HER2-overexpressing Cells Research Articles

Abstract 2112: Chimeric antigen receptor macrophages (CAR-M) sensitize solid tumors to anti-PD1 immunotherapy

Abstract Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, achieving efficacy against solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Given that CAR-M are M1-polarized macrophages with the potential to remodel the tumor microenvironment (TME) and act as professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the interaction of CAR-M with the endogenous immune system and to study the combinatorial approach of CAR-M with blockade of the PD1/PDL1 T cell checkpoint axis. Murine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. In addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. Anti-HER2 CAR-M, but not control macrophages, phagocytosed and killed HER2-overexpressing tumor cell lines. CAR-M induced MHC-I expression on tumor cells and enhanced the cytotoxicity of CD8+ T cells. To evaluate the safety and efficacy of CAR-M therapy, immunocompetent mice were engrafted with HER2+ tumors and treated with syngeneic HER2-CAR or untransduced (UTD) macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cell responsiveness to tumor-associated antigens, indicating enhanced epitope spreading. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with anti-PD1 in tumors resistant to anti-PD1 monotherapy and found that the combination further reprogrammed the TME, significantly enhanced tumor control, and improved overall survival compared to monotherapy with either agent. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2-negative rechallenge model, indicating long-term anti-tumor immunity. Finally, the combination of CAR-M with anti-PD1 did not trigger sustained elevations of serum analytes associated with cytokine release syndrome (CRS) and was well tolerated across numerous safety assessments. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Alison Worth, Ilyssa Ramos, Daniel Blumenthal, Yumi Ohtani, Linara Gabitova, Michael Ball, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) sensitize solid tumors to anti-PD1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2112.

Newly identified breast luminal progenitor and gestational stem cell populations likely give rise to HER2-overexpressing and basal-like breast cancers

Breast Cancer (BrC) is a common malignancy with genetically diverse subtypes. There is evidence that specific BrC subtypes originate from particular normal mammary cell populations. However, the cell populations that give rise to most BrC subtypes are unidentified. Several human breast scRNAseq datasets are available. In this research, we utilized a robust human scRNAseq dataset to identify population-specific marker genes and then identified the expression of these marker genes in specific BrC subtypes. In humans, several BrC subtypes, HER2-enriched, basal-like, and triple-negative (TN), are more common in women who have had children. This observation suggests that cell populations that originate during pregnancy give rise to these BrCs. The current human datasets have few normal parous samples, so we supplemented this research with mouse datasets, which contain mammary cells from various developmental stages. This research identified two novel normal breast cell populations that may be the origin of the basal-like and HER2-overexpressing subtypes, respectively. A stem cell-like population, SC, that expresses gestation-specific genes has similar gene expression patterns to basal-like BrCs. A novel luminal progenitor cell population and HER2-overexpressing BrCs are marked by S100A7, S100A8, and S100A9 expression. We bolstered our findings by examining SC gene expression in TN BrC scRNAseq datasets and S100A7-A9 gene expression in BrC cell lines. We discovered that several potential cancer stem cell populations highly express most of the SC genes in TN BrCs and confirmed S100A8 and A9 overexpression in a HER2-overexpressing BrC cell line. In summary, normal SC and the novel luminal progenitor cell population likely give rise to basal-like and HER2-overexpressing BrCs, respectively. Characterizing these normal cell populations may facilitate a better understanding of specific BrCs subtypes.

Photothermal Therapy with HER2-Targeted Silver Nanoparticles Leading to Cancer Remission.

Nanoparticles exhibiting the localized surface plasmon resonance (LSPR) phenomenon are promising tools for diagnostics and cancer treatment. Among widely used metal nanoparticles, silver nanoparticles (Ag NPs) possess the strongest light scattering and surface plasmon strength. However, the therapeutic potential of Ag NPs has until now been underestimated. Here we show targeted photothermal therapy of solid tumors with 35 nm HER2-targeted Ag NPs, which were produced by the green synthesis using an aqueous extract of Lavandula angustifolia Mill. Light irradiation tests demonstrated effective hyperthermic properties of these NPs, namely heating by 10 °C in 10 min. To mediate targeted cancer therapy, Ag NPs were conjugated to the scaffold polypeptide, affibody ZHER2:342, which recognizes a clinically relevant oncomarker HER2. The conjugation was mediated by the PEG linker to obtain Ag-PEG-HER2 nanoparticles. Flow cytometry tests showed that Ag-PEG-HER2 particles successfully bind to HER2-overexpressing cells with a specificity comparable to that of full-size anti-HER2 IgGs. A confocal microscopy study showed efficient internalization of Ag-PEG-HER2 into cells in less than 2 h of incubation. Cytotoxicity assays demonstrated effective cell death upon exposure to Ag-PEG-HER2 and irradiation, caused by the production of reactive oxygen species. Xenograft tumor therapy with Ag-PEG-HER2 particles in vivo resulted in full primary tumor regression and the prevention of metastatic spread. Thus, for the first time, we have shown that HER2-directed plasmonic Ag nanoparticles are effective sensitizers for targeted photothermal oncotherapy.

Abstract P4-01-12: Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models

Abstract Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.

Abstract P4-01-14: The RXR agonist, IRX4204, increases the anti-tumor activity of HER2-targeted therapies in HER2-amplified breast cancer

Abstract Background: HER2 targeted monoclonal antibodies and tyrosine kinase inhibitors (TKI) are effective treatments for HER2-amplified primary tumors. However, the challenge persists that primary and acquired resistance to anti-HER2 therapy is common, and that anti-HER2 targeted therapies fail to achieve a cure in the metastatic setting. There is a critical need for safe and effective therapies that can overcome anti-HER2 drug resistance and eliminate breast cancer metastases. Here, we present data demonstrating the activity of IRX4204, a highly specific agonist of the nuclear retinoid X receptor (RXR), to inhibit the growth of HER2-amplified breast cancer cell lines alone and in combination with current anti-HER2 therapies. Methods: In vitro cell growth assays were conducted to evaluate the efficacy of IRX4204 alone and in combination with trastuzumab, tucatinib, neratinib or T-DM1, using a panel of breast cancer cell lines expressing various levels of HER2. A calculated coefficient of drug interaction (CDI) and isobologram analysis was used to assess the additive, synergistic or antagonistic effects of IRX4204 with each anti-HER2 targeted therapy. Results: IRX4204 treatment alone inhibited the growth of HER2-overexpressing cell lines, SkBr3, AU565, HCC1419 and ZR75-1 in vitro, but not the growth of HER2-normal cell lines MCF7, MDA-MB-231, or HCC1143. When combined with trastuzumab, tucatinib, neratinib or T-DM1, additive or synergistic effects were observed in all of the drug-sensitive cell lines. Moreover, IRX4204 in combination with neratinib or tucatinib inhibited the growth of a TKI-resistant breast cancer cell line (HCC1954). Conclusion: These data demonstrate a novel use of an RXR agonist, IRX4204, to inhibit the growth of HER2-amplified breast cancer cell lines and to enhance the activity of anti-HER2 therapy in vitro. These results also demonstrate that IRX4204 can overcome anti-HER2 resistance to inhibit the growth of HER2-positive cells. These data demonstrate that IRX4204 can enhance the anti-cancer activity of anti-HER2 therapies even in TKI-resistant cells. In vivo experiments and mechanistic studies are ongoing. Citation Format: Cassandra L Moyer, Abhijit Mazumdar, Jamal Hill, Martin E Sanders, Powel Brown. The RXR agonist, IRX4204, increases the anti-tumor activity of HER2-targeted therapies in HER2-amplified breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-14.

A highly sensitive nanobiosensor based on aptamer-conjugated graphene-decorated rhodium nanoparticles for detection of HER2-positive circulating tumor cells

AbstractAssessment of human epidermal growth factor receptor-2 (HER2) tumor marker status is an impressive factor in screening, diagnosing and monitoring breast cancer (BC). The electrochemical biosensor is a revolutionary method in cancer diagnosis, which is used in this research to detect HER2+circulating tumor cells. The electrochemical activity, size, shape, and morphology of the synthesized nanomaterials were analyzed. The hybrid nanocomposite established by the coupling of reduced graphene oxide nanosheets (rGONs) and rhodium nanoparticles (Rh-NPs) on the surface of graphite electrode resulted in improved surface area, electrochemical activity, and biocompatibility. The graphite electrode-based aptasensor (g-aptasensor) demonstrated exceptional performance against HER2-overexpressed SKBR3 cancer cells, with a linear dynamic range of 5.0 to 10.0 × 104 cells/mL, an analytical limit of detection (LOD) as low as 1.0 cell/mL, and a limit of quantification (LOQ) of 3.0 cells/mL. The G-rich DNA aptamers can fold into an intermolecular G-quadruplex, which specifically bind to the target molecule. Consequently, the advantages of this highly efficient nanocomposite platform include broad dynamic range, high specificity, selectivity, stability, reproducibility, and low cost. These characteristics indicate that the fabricated nanobiosensor has a high potential for use in detecting and monitoring HER2 level for the care of BC patients and clinical diagnosis.

Near-infrared PbS quantum dots functionalized with affibodies and ZnPP for targeted imaging and therapeutic applications

We report a new theranostic device based on lead sulfide quantum dots (PbS QDs) with optical emission in the near infrared wavelength range decorated with affibodies (small 6.5 kDa protein-based antibody replacements) specific to the cancer biomarker human epidermal growth factor receptor 2 (HER2), and zinc(II) protoporphyrin IX (ZnPP) to combine imaging, targeting and therapy within one nanostructure. Colloidal PbS QDs were synthesized in aqueous solution with a nanocrystal diameter of ∼5 nm and photoluminescence emission in the near infrared wavelength range. The ZHER2:432 affibody, mutated through the introduction of two cysteine residues at the C-terminus (Afb2C), was used as capping ligand to form Afb2C-PbS QDs that have a high binding affinity for HER2, which is overexpressed in several types of cancer including breast cancer. Afb2C-PbS QDs were further modified by conjugation with ZnPP, which acts as an anticancer agent. The biological activity of these QDs was tested against SKBR3 (HER2-positive) and MDA-MB-231 (HER2-normal) breast cancer cells, with results showing that ZnPP-Afb2C-functionalized PbS QDs were successfully targeted to the HER2-overexpressing cancer cells and induced cell apoptosis thanks to the conjugation with ZnPP. These results expand the use of the QD nanoplatform with the formulation of novel nanomaterials for targeted delivery and combined imaging and therapy via direct surface-protein interaction.

Toward Understanding the Binding Synergy of Trastuzumab and Pertuzumab to Human Epidermal Growth Factor Receptor 2

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast, gastric, esophageal, ovarian, and endometrial cancer. Combination therapy using trastuzumab and pertuzumab antibodies targeting HER2 has shown better survival outcomes in breast cancer patients. In the quest to understand the synergistic effect observed due to combination therapy, trastuzumab, pertuzumab, and their F(ab')2 fragments were labeled with radioisotope and fluorescent probes. Detailed in vitro studies to understand binding synergism in HER2 overexpressing cell lines were done. Antibodies and their F(ab')2 fragments prepared by enzyme digestion with pepsin were radiolabeled with iodine-125. In vitro binding studies to evaluate immunoreactivity, specificity, affinity, and binding synergism between radiolabeled trastuzumab, pertuzumab, and their F(ab')2 fragments were carried out. Synergism was observed by 20-30% enhanced uptake of radiolabeled pertuzumab and its F(ab')2 fragments in the presence of excess of unlabeled trastuzumab or F(ab')2-trastuzumab. However, uptake of radiolabeled trastuzumab was not enhanced in the presence of excess pertuzumab or its fragments; rather inhibition or competition in binding to HER2 was observed. Studies using fluorescent antibodies by flow cytometry confirmed enhanced binding of pertuzumab in the presence of trastuzumab. Live cell tracking was done to give insights into the binding synergy and fate of fluorescent antibodies . Colocalization of antibodies on HER2 followed by internalization in the cells was observed. The radiolabeled immunoconjugates served as an important tool for experimental characterization of interaction between pertuzumab and trastuzumab to HER2. Studies with fluorescent antibodies corroborated the binding data and provided evidence of colocalization and internalization of both the antibodies in HER2-positive cells.

Affibody-conjugated 5-fluorouracil prodrug system preferentially targets and inhibits HER2-expressing cancer cells

Overexpression of HER2 is associated with cancer phenotypes, such as proliferation, survival, metastasis and angiogenesis, and has been validated as a therapeutic target. However, only a portion of patients benefited from anti-HER2 treatments, and many would develop resistance. A more effective HER2 targeted therapeutics is needed. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and a HER2-targeting scaffold protein, ZHER2:2891, fused with yeast cytosine deaminase (Fcy) to target HER2-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned the coding gene of ZHER2:2891 and fused with those of ABD (albumin-binding domain) and Fcy. The purified ZHER2:2891-ABD-Fcy fusion protein specifically binds to HER2 with a Kd value of 1.6 nM ZHER2:2891-ABD-Fcy binds to MDA-MB-468, SKOV-3, BT474, and MC38-HER2 cells, which overexpress HER2, whereas with a lower affinity to HER2 non-expresser, MC38. Correspondingly, the viability of HER2-expressing cells was suppressed by relative low concentrations of ZHER2:2891-ABD-Fcy in the presence of 5-FC, and the IC50 values of ZHER2:2891-ABD-Fcy for HER2 high-expresser cells were approximately 10–1000 fold lower than those of non-HER2-targeting Fcy, and ABD-Fcy. This novel prodrug system, ZHER2:2891-ABD-Fcy/5-FC, might become a promising addition to the existing class of therapeutics specifically target HER2-expressing cancers.

The effects of T-DXd on the expression of HLA class I and chemokines CXCL9/10/11 in HER2-overexpressing gastric cancer cells

Trastuzumab deruxtecan (T-DXd), a HER2-targeting antibody–drug conjugate with a topoisomerase I inhibitor deruxtecan (DXd), exhibits an excellent anti-tumor effect in previously treated HER2-positive tumors. A recent study demonstrated that T-DXd not only suppressed tumor growth but also enhanced anti-tumor immunity through increasing the number of tumor-infiltrating CD8+ T cells and enhancement of major-histocompatibility-complex class I expression on tumor cells in a mouse model. However, the effect of T-DXd on anti-tumor immune responses in human cancers is largely unknown. We investigated the effect of T-DXd on the expression of HLA class I and CXCL9/10/11, T-cell chemoattractants, in HER2-positive human gastric cancer (GC) cells. We found that T-DXd significantly inhibited GC cell proliferation in a HER2-dependent manner, while it slightly increased the expression of HLA class I in HER2-positive GC cells. Moreover, we revealed that T-DXd significantly induced mRNA expression of CXCL9/10/11 in HER2-positive GC cells. T-DXd-triggered up-regulation of these chemokines was mediated through the activation of DNA damage signaling pathways. These results suggest that T-DXd triggers anti-tumor immune responses at least in part through induction of the expression of HLA class I and CXCL9/10/11 on HER2-positive GC cells, resulting in the enhancement of anti-tumor immunity in human GC.

An Anti-HER2 Monoclonal Antibody H2Mab-41 Exerts Antitumor Activities in Mouse Xenograft Model Using Dog HER2-Overexpressed Cells.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in a variety of cancer types, including breast, lung, gastric, pancreatic, and colorectal cancers. Trastuzumab, a humanized anti-HER2 monoclonal antibody (mAb), has been shown to provide significant survival benefits in HER2-overexpressing breast cancer and gastric cancer patients. Previously, an anti-HER2 mAb, H2Mab-41 (IgG2b, kappa), was developed in our laboratory and its antitumor activity was demonstrated in mouse xenograft models of human colon cancer. The present study aimed to investigate the ability of H2Mab-41 to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dog HER2 (dHER2)-overexpressed cell lines, and thus exert its antitumor activity against dHER2-overexpressed tumors in vivo. Flow cytometry results demonstrated the cross-reactivity of H2Mab-41 with dHER2. Further evaluation of interaction between H2Mab-41 and dHER2-overexpressed CHO-K1 (CHO/dHER2) cells indicated moderate binding affinity of H2Mab-41 toward dHER2, with a dissociation constant (KD) of 2.6 × 10-8 M. In vitro analysis revealed that the administration of H2Mab-41 induced high levels of ADCC and CDC in CHO/dHER2 cells. Furthermore, intraperitoneal administration of H2Mab-41 in mouse xenograft models of CHO/dHER2 resulted in significant inhibition of tumor development compared to the control mouse IgG. Thus, the findings of the present study demonstrated the in vivo safety and efficacy of H2Mab-41, highlighting its suitability to be included as a part of a therapeutic regimen for dHER2-expressing canine cancers.

Abstract 1078: Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features

Abstract Trastuzumab and trastuzumab emtansine (T-DM1) targeted therapies are the main choice for the treatment of HER2-overexpressing breast cancer patients. However, de novo or acquired resistance is still the major obstacle in clinical practice. It has been shown that several pathways, including HER2, can lead to HIF-α stabilization in breast cancer cells. Thus, the purpose of our study was to analyse the effect of hypoxia in acquired resistance to anti-HER2 therapies. We used HER2-overexpressing BT-474 and non-overexpressing MCF-7 human breast cancer cell lines. As an acute hypoxia model, we added CoCl2 (100 µM) to cell culture medium. The hypoxic status of the cells was evaluated by a Western blot analysis, showing a peak of HIF-1α expression after 6 hours of CoCl2 exposure. This result correlated with VEGF induction, as measured by RT-qPCR (p<0.05). Interestingly, under hypoxia, BT-474 cells treated with increasing concentrations of trastuzumab and T-DM1 for 72 hours presented a significantly higher viability compared with non-hypoxic cells (p<0.05). However, although the drugs decreased membrane HER2 protein expression (p<0.01), HER2 levels did not change under hypoxic conditions, as shown by flow cytometry and Western blot assays. Moreover, drug effects on MCF-7 cells did not change under hypoxic conditions. To further analyse this cytoprotective effect of hypoxia, proliferation-associated markers such as PCNA expression and AKT activation were evaluated by Western blot, while apoptosis frequency was measured by flow cytometry in BT-474 cells. PCNA and pAKT expression remained unaltered, while T-DM1-mediated apoptosis was significantly reduced under hypoxia (p<0.05). Then, using different open gene expression datasets, we identified a group of four genes (COL12A1, DKK1, CEACAM5 and ANTXR1) differentially expressed in T-DM1 resistant HER2-overexpressing breast cancer BT-474 and KPL-4 cell lines (p<0.05), which were coexpressed with hypoxia-related genes (HIF1A, EPAS1 and VEGFA) in HER2-overexpressing breast cancer tumours and cell lines from TCGA (p<0.01). Further in silico analyses revealed a direct association between those genes and the fibronectin gene (FN1, p<0.05). Remarkably, they were involved in TGF-β and angiogenesis pathways, as shown by a functional enrichment analysis (p<0.01). In addition, we found that BT-474 cells under hypoxia presented a higher mammosphere formation efficiency than non-hypoxic cells (p<0.05), showing a tendency towards a cancer stem cell phenotype. It is known that hypoxia represents a common feature of metastatic tumours and has a role in regulating stem cell behaviour. According to this, our results highlight a link between anti-HER2 therapy resistance and epithelial-to-mesenchymal transition characteristics, where hypoxia plays a key role which deserves further study. Citation Format: Virginia Judith Wolos, Ivana Jaqueline Tapia, Marianela Abrigo, Ezequiel Lacunza, Elisa Dora Bal de Kier Joffé, Gabriel León Fiszman. Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1078.

Abstract 1005: The bispecific antibody zanidatamab's (ZW25's) unique mechanisms of action and durable anti-tumor activity in HER2-expressing cancers

Abstract HER2-directed therapies have improved clinical outcomes for many patients with HER2-positive breast and gastric cancer. Despite these successes, there remains a need to develop improved HER2-targeted therapies for these and other HER2-expressing tumors, particularly in the setting of recurrent or metastatic disease. Zanidatamab (ZW25) is a humanized, bispecific, immunoglobulin (Ig) G1-like antibody directed against the juxtamembrane extracellular domain (ECD4) and the dimerization domain (ECD2) of human epidermal growth factor receptor 2 (HER2), the same domains targeted by trastuzumab (T) and pertuzumab (P), respectively. Data from the ongoing phase 1 study (NCT02892123) demonstrate that zanidatamab is well tolerated and has single agent activity in patients with advanced HER2-expressing cancers that have progressed after standard of care (SOC) therapies, including HER2-targeted agents such as T, P, and trastuzumab emtansine.1,2 We have previously shown that the unique design and bispecific binding of zanidatamab results in multiple mechanisms of action including increased antibody binding density, potent effector function, improved receptor internalization and HER2 downregulation relative to T.3 To better understand the mechanism by which zanidatamab differentiates itself from T, P and T+P, we recently expanded our mechanistic evaluations including cell surface HER2 aggregation, complement-dependant cytotoxicity (CDC) and inhibition of both tumor cell growth and intracellular signaling. Single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM) was used to map HER2 receptor distribution and quantitate the size, density, and frequency of receptor clusters induced by antibody binding. In vitro assessments were performed in a panel of HER2-expressing cell lines using standard assays including CDC with human complement serum and inhibition of both tumor cell growth and intracellular signaling. Using dSTORM, we observed that zanidatamab binding resulted in enhanced HER2 aggregation and distinct HER2 capping on the tumor cell surface compared to T, P or T+P. Evaluation of CDC activity in HER2-overexpressing tumor cells demonstrated that zanidatamab, but not T, P or T+P, elicited CDC suggesting that the enhanced HER2 aggregation and capping on the tumor cell surface provides high avidity docking sites to which C1 binds and is activated. Zanidatamab showed further differentiation in the inhibition of both tumor growth and intracellular signaling of HER2-overexpressing cells compared to T, P and T+P. Zanidatamab has novel cell surface binding and additional mechanisms of action compared to T, P and T+P. Zanidatamab is actively being evaluated in clinical trials in multiple HER2-expressing solid tumors, including a registration-enabling clinical trial in HER2 gene amplified biliary tract cancer (NCT04466891). Citation Format: Nina E. Weisser, Grant Wickman, Libin Abraham, Jason O'Toole, Bryant Harbourne, Joy Guedia, Chi Wing Cheng, Peter Chan, Duncan Browman, Michael R. Gold, Neil Josephson, Surjit Dixit, Gerry Rowse. The bispecific antibody zanidatamab's (ZW25's) unique mechanisms of action and durable anti-tumor activity in HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1005.

Abstract 1530: Anti-HER2 CAR monocytes demonstrate targeted anti-tumor activity and enable a single day cell manufacturing process

Abstract Recent advances in cell therapy have led to significant efficacy in hematologic malignancies, but solid tumors remain an intractable challenge. We have previously developed a CAR macrophage (CAR-M) adoptive cell therapy platform and demonstrated potent anti-tumor activity in pre-clinical models. CAR-M overcome several of the barriers to efficacy in the solid tumor setting - trafficking, immunosuppression, lymphocyte exclusion, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. In order to streamline the manufacturing process, improve cell yields, and potentially improve tumor infiltration, we sought to evaluate the feasibility of directly engineering CD14+ monocytes to express CAR. Using the chimeric adenoviral vector Ad5f35, we engineered primary human CAR-monocytes to target tumors overexpressing HER2. CAR expression and viability both exceeded 90%. Anti-HER2 CAR-monocytes produced pro-inflammatory cytokines in response to antigen, specifically phagocytosed HER2 overexpressing target cells, and eradicated HER2-overexpressing tumor cells in a time and dose-dependent manner. CAR-monocytes efficiently differentiated into CAR-expressing macrophages in response to various maturation factors and cytokines within 3-5 days. Adenoviral based gene modification resulted in the upregulation of several pro-inflammatory markers on CAR-monocytes and pre-conditioned these cells to differentiate into M1 macrophages in the absence of exogenous M1 polarization factors. The M1 phenotype was maintained when CAR-monocytes were challenged with immunosuppressive cytokines in vitro. These cells demonstrated potent effector function after differentiation into macrophages, regardless of exposure to GM-CSF, M-CSF, or immunosuppressive factors. Anti-HER2 CAR monocytes carried broadly expressed myeloid chemokine receptors and responded to a panel of chemotactic factors. In order to optimize the cell manufacturing process associated with CAR-M, we established a closed-system, ultra-rapid CAR monocyte process. This process enabled same day manufacturing of the final CAR monocyte cell product, significantly reducing the cost of goods associated with autologous cell therapy as well as “vein to vein” time. This study demonstrated the successful development of CAR-monocytes with direct anti-tumor activity and capacity to differentiate into M1 polarized CAR-M. In addition, we established an ultra-rapid same-day CAR monocyte manufacturing process. The CAR-monocyte platform represents an advance in the field of adoptive cell therapy. Citation Format: Linara Gabitova, Brett Menchel, Rashid Gabbasov, Stefano Pierini, Andrew Best, Kayleigh Ross, Yumi Ohtani, Daniel Blumenthal, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Anti-HER2 CAR monocytes demonstrate targeted anti-tumor activity and enable a single day cell manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1530.

ADCC-Inducing Antibody Trastuzumab and Selection of KIR-HLA Ligand Mismatched Donors Enhance the NK Cell Anti-Breast Cancer Response.

Simple SummaryNatural killer (NK) cells are potent killers of tumor cells. Many tumors, including breast cancers, develop mechanisms to suppress anti-tumor immune responses, requiring the development of strategies to overcome suppression. Here, we tested a combination therapy that aims to (1) enhance NK cell activation and (2) reduce NK cell inhibition mediated by suppressive factors in tumors or in the tumor microenvironment. We cultured cell lines under hypoxia to mimic the tumor microenvironment or used patient-derived breast cancer cells that were primed by the patient’s tumor environment. Our results demonstrated that cytokine-activated NK cells remained active under hypoxia and that tumor-targeting antibodies enhanced the NK cell anti-breast cancer response. Moreover, we observed that NK cell suppression by inhibitory ligands on the tumor cells can be reduced by the selection of NK cell donors with NK receptors that are incompatible with these ligands. Collectively, we present two powerful strategies to enhance the NK cell responses against breast cancer.Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells’ full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.

PLGA Nanoparticles Decorated with Anti-HER2 Affibody for Targeted Delivery and Photoinduced Cell Death.

The effect of enhanced permeability and retention is often not sufficient for highly effective cancer therapy with nanoparticles, and the development of active targeted drug delivery systems based on nanoparticles is probably the main direction of modern cancer medicine. To meet the challenge, we developed polymer PLGA nanoparticles loaded with fluorescent photosensitive xanthene dye, Rose Bengal, and decorated with HER2-recognizing artificial scaffold protein, affibody ZHER2:342. The obtained 170 nm PLGA nanoparticles possess both fluorescent and photosensitive properties. Namely, under irradiation with the green light of 540 nm nanoparticles, they produced reactive oxygen species leading to cancer cell death. The chemical conjugation of PLGA with anti-HER2 affibody resulted in the selective binding of nanoparticles only to HER2-overexpressing cancer cells. HER2 is a receptor tyrosine kinase that belongs to the EGFR/ERbB family and is overexpressed in 30% of breast cancers, thus serving as a clinically relevant oncomarker. However, the standard targeting molecules such as full-size antibodies possess serious drawbacks, such as high immunogenicity and the need for mammalian cell production. We believe that the developed affibody-decorated targeted photosensitive PLGA nanoparticles will provide new solutions for ongoing problems in cancer diagnostics and treatment, as well in cancer theranostics.

Comparative Evaluation of Engineered Polypeptide Scaffolds in HER2-Targeting Magnetic Nanocarrier Delivery.

Targeted drug delivery is one of the most intriguing and challenging issues in modern biomedicine. For active targeting, full-size IgG molecules (150 kDa) are usually used. Recent studies have revealed that small artificial polypeptide scaffolds such as DARPins (14 kDa) and affibodies (8 kDa) are much more promising tools for drug delivery due to their small size, artificial nature, low immunogenicity, and many other properties. However, there is no comparative information on the targeting abilities of scaffold polypeptides, which should be taken into account when developing drug delivery systems (DDSs). The present work is the first comprehensive study on the comparison of the effectiveness of different HER2-targeting proteins within the architecture of nanoparticles. Namely, we synthesized trimodal nanoparticles: magnetic, fluorescent, and directed toward HER2 oncomarker on cancer cells. The magnetic particles (MPs) were covalently modified with (i) full-size IgG, 150 kDa, (ii) DARPin_G3, 14 kDa, and (iii) affibody ZHER2:342, 8 kDa. We showed that the number of DARPin_G3 and affibody ZHER2:342 molecules conjugated to the nanoparticle surface are 10 and 40 times higher, respectively, than the corresponding value for trastuzumab. Using the methods of magnetic particle quantification (MPQ)-cytometry and confocal microscopy, we showed that all types of the obtained magnetic conjugates specifically labeled HER2-overexpressing cells. Namely, we demonstrated that particle binding to HER2-positive cells is 1113 ± 39 fg/cell for MP*trastuzumab, 1431 ± 186 fg/cell for MP*ZHER2:342, and 625±21 fg/cell for MP*DARPin_G3, which are 2.77, 2.75, and 2.30 times higher than the corresponding values for control HER2-negative cells. Thus, we showed that the smallest HER2-recognizing polypeptide affibody ZHER2:342 is more effective in terms of specificity and selectivity in nanoparticle-mediated cell labeling.

Development of HER2-Targeting-Ligand-Modified Albumin Nanoparticles Based on the SpyTag/SpyCatcher System for Photothermal Therapy.

The successful development of targeted nanoparticle (NP)-based therapeutics depends on the effective conjugation of targeting ligands to the NP. However, conventional methods based on chemical reactive groups such as N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and maleimide have several limitations, including low binding efficiency, complex reaction methods, long reaction times, and reduced activity of the targeting ligand. In this study, we developed a novel method for conjugating targeting ligands to albumin NPs using the recently developed bacterial superglue the SpyTag/SpyCatcher (ST/SC) ligation system. This method involves a rapid one-step conjugation process with almost 100% efficiency. Albumin NPs conjugated to human epidermal growth factor receptor 2 (HER2) affibody molecules using the ST/SC system showed strong binding to HER2-overexpressing cells. In addition, NPs encapsulated with indocyanine green accumulated in cells overexpressing HER2 and exhibited superior photothermal treatment effects. Thus, surface functionalization of NPs using the ST/SC reaction may be used to develop new nanosystems that exhibit improved therapeutic benefits.

Axially Substituted Silicon Phthalocyanine Payloads for Antibody–Drug Conjugates

AbstractIR700, a silicon phthalocyanine (SiPc) photosensitizer, is an antibody–drug conjugate payload used clinically. It is, however, the sole SiPc payload to date, possibly due to the difficulty of its synthesis, resulting from its asymmetric phthalocyanine skeleton. Here, we report a new axially substituted SiPc payload that is more easily synthesized. Trastuzumab conjugated with the SiPc showed light- and antigen-dependent cytotoxicity in HER2-overexpressing cancer cell lines.

Nature-Inspired Nanoparticles as Paclitaxel Targeted Carrier for the Treatment of HER2-Positive Breast Cancer.

Simple SummaryGreat advances have been made in the treatment of an aggressive subtype of breast cancer, known as HER2-positive subtype. However, the antitumor therapy most widely employed in the clinical fight against it has severe side effects, and the apparition of treatment resistances is frequent. To overcome these drawbacks, nano-sized particles had already been developed as targeted vehicles for the drug and the antibody that are usually administered as first-line treatment for this subtype of breast cancer. These nanoparticles showed better results than the drug that they transported. Nevertheless, to further reduce the drug effective dose and its toxicity to normal tissues, other nanoparticles, more advantageous than the previous ones, were developed in the current work. Compared to the previous nanoparticles, those prepared here proved to be more efficacious, and their potential administration may constitute an excellent approach to improve the outcomes of patients suffering from HER2-positive breast cancer. Despite the advances made in the fight against HER2-positive breast cancer, the need for less toxic therapies and strategies that avoid the apparition of resistances is indisputable. For this reason, a targeted nanovehicle for paclitaxel and trastuzumab, used in the first-line treatment of this subtype of breast cancer, had already been developed in a previous study. It yielded good results in vitro but, with the aim of further reducing paclitaxel effective dose and its side effects, a novel drug delivery system was prepared in this work. Thus, polydopamine nanoparticles, which are gaining popularity in cancer nanomedicine, were novelty loaded with paclitaxel and trastuzumab. The effectiveness and selectivity of the nanoparticles obtained were validated in vitro with different HER2-overexpressing tumor and stromal cell lines. These nanoparticles showed more remarkable antitumor activity than the nanosystem previously designed and, in addition, to affect stromal cell viability rate less than the parent drug. Moreover, loaded polydopamine nanoparticles, which notably increased the number of apoptotic HER2-positive breast cancer cells after treatment, also maintained an efficient antineoplastic effect when validated in tumor spheroids. Thereby, these bioinspired nanoparticles charged with both trastuzumab and paclitaxel may represent an excellent approach to improve current HER2-positive breast cancer therapies.