HER2-positive Cancer Cell Lines Research Articles

Abstract 2633: Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer

Abstract Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2633.

Abstract 1803: GNP101, anti-HER2-IFN-β mutein immunocytokine, for the treatment of Trastuzumab deruxtecan-resistant HER2-positive cancers

Abstract Background: Trastuzumab deruxtecan (T-DXd) has been approved in various types cancer and recently expanded its indication to cancers with various HER2 expressions, such as HER2-low breast cancer. Since the effect of T-DXd is based on toxin payload, topoisomerase I inhibitor, the emergence of toxin-resistant cancer cells is unavoidable. Clinical cases of Primary, or secondary resistance to T-DXd have been reported. Therefore, it is necessary to develop novel drugs that can effectively exhibit antitumor effects against T-DXd-resistant carcinoma. Interferon beta (IFN-β), a promising potent cytokine, has been attracting attention for treatment of cancer. It is one of the few cytokines that induce tumor cell killing directly. Importantly, it could be an alternative to conventional drug-resistant cancer, because it regulates tumor growth in a way different from cytotoxic agents. Here, GNP101, an IFN-β mutein-based immunocytokine, exhibits antitumor properties against HER2-positive cancers, including T-DXd-resistant cancer. Method: A panel of human HER2-positive cancer cell lines derived from breast, stomach and lung were treated with GNP101 or HER2 therapeutics (trastuzumab, T-DM1, T-DXd) to compare direct antitumor effects. To generate a T-DXd-resistant cells, HCC1954, NCI-N87 and Calu-3 were exposed to the IC50, IC80 or IC90 of T-DXd by cyclic treatment. After 4 months, HCC1954-R cells were established and IFN-β signaling was assessed by STAT1-phosphorylation. The direct antitumor effect of GNP101 in vivo was tested in HER2-positive cancer xenograft models. Result: GNP101 showed superior potency compared to existing HER2 therapies (trastuzumab, T-DM1, and T-DXd) in all HER2-positive cancers regardless of tissue origin. To investigate the antitumor effect of GNP101 against T-DXd resistant cancer, we first generated T-DXd-resistant cell line (HCC1954-R). These HCC1954-R cells were strongly resistant to T-DXd, and showed no antitumor effect even at 100 nM of T-DXd. However, they were sensitive to GNP101, suggesting that GNP101 maintained its antitumor effect in T-DXd resistant cells. In addition, there was no difference in IFN-β-induced STAT1 phosphorylation between the parent cell and HCC1954-R. Taken together, these results indicate that HCC1954-R cells maintain IFN-β signaling and are selectively resistant to T-DXd. The activities of GNP101, T-DM1 and T-DXd on HER2-positive cancer cells were compared in vivo. GNP101 inhibited the growth of tumor by 90%, with no difference from that of T-DXd or T-DM1. Conclusion: We have evaluated the antitumor properties of GNP101, a trastuzumab-IFN-β mutein in HER2-expressing cancer. Given that GNP101 retains its antitumor properties in T-DXd-resistant cancer cells, the study suggests that anti-HER2-IFN-β mutein is a promising candidate for the treatment of HER2-positive and T-DXd resistant cancers. Citation Format: Chan Gyu Lee, Minkyung Park, Hyun Kyung Lee, Na Young Kim, Jun Young Choi, Sungyoul Hong, Young Kee Shin, Hae Min Jeong. GNP101, anti-HER2-IFN-β mutein immunocytokine, for the treatment of Trastuzumab deruxtecan-resistant HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1803.

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5 h in human serum compared to the half-life of 2 h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.

An optimised synthesis of SG3376, a non-cleavable antibody-drug conjugate pyrrolobenzodiazepine drug-linker

In recent years, antibody-drug conjugates (ADCs) have been evaluated in a growing number of clinical trials. Preclinical evaluation and the synthetic accessibility of cleavable pyrrolobenzodiazepine drug-linkers, such as talirine and tesirine, have been previously described. This article details how the synthesis of SG3376, a non-cleavable pyrrolobenzodiazepine drug-linker, has been optimised to provide access to late-stage versatile intermediates in a robust and scalable fashion. Of particular importance was the selective deprotection of primary N-Boc in the presence of secondary N-Boc and secondary O-TBS. SG3376 was conjugated to trastuzumab and the resulting ADC was found to have potent cytotoxic activity in a number of HER2-positive cancer cell lines.

Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.

Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK), to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin) for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3) indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells.

HSP90 expression and its association with wighteone metabolite response in HER2-positive breast cancer cells

It is well known that heat shock protein 90 (HSP90) overexpression is correlated with poor prognosis and chemo-resistance in human malignant cancers. At the same time, wighteone, or 6-prenyl-5,7,4'-trihydroxyisoflavone, a major isoflavone component of the ornamental tall tree Erythrina suberosa, has been demonstrated to exhibit a potent anti-proliferative effect on human leukemia HL-60 cancer cell lines. In this study, the effects of wighteone on the proliferation of HER2-positive breast cancer cells were investigated, and the action mechanism was explored. MCF-7 HER2-positive breast cancer cells were treated with various concentrations of wighteone. The growth inhibitory rate of the cells was calculated by MTT assay, apoptosis was detected by flow cytometry, and the expression level of HSP90 was assessed by western blot analysis. The addition of wighteone at concentrations ranging from 1-10 g/ml in the medium for 48 h had a marked inhibition on the proliferation of HER2-positive cancer cell lines. The growth inhibitory rates with 0.5, 2 or 8 mM wighteone were significantly higher compared with the control group. Apoptosis in the wighteone-treated cells was also significantly higher compared with the control group. The expression level of HSP90 in the wighteone group was significantly lower than that in the control group. Our findings demonstrated that wighteone effectively inhibited the proliferation of HER2-positive cancer cell lines, and this is considered to be the result of downregulating HSP90 receptor and downstream signaling.

Therapeutic siRNA for drug-resistant HER2-positive breast cancer.

HER2 is overexpressed in about 20% of breast cancers and contributes to poor prognosis. Unfortunately, a large fraction of patients have primary or acquired resistance to the HER2-targeted therapy trastuzumab, thus a multi-drug combination is utilized in the clinic, putting significant burden on patients. We systematically identified an optimal HER2 siRNA from 76 potential sequences and demonstrated its utility in overcoming intrinsic and acquired resistance to trastuzumab and lapatinib in 18 HER2-positive cancer cell lines. We provided evidence that the drug-resistant cancer maintains dependence on HER2 for survival. Importantly, cell lines did not readily develop resistance following extended treatment with HER2 siRNA. Using our recently developed nanoparticle platform, systemic delivery of HER2 siRNA to trastuzumab-resistant tumors resulted in significant growth inhibition. Moreover, the optimal HER2 siRNA could also silence an exon 16 skipped HER2 splice variant reported to be highly oncogenic and linked to trastuzumab resistance.

Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads.

Trastuzumab conjugates consisting of exatecan derivatives were prepared and their biological activities and physicochemical properties were evaluated. The ADCs showed strong efficacy and a low aggregation rate. The exatecan derivatives were covalently connected via a peptidyl spacer (Gly-Gly-Phe-Gly), which is assumed to be stable in circulation, and were cleaved by lysosomal enzymes following ADC internalization into tumor tissue. These anti-HER2 ADCs exhibited a high potency, specifically against HER2-positive cancer cell lines in vitro. The ADCs, bearing exatecan derivatives which have more than two methylene chains, exhibited superior cytotoxicity. It was speculated that steric hindrance of the cleavable amide moiety could be involved in the drug release. The adequate alkyl lengths of exatecan derivatives (13, 14, 15) were from two to four in terms of aggregation rate. The ADC having a hydrophilic moiety showed good efficacy in a HER2-positive and Trastuzumab-resistant breast carcinoma cell model in mice.

Abstract 4461: Anti-cancer activity of immunoliposomes encapsulated effective amount of glycosylated paclitaxel with novel loading strategy

Abstract Targeted liposomal drug delivery is considered to enhance the therapeutic effects of anti-cancer agents because high amount of drug could be administered with least side effects. However, significant hydrophobic compounds such as paclitaxel (PTX) are not suitable for efficient encapsulating. We conferred water-solubility of PTX by coupling glucose at 7-OH to produce glycosylated PTX (gPTX). gPTX appeared to have a 10-fold higher solubility than PTX in 40% ethylene glycol (EG) and in Cremophor® EL/Ethanol/PBS (12:12:76 volume%) (CEP). The maximal solubility of gPTX in these solvents was 1.1 and 20.0 mg/ml, respectively. An anti-cancer activity of PTX is due to inhibit tubulin depolymerization. We confirmed tubulin polymerization activity of gPTX in vitro. The activity of gPTX to stabilize tubulin polymerization was lower than that of PTX by only 10%. We successfully prepared gPTX-encapsulated liposome (gPTX-L) with the remote loading method by solubility gradient between 40% EG and CEP. The encapsulation efficiency and loading efficiency were 70.8 % and 8.0 mol% (drug mol / initial lipid mol). Then, trastuzumab was conjugated on the surface of gPTX-L to prepare immunoliposome (gPTX-IL) for targeting HER2 positive cancer cells. The particle size of gPTX-IL was around 150 nm and Z potential was -8.1 mV. We evaluated cellular cytotoxicity of PTX, gPTX, gPTX-L and gPTX-IL in HER2 positive cancer cell lines, SK-BR-3 and HT-29, and HER2 negative cancer cell line, MDA-MB-231, by MTT assay. The IC50 values after 72 hours drug-exposure showed gPTX-L exhibited lower IC50 values than free-gPTX in all cell line, which indicated gPTX-L internalized with liposomal formulation contained high concentration of gPTX. Furthermore, gPTX-IL exhibited lower IC50 values than gPTX-L in HER2 positive cancer cell lines but no significant statically difference in this assay condition. We then evaluated the time required to inhibit the growth at the half maximal level (IT50) at the minimum concentration to show maximal cytotoxicity after 72 hours drug-exposure. gPTX-IL showed the shortest IT50 values of 6.4 and 4.7 h in SK-BR-3 and HT-29 cells, respectively. On the other hand, gPTX-IL showed the same IT50 value of gPTX-L in MDA-MB-231 cells. We then evaluated the anti-cancer efficacy of the formula in vivo using tumor bearing ICR-nu/nu mice. The mice were intravenously injected at the dose of 150 mg/kg of gPTX in a day, which was the lethal dose of free-gPTX. While gPTX-L and free-trastuzumab did not inhibit tumor growth, gPTX-IL significantly decreased tumor volume with low side effects. We further evaluated dose dependency of gPTX-IL using tumor bearing BALB/c-nu/nu mice. The doses of over 100 mg/kg decreased tumor volume and the dose of 150 mg/kg showed the most efficient anti-cancer activity. Collectively, the gPTX-IL with novel encapsulation strategy should be a candidate of highly efficient targeting drug delivery system. Citation Format: Tsukasa Shigehiro, Tomonari Kasai, Akifumi Mizutani, Hiroshi Murakami, Katsuhiko Mikuni, Tadakatsu Mandai, Hiroki Hamada, Masaharu Seno. Anti-cancer activity of immunoliposomes encapsulated effective amount of glycosylated paclitaxel with novel loading strategy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4461. doi:10.1158/1538-7445.AM2014-4461

The mitochondrial transport protein SLC25A43 affects drug efficacy and drug-induced cell cycle arrest in breast cancer cell lines

The mitochondria have been identified as key players of apoptosis, cell proliferation and cell cycle regulation. However, the role of mitochondria in breast cancer and treatment failure remains unclear. We have previously shown a common deletion of the gene SLC25A43 in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This gene is coding for a mitochondrial inner membrane transporter and, to date, little is known about the function of this protein. We have also found that low protein expression of SLC25A43 significantly correlates with a lower S phase fraction in HER2-positive breast cancer. The aim of this study was to investigate whether knockdown (KD) of SLC25A43 could have an effect on the cytotoxicity of different cytostatic drugs using MCF10A, MCF7 and BT-474 cells. Following siRNA-mediated KD of SLC25A43, one non-malignant and two breast cancer cell lines were exposed to the anthracycline epirubicin or the taxane paclitaxel. The HER2-positive breast cancer cells were also exposed to the targeted therapy trastuzumab and dual exposure to trastuzumab and paclitaxel. We found that KD of SLC25A43 resulted in a decreased cytotoxic effect of paclitaxel in the two cancer cell lines (P<0.05). Further analysis of cell cycle phase distribution showed that KD increased the paclitaxel-induced G2/M block in these two cell lines (P<0.05). KD of SLC25A43 also reduced the inhibitory effect of trastuzumab on cell proliferation in the HER2-positive cancer cell line BT-474 (P<0.05), and the drug-induced G0/G1 block (P<0.05). Moreover, SLC25A43 influenced the percentage of Ki-67-positive cells. Our findings demonstrate that the mitochondrial protein SLC25A43 affects drug efficacy and cell cycle regulation following drug exposure in breast cancer cell lines.

Vaccination with human anti-trastuzumab anti-idiotype scFv reverses HER2 immunological tolerance and induces tumor immunity in MMTV.f.huHER2(Fo5) mice

IntroductionNovel adjuvant therapies are needed to prevent metastatic relapses in HER2-expressing breast cancer. Here, we tested whether trastuzumab-selected single-chain Fv (scFv) could be used to develop an anti-idiotype-based vaccine to inhibit growth of HER2-positive tumor cells in vitro and in vivo through induction of long-lasting HER-specific immunity.MethodsBALB/c mice were immunized with anti-trastuzumab anti-idiotype (anti-Id) scFv (scFv40 and scFv69), which mimic human HER2. Their sera were assessed for the presence of HER2-specific Ab1' antibodies and for their ability to reduce viability of SK-OV-3 cells, a HER2-positive cancer cell line, in nude mice. MMTV.f.huHER2(Fo5) transgenic mice were immunized with scFv40 and scFv69 and, then, growth inhibition of spontaneous HER2-positive mammary tumors, humoral response, antibody isotype as well as splenocyte secretion of IL2 and IFN-γ were evaluated.ResultsAdoptively-transferred sera from BALB/c mice immunized with scFv40 and scFv69 contain anti-HER2 Ab1' antibodies that can efficiently inhibit growth of SK-OV-3 cell tumors in nude mice. Similarly, prophylactic vaccination with anti-Id scFv69 fully protects virgin or primiparous FVB-MMTV.f.huHER2(Fo5) females from developing spontaneous mammary tumors. Moreover, such vaccination elicits an anti-HER2 Ab1' immune response together with a scFv69-specific Th1 response with IL2 and IFN-γ cytokine secretion.ConclusionsAnti-trastuzumab anti-Id scFv69, used as a therapeutic or prophylactic vaccine, protects mice from developing HER2-positive mammary tumors by inducing both anti-HER2 Ab1' antibody production and an anti-HER2 Th2-dependent immune response. These results suggest that scFv69 could be used as an anti-Id-based vaccine for adjuvant therapy of patients with HER2-positive tumors to reverse immunological tolerance to HER2.

Abstract 1387: Development of novel antiangiogenic biologics: multifunctional VEGF traps

Abstract Current anti-VEGF biologics have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, side effects, long-term toxicities, such as proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this study was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of antibody-based recombinant proteins were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup of proteins, “shV-H traps”, is comprised of a Fc domain shortened form of VEGF-trap linked to a sequence of hydrophobic amino acids, with various affinities for heparin sulfate proteoglycans of the ECM, designed to have a short systemic half-life. The second subgroup of molecules, “V-lassos”, is composed of a C-terminus positioned form of VEGF-trap linked to single-chain variable domain antibodies specific for either HER-2 or fibronectin extra domain B (EDB), expressed on tumour cell surfaces or in the vascular bed of solid tumours, respectively. Recombinant proteins were expressed in transgenic cancer cell lines in a doxycycline inducible manner and were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the shV-H traps adhered to the ECM and the HER-2 V-lasso inhibited the proliferation of HER-2 positive cancer cell lines. Xenograft studies were performed using the aforementioned transgenic cancer cell lines in order to determine the efficacy of shV-H traps and Vlasso. shV-H traps were able to inhibit or delay tumour growth of A-673, Pc-3 and HT-29 xenografts. In contrast to soluble VEGF-trap, sh-VH traps were retained at the tumour site and were undetectable in the circulation. Moreover, sh-VH traps did not cause any of the side-effects observed with soluble VEGF-trap, such as delay of wound healing and regression of trachea blood vessels. Both Vlassos were able to inhibit or delay the tumour growth of SKOV-3 and A-673 xenografts. Furthermore, the transgenic approach for inducible expression indicated that HER-2 Vlasso is more effective compared to anti-HER-2 Ab and VEGF-trap used alone or in combination. Preliminary results suggest that high recombinant protein levels can be generated via large-scale production in 293F cells, indicating that the new multifunctional VEGF-traps can be produced in large amounts for clinical studies. These novel classes of anti-angiogenic molecules could potentially be advantageous in a clinical setting. Furthermore, they can be useful in order to further study and understand the mechanisms involved in VEGF inhibition. Finally, similar dual function proteins can be designed for inhibition of other molecules with disease relevance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1387.