as in vivo antitumor effects (tumor volume ratio of 0.50 on 14days post-injection) on HER2-expressing breast and gastric cancer models. This study proves that high-precision treatment regimen using dual-targeted CLET-based PDT is feasible for HER2-expressing cancers. Furthermore, the results offer great potential for clinical translation to the dual-targeted CLET-based PDT because the treatment regimen uses components, 5-ALA and 64Cu-DOTA-trastuzumab, which are already in clinical uses.

Leveraging a Dual Variable Domain Immunoglobulin to Create a Site-Specifically Modified Radioimmunoconjugate.

Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a 177Lu-labeled radioimmunoconjugate based on HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A″-DTPA (PODS-CHX-A''-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [177Lu]Lu3+ to produce the completed radioimmunoconjugate, [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD, in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/μmol. [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetalation over a 7 days of incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD to that of a 177Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [177Lu]Lu-CHX-A″-DTPAPODS-HER2DVD matched that of 177Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8, respectively, at the same time point. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.

Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with 99mTc for Radionuclide Imaging of HER2 Expression in Cancer.

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [99mTc]Tc-(HE)3-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)3), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection. The goal of this study was to evaluate if the use of cysteine-containing peptide-based chelators Glu-Glu-Glu-Cys (E3C), Gly-Gly-Gly-Cys (G3C), and Gly-Gly-Gly-Ser-Cys connected via a (Gly-Gly-Gly-Ser)3-linker (designated as G3-(G3S)3C) would further improve the contrast of imaging using 99mTc-labeled derivatives of G3. The labeling of the new variants of G3 provided a radiochemical yield of over 95%. Labeled G3 variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 1.9-5 nM. Biodistribution of [99mTc]Tc-G3-G3C, [99mTc]Tc-G3-(G3S)3C, and [99mTc]Tc-G3-E3C in mice was compared with the biodistribution of [99mTc]Tc-(HE)3-G3. It was found that the novel variants provide specific accumulation in HER2-expressing human xenografts and enable discrimination between tumors with high and low HER2 expression. However, [99mTc]Tc-(HE)3-G3 provided better contrast between tumors and the most frequent metastatic sites of HER2-expressing cancers and is therefore more suitable for clinical applications.

Effect of Trastuzumab Deruxtecan on QT/QTc Interval and Pharmacokinetics in HER2-Positive or HER2-Low Metastatic/Unresectable Breast Cancer.

HER2-targeted anticancer therapies may be associated with cardiovascular adverse events. This study evaluated effects of the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan (T-DXd, DS-8201a) on QT/QTc interval and its pharmacokinetics. Patients with heavily pretreated, metastatic HER2-expressing breast cancer were enrolled at seven study sites in Japan. T-DXd was administered intravenously at 6.4 mg/kg on day 1 of each 21-day cycle. Primary end points were baseline-adjusted QTcF interval and pharmacokinetics parameters. Key secondary end points included safety events, serum concentration of T-DXd and DXd at the time of electrocardiographic measurements, and antitumor activity parameters. Among 51 total patients, 47 (92.2%) had HER2-low breast cancer (immunohistochemistry 1+ or 2+ and in situ hybridization-negative/equivocal/missing). Pharmacokinetic parameters after a single dose of T-DXd were consistent with previous studies. After multiple doses, T-DXd showed moderate accumulation (accumulation ratio (cycle 3/cycle 1), 1.35), but DXd showed minimal accumulation (1.09). The upper bound of the 90% confidence interval for mean ΔQTcF interval was < 10 ms at all timepoints, and at mean maximum serum concentration was also < 10 ms. Based on concentration-QT analysis, ΔQTcF increased with increasing concentrations of T-DXd and DXd. No clinically meaningful QTcF prolongation was observed. T-DXd had a manageable safety profile and showed antitumor activity in HER2-low breast cancer. In this study, a T-DXd dose of 6.4 mg/kg, higher than the 5.4-mg/kg dose currently approved for breast cancer, was not associated with clinically relevant QTcF prolongation in heavily pretreated patients with HER2-expressing metastatic breast cancer. This study adds to our understanding of T-DXd for treatment of HER2-low breast cancer.

Possible Involvement of the Upregulation of ΔNp63 Expression Mediated by HER2-Activated Aryl Hydrocarbon Receptor in Mammosphere Maintenance.

Cancer stem cells (CSCs) contribute to the drug resistance, recurrence, and metastasis of breast cancers. Recently, we demonstrated that HER2 overexpression increases mammosphere formation via the activation of aryl hydrocarbon receptor (AHR). In this study, the objective was to identify the mechanism underlying mammosphere maintenance mediated by HER2 signaling-activated AHR. We compared the chromatin structure of AHR-knockout (AHRKO) HER2-overexpressing MCF-7 (HER2-5) cells with that of wild-type HER2-5 cells; subsequently, we identified TP63, a stemness factor, as a potential target gene of AHR. ΔNp63 mRNA and protein levels were higher in HER2-5 cells than in HER2-5/AHRKO cells. Activation of HER2/HER3 signaling by heregulin treatment increased ΔNp63 mRNA levels, and its induction was decreased by AHR knockdown in HER2-5 cells. The results of the chromatin immunoprecipitation assay revealed an interaction between AHR and the intronic region of TP63, which encodes ΔNp63. A luciferase reporter gene assay with the intronic region of TP63 showed that AHR expression increased reporter activity. Collectively, our findings suggest that HER2-activated AHR upregulates ΔNp63 expression and that this signaling cascade is involved in CSC maintenance in HER2-expressing breast cancers.

Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates.

The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN3) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide-alkyne cycloaddition. This methodology was used to create [89Zr]Zr-SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (89Zr). [89Zr]Zr-SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([89Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([89Zr]Zr-malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and 89Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to ∼55.5 GBq/μmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of 89Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.

In Vivo and In Vitro Efficacy of Enhertu in Uterine Serous Carcinoma (047)

Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Graphical Abstract View Large Image Figure Viewer Download Hi-res image Graphical Abstract View Large Image Figure Viewer Download Hi-res image Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted. Objectives: Uterine serous carcinoma (USC) is an aggressive subtype of epithelial endometrial cancer, comprising approximately 40% of endometrial cancer-related deaths. HER2 overexpression is detected in about 35% of patients with USC. Enhertu is a novel humanized monoclonal anti-HER2 antibody (trastuzumab) linked to the topoisomerase I inhibitor payload deruxtecan by a cleavable linker. Enhertu is FDA-approved for HER2-expressing metastatic breast and gastric cancers; however, its efficacy has not been tested in endometrial cancer. The objective of this study was to study the in vitro and in vivo efficacy of Enhertu in USC. Methods: HER2 expression was analyzed with flow cytometry in primary USC cell lines: ARK2, ARK4, ARK7, and ARK20. Cell lines were treated with 0.05, 0.010, 0.025, 0.05, 0.2 μg/ml of Enhertu or Control antibody-drug conjugate (Control ADC). The inhibitory concentration 50 (IC50) was assessed by counting the viable cells using a flow cytometer. Apoptosis and necrosis were assessed with Annexin V and propidium iodide (PI) staining. GFP-expressing ARK4 (ARK4-GFP) cells were co-cultured with ARK2 cells for the bystander effect assay. For in vivo experiments, eight million ARK2 cells were injected subcutaneously into SCID mice. Animals were randomized to intravenous injection of PBS (n=5), control ADC (4 mg/kg, n=6) or Enhertu (4 mg/kg, n=5) treatment. Kruskal Wallis, Friedman, and Log Rank tests were used for statistical analysis. Conclusions: Enhertu showed significant preclinical efficacy against HER2 overexpressing uterine serous carcinoma cell lines. Further clinical trials in USC are warranted.

Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors

BackgroundChimeric antigen receptor (CAR) T cell therapy has proven its clinical utility in hematological malignancies. Optimization is still required for its application in solid tumors. Here, the lack of cancer-specific...