HER2-overexpressing Tumor Cells Research Articles

Blockade of exosome release alters HER2 trafficking to the plasma membrane and gives a boost to Trastuzumab.

Exosomal HER2 has been evidenced to interfere with antibody-induced anti-tumor effects. However, whether the blockade of HER2+ exosomes release would affect antibody-mediated tumor inhibition has yet to be investigated. Exosomes derived from BT-474, SK-BR3 and SK-OV3 (HER2-overexpressing tumor cells) and MDA-MB-231 cells (HER2 negative) were purified and characterized by bicinchoninic acid (BCA) assay, western blotting and Transmission electron microscopy (TEM). Inhibition of exosome release was achieved by neutral sphingomyelinase-2 (nSMase-2) inhibitor, GW4869. The effects of exosome blockade on the anti-proliferative effects, apoptosis induction, and antibody-mediated cellular cytotoxicity (ADCC) activity of Trastuzumab were examined using MTT, flow cytometry, and LDH release assays. Also, the effects of exosome inhibition on the surface expression and endocytosis/internalization of HER2 were studied by flow cytometry. Purified exosomes derived from HER2 overexpressing cancer cells were positive for HER2 protein. Blockade of exosome release was able to significantly improve apoptosis induction, anti-proliferative and ADCC responses of Trastuzumab dose dependently. The pretreatment of Trastuzumab/purified NK cells, but not PBMCs, with HER2+ exosomes could also decrease the ADCC effects of Trastuzumab. Exosome inhibition also remarkably downregulated surface HER2 levels in a time-dependent manner, but does not affect its endocytosis/internalization. Based on our findings, HER2+ exosomes may benefit tumor progression by dually suppressing Trastuzumab-induced tumor growth inhibition and cytotoxicity of NK cells. It seems that concomitant blocking of exosome release might be an effective approach for improving the therapeutic effects of Trastuzumab, and potentially other HER2-directed mAbs. In addition, the exosome secretion pathway possibly contributes to the HER2 trafficking to plasma membrane, since the blockade of exosome secretion decreased surface HER2 levels.

Potent anti-tumor immune response and tumor growth inhibition induced by HER2 subdomain fusion protein in a mouse tumor model.

Several approaches have so far been employed to establish anti-tumor immunity by targeting HER2 protein. Active immunization with recombinant HER2 subdomains has previously been demonstrated to induce potent immune response and tumor growth inhibition. In the present study, we investigated the immunogenicity and tumor inhibitory effect of a fusion protein consisting of human HER2 extracellular subdomain (ECD-DI + II) together with T-helper cell epitopes of Tetanus toxin (p2 and p30). BALB/c mice were immunized with two recombinant proteins (DI + II and p2p30-DI + II) emulsified in 4 different adjuvants. Anti-DI + II antibody response, cytokine profile, frequency of splenic CD25+FOXP3+ regulatory T cells (Tregs) and CD8+CD107a+ cytotoxic T lymphocytes (CTLs) were assessed in the immunized mice. To assess the anti-tumor effect, the immunized mice were subcutaneously challenged with HER2-overexpressing tumor cells and the tumor growth was determined. Both recombinant proteins were able to induce comparable levels of ECD-DI + II-specific antibodies. Immunization with p2p30-DI + II resulted in a significant increase in the level of Interferon-gamma (IFN-γ) secretion compared to DI + II protein and significantly higher frequency of CTLs and lower frequency of Tregs. The number of mice that remained tumor-free until day 120 was significantly higher in p2p30-DI + II vaccinated groups. Our data suggest that the p2p30-DI + II fusion protein together with CpG adjuvant induces more potent anti-tumor immune responses in a mouse tumor model. Accordingly, this formulation might be considered as a potential immunotherapeutic approach in HER2+ cancers.

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.

Purification and In Vitro Evaluation of an Anti-HER2 Affibody-Monomethyl Auristatin E Conjugate in HER2-Positive Cancer Cells.

Simple SummaryAntibody-drug conjugates (ADCs) represent an innovative class of anticancer agents specifically aimed at targeting cancer cells, reducing damage to healthy tissues but showing some weaknesses. A promising approach for the development of high-affinity tumor targeting ADCs is the use of engineered protein drugs, such as affibody molecules. Our aim was to develop a more efficient purification method for the cytotoxic conjugate ZHER2:2891DCS-MMAE that targets human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. The conjugate is based on ZHER2:2891 affibody and a drug conjugation sequence (DCS), which allowed for site-specific conjugation of the cytotoxic auristatin E molecule (MMAE) to the affibody. We tested the in vitro efficacy of ZHER2:2891DCS-MMAE on several parameters, such as cell viability, proliferation, migration, and apoptosis. Our results confirmed that the cytotoxic conjugate efficiently interacts with high affinity with HER2 positive cancer cells, allowing the selective and specific delivery of the cytotoxic payload.A promising approach for the development of high-affinity tumor targeting ADCs is the use of engineered protein drugs, such as affibody molecules, which represent a valuable alternative to monoclonal antibodies (mAbs) in cancer-targeted therapy. We developed a method for a more efficient purification of the ZHER2:2891DCS affibody conjugated with the cytotoxic antimitotic agent auristatin E (MMAE), and its efficacy was tested in vitro on cell viability, proliferation, migration, and apoptosis. The effects of ZHER2:2891DCS-MMAE were compared with the clinically approved monoclonal antibody trastuzumab (Herceptin®). To demonstrate that ZHER2:2891DCS-MMAE can selectively target HER2 overexpressing tumor cells, we used three different cell lines: the human adenocarcinoma cell lines SK-BR-3 and ZR-75-1, both overexpressing HER2, and the triple-negative breast cancer cell line MDA-MB-231. MTT assay showed that ZHER2:2891DCS-MMAE induces a significant time-dependent toxic effect in SK-BR-3 cells. A 30% reduction of cell viability was already found after 10 min exposure at a concentration of 7 nM (IC50 of 80.2 nM). On the contrary, MDA-MB-231 cells, which express basal levels of HER2, were not affected by the conjugate. The cytotoxic effect of the ZHER2:2891DCS-MMAE was confirmed by measuring apoptosis by flow cytometry. In SK-BR-3 cells, increasing concentrations of conjugated affibody induced cell death starting from 10 min of treatment, with the strongest effect observed after 48 h. Overall, these results demonstrate that the ADC, formed by the anti-HER2 affibody conjugated to monomethyl auristatin E, efficiently interacts with high affinity with HER2 positive cancer cells in vitro, allowing the selective and specific delivery of the cytotoxic payload.

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.

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.

Abstract 6352: Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells

Abstract Background/Significance: Exosomes are 30-100 nm membrane vesicles secreted by many cell types. Tumors secrete large numbers of exosomes that transport oncoproteins and immune suppressive molecules, supporting tumor growth and metastasis. The role of exosomes in intercellular signaling is shown by human epidermal growth factor receptor type 2 (HER2)-expressing breast tumors, where exosomes with HER2 stimulate tumor growth and interfere with activity of the therapeutic antibody Herceptin, which binds to the extracellular domain of HER2 and prolongs the overall survival of HER2-positive breast cancer patients. Exosome release is modulated by growth factors EGF and heregulin, HER2 receptor-activating ligands naturally present in the surrounding tumor microenvironment. The SMRwt peptide reduces secretion of HER2 exosomes; thus, it can potentially be exploited as an anticancer agent. Methods: BT-474 and HCC1569 HER2-positive breast cancer cells overexpressing HER2 and mortalin were used in these experiments. Exosome size and concentration were measured by NanoSight. Purified tumor exosomes were prepared using paramagnetic beads coated with antibodies against tumor specific proteins and confirmed by flow cytometry and electron microscopy. To identify biological functions, we cultured tumor exosomes with breast cancer cells, which increased tumor cell proliferation. To determine SMRwt peptide inhibition effects, we assayed tumor exosome secretion and cell proliferation by NanoSight, Western blot and MTT. Results: We characterized exosomes secreted by mortalin and HER2-overexpressing breast cancer cell lines and analyzed SMRwt peptide's role in reducing tumor exosome release and cell proliferation. Exosome secretion increased in HER2-overexpressing tumor cell lines HCC1596 and BT474. Exosomes from HER2-positive tumor cell-conditioned supernatants stimulated tumor cell growth and tumor exosome release and inhibited Herceptin activity on HCC1569 and BT474 cell proliferation. Addition of SMRwt peptide reduced tumor cell proliferation and tumor exosome release. Conclusions: These data show the role of HER2-positive exosomes in modulating sensitivity to Herceptin and thus, to HER2-driven tumor aggressiveness; and indicate that the SMRwt peptide has potential anticancer functions in HER2-positive HCC1569 and BT474 human breast cancer cells. The data also indicate tumor exosomes affect the proliferation of parental cells. Tumor exosomes derived from BT-474 cells and HCC1569 cells stimulated the proliferation of BT-474 cells and HCC1569 cells, suggesting a biological function for tumor exosomes. Citation Format: Ming Bo Huang. Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6352.

Selective targeting of HER2-overexpressing solid tumors with a next-generation CAR-T cell therapy.

3041 Background: Conventional chimeric antigen receptor T cell (CAR-T) therapies have achieved limited clinical success in the treatment of solid tumors, in part due to the challenges of identifying tumor antigen(s) that are uniquely expressed on tumor cells. The dearth of such targets requires that current CAR-T therapies be re-engineered to preferentially target tumor cells thereby mitigating potential on-target off-tumor toxicity to normal cells. Herein we describe a novel cell therapy platform comprising Antigen Receptor Complex T (ARC-T) cells that are readily activated, silenced, and reprogrammed in vivo by administration of a novel tumor-targeting soluble protein antigen-receptor X-linker (sparX). The formation of the ARC-T, sparX, and tumor complex is required for the ARC-T to kill the tumor. Because ARC-T activity is entirely dependent on the dose of sparX administered, therapeutic doses of sparX may be defined that preferentially target cells over-expressing a target antigen and thus limit coincident kill of normal cells expressing lower levels of target antigen. Methods: We have created a library of sparX that bind different cell surface antigens, including HER2. The HER2 sparX was tested as both monovalent and bivalent constructs in vitro by assessing ARC-T cell activation, cytokine release and target cell cytotoxicity. In vivo efficacy models utilized NSG mice and incorporated tumor volume measurements and histopathologic assessments to evaluate tumor clearance. Results: In vitro studies demonstrate that co-culture of ARC-T cells, sparX-HER2 and HER2-expressing target cells drives T cell activation, expansion, cytokine secretion and cytotoxicity of target cells in a dose-dependent manner. Furthermore, by affinity tuning the HER2 binding domain and bivalent formatting of sparX-HER2, we achieved selective killing of HER2-overexpressing breast cancer cells with minimal effect on cells expressing HER2 levels representative of normal tissues. In vivo proof-of-principal studies with ARC-T/sparX-HER2 similarly demonstrate complete eradication of HER2-overexpressing solid tumor cells. Conclusions: These results demonstrate that a single intravenous dose of ARC-T cells can traffic to a solid tumor site and induce tumor eradication upon systemic administration and co-localization of tumor-targeting sparX in a mouse model. Bivalent formatting of sparX-HER2 further enabled ARC-T sensitivity to target antigen density to avoid the on-target off-tumor toxicity that has hindered conventional monovalent CAR-T treatments.

Herceptin-conjugated paclitaxel loaded PCL-PEG worm-like nanocrystal micelles for the combinatorial treatment of HER2-positive breast cancer

We have constructed Herceptin-conjugated, paclitaxel (PTX) loaded, PCL-PEG worm-like nanocrystal micelles (PTX@PCL-PEG-Herceptin) for the combinatorial therapy of HER2-positive breast cancer that exploit the specific targeting of Herceptin to HER2-positive breast cancer cells. Firstly, amphiphilic PCL2000-MPEG2000 and PCL5000-PEG2000-CHO were selected as the optimized matrix to wrap PTX that self-assembled into worm-like micelles with internal nanocrystal structures (PTX@PCL-PEG). Then the aldehydes of PCL5000-PEG2000-CHO exposed on the outside surface of PTX@PCL-PEG were utilized to react with the primary amines of Herceptin and formed stable, carbon-nitrogen single linkers (–C–N–) between the antibodies and nanoparticles. This study shows PTX@PCL-PEG-Herceptin remained relatively stable in the circulation and in the tumor microenvironment, and rapidly targeted and entered into the HER2-overexpressing tumor cells while sparing normal tissues from the toxic effects. PTX@PCL-PEG-Herceptin shrank the tumors and prolonged survival time in a SKBR-3-tumor-xenograft, nude mice model more effectively than TAXOL®, PTX@PCL-PEG, Herceptin+TAXOL® and Herceptin+PTX@PCL-PEG. Mechanistic studies showed that PTX@PCL-PEG-Herceptin entered into the HER2-positive tumor cells through the caveolin-mediated pathway. The conjugated Herceptin greatly enhanced the binding ability of the nanoparticle to the targeted SKBR-3 cells. This novel strategy provides a rational and simple antibody-conjugated-nanoparticle platform for the clinical application of combinatorial anticancer treatment.

Design, Expression, Purification and Characterization of the Recombinant Immunotoxin 4D5 scFv-TRAIL

Immunotoxins are chimeric proteins that combine antibodies and toxins in targeted cancer therapy. Despite the promising application prospects, immunogenicity and nonspecific cytotoxicity of immunotoxins with toxins from bacterial or plant origin have hindered their clinical application. In this study, an anti-HER2 single-chain antibody fragment 4D5, was fused to a human-origin antitumor drug, TRAIL to produce a humanized immunotoxin in Escherichia coli BL21 (DE3). An appropriate linker between the domains was selected using the software Antheprot 5.0. The 4D5 scFv-TRAIL was obtained using a SUMO tag for solubility-enhancing and purified by Ni–NTA affinity chromatography after tag removing. The purity and yield of 4D5 scFv-TRAIL reached 92% and 11.6 mg/L in shake flasks, respectively. Additionally, MTT assays showed 4D5 scFv-TRAIL exhibited selective in vitro cytotoxicity on the breast cancer cell line MDA-MB-231 without cytotoxic effect on the normal cell FBHE. Therefore, 4D5 scFv-TRAIL is a potential humanized immunotoxin for treatment of HER2-overexpressing tumor cells.

Paclitaxel-Trastuzumab Mixed Nanovehicle to Target HER2-Overexpressing Tumors.

Paclitaxel is one of the most widely used chemotherapeutic agents thanks to its effectiveness and broad spectrum of antitumor activity. However, it has a very poor aqueous solubility and a limited specificity. To solve these handicaps, a novel paclitaxel-trastuzumab targeted transport nanosystem has been developed and characterized in this work to specifically treat cancer cells that overexpress the human epidermal growth factor receptor-2 (HER2). Methods: Alginate and piperazine nanoparticles were synthetized and conjugated with paclitaxel:β-cyclodextrins complexes and trastuzumab. Conjugated nanoparticles (300 nm) were characterized and their internalization in HER2-overexpressing tumor cells was analyzed by immunofluorescence. Its specific antitumor activity was studied in vitro using human cell lines with different levels of HER2-expression. Results: In comparison with free paclitaxel:β-cyclodextrins complexes, the developed conjugated nanovehicle presented specificity for the treatment of HER2-overpressing cells, in which it was internalized by endocytosis. Conclusions: It seems that potentially avoiding the conventional adverse effects of paclitaxel treatment could be possible with the use of the proposed mixed nanovehicle, which improves its bioavailability and targets HER2-positive cancer cells.

Abstract 2850: FGFR signaling facilitates recurrence of minimal residual disease post trastuzumab emtansine treatment in breast cancer

Abstract The Her2 subtype of breast cancer (BC) accounts for about 20-25% of all the BC patients. This subtype is defined by the overexpression of human epidermal growth factor receptor 2 (Her2) which drives elevated downstream signaling promoting tumorigenesis. Trastuzumab emtansine (T-DM1) is an antibody drug conjugate in which an anti-Her2 antibody targets Her2 overexpressing tumor cells and delivers emtansine, a highly potent microtubule inhibitor. Metastatic BC patients who have progressed on Trastuzumab show improved overall survival with T-DM1 treatment. However, there is an unmet medical need in a group of patients where their initial response is followed by disease recurrence due to the development of acquired resistance. Therefore, it is important to investigate the molecular mechanisms of resistance to T-DM1. In this study, we established minimal residual disease (MRD) following T-DM1 treatment of HMLE-Her2 xenografts. Upon removal of the drug, tumors relapsed and were no longer responsive to T-DM1. In addition, immunohistochemistry staining revealed that these T-DM1 drug-resistant tumors lost their Her2 expression. In contrast to these in vivo results, continuous treatment of HMLE-Her2 cells with T-DM1 failed to show any acquired resistance in vitro. However, induction of epithelial-mesenchymal transition (EMT) via pretreatment with TGF-β facilitated acquisition of drug resistance to T-DM1. Similar to our in vivo resistant model, these in vitro resistant cells showed diminution in Her2 expression. Flow cytometry analysis suggested that TGF-β treatment may promote a heterogeneous expression of Her2, which helps the selection of a low Her2 expressing, T-DM1 resistant population. Next, we performed cell viability assays in the presence various kinase inhibitors. While the T-DM1 resistant cells were not sensitive to Lapatinib and Afatinib; they showed a dramatic response to FIIN-4, a covalent inhibitor of FGFR. Indeed, we observed elevated expression of FGFR1 at both the mRNA and protein levels in the T-DM1 resistant models. Moreover, we have found that ectopic overexpression of a truncated FGFR1 splice variant, FGFR1β, was sufficient to increase cell survival in response to T-DM1 treatment. Along these lines, orthotopic HMLE-Her2 tumors were less responsive to T-DM1 upon FGFR1β overexpression. Furthermore, overexpression of FGFR1β also leads to enhanced tumor recurrence following T-DM1 treatment. In line with these observations, we found that a patient-derived xenograft (PDX) from a Her2+ BC patient who had progressed on trastuzumab, were responsive to FIIN-4 while showing limited response to T-DM1. Currently, we are investigating potential mechanisms of FGFR mediated survival of T-DM1 resistant cells. Finally, we are exploring combination therapeutics using T-DM1 and FGFR inhibitors in various models of Her2 BC. Citation Format: Saeed Salehin Akhand, Connor Purdy, Michael Wendt. FGFR signaling facilitates recurrence of minimal residual disease post trastuzumab emtansine treatment in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2850.

Mechanisms of Acquired Resistance to Trastuzumab Emtansine in Breast Cancer Cells.

The receptor tyrosine kinase HER2 is overexpressed in approximately 20% of breast cancer, and its amplification is associated with reduced survival. Trastuzumab emtansine (Kadcyla, T-DM1), an antibody-drug conjugate that is comprised of trastuzumab covalently linked to the antimitotic agent DM1 through a stable linker, was designed to selectively deliver DM1 to HER2-overexpressing tumor cells. T-DM1 is approved for the treatment of patients with HER2-positive metastatic breast cancer following progression on trastuzumab and a taxane. Despite the improvement in clinical outcome, many patients who initially respond to T-DM1 treatment eventually develop progressive disease. The mechanisms that contribute to T-DM1 resistance are not fully understood. To this end, we developed T-DM1-resistant in vitro models to examine the mechanisms of acquired T-DM1 resistance. We demonstrate that decreased HER2 and upregulation of MDR1 contribute to T-DM1 resistance in KPL-4 T-DM1-resistant cells. In contrast, both loss of SLC46A3 and PTEN deficiency play a role in conferring resistance in BT-474M1 T-DM1-resistant cells. Our data suggest that these two cell lines acquire resistance through distinct mechanisms. Furthermore, we show that the KPL-4 T-DM1 resistance can be overcome by treatment with an inhibitor of MDR1, whereas a PI3K inhibitor can rescue PTEN loss-induced resistance in T-DM1-resistant BT-474M1 cells. Our results provide a rationale for developing therapeutic strategies to enhance T-DM1 clinical efficacy by combining T-DM1 and other inhibitors that target signaling transduction or resistance pathways. Mol Cancer Ther; 17(7); 1441-53. ©2018 AACR.

An open-label, multicenter, phase Ib study to evaluate RC48-ADC in patients with HER2-positive metastatic breast cancer.

1028Background: RC48-ADC is a novel HER2-targeting antibody–drug conjugate (ADC) that selectively delivers anticancer agent MMAE into HER2-overexpressing tumor cells. A Phase I study (NCT02881138) ...

Developing anti-HER2 vaccines: Breast cancer experience.

Breast cancer accounts for more than one million new cases annually and is the leading cause of death in women globally. HER2 overexpression induces cellular and humoral immune responses against the HER2 protein and is associated with higher tumor proliferation rates. Trastuzumab-based therapies are effectively and widely used as standard of care in HER2-amplified/overexpressed breast cancer patients; one cited mechanism of action is the induction of passive immunity and antibody-dependent cellular cytotoxicity against malignant breast cancer cells. These findings drove the efforts to generate antigen-specific immunotherapy to trigger the patient's immune system to target HER2-overexpressing tumor cells, which led to the development of various vaccines against the HER2 antigen. This article discusses the various anti-HER2 vaccine formulations and strategies and their potential role in the metastatic and adjuvant settings.

Hersintuzumab: A novel humanized anti-HER2 monoclonal antibody induces potent tumor growth inhibition

Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.

Antibody Response to Human Extracellular HER2 Subdomain Proteins in Mice.

In addition to passive immunotherapy using anti-HER2 monoclonal antibodies, active immunotherapy via HER2 targeting is an interesting approach to inducing specific anti-tumor immune responses. We have recently reported the immunogenicity of HER2 subdomains following DNA immunization and HER2 protein boosting. In the present study, we evaluated the immunogenicity of different HER2 extracellular subdomains for the induction of anti-HER2 antibody response in BALB/c mice. To investigate and characterize antibody responses to human recombinant proteins of HER2 extracellular subdomains in immunized mice. Four subdomains of HER2 extracellular domain were expressed in E.coli; subsequently, purified recombinant proteins were intraperitoneally injected in BALB/c mice with Freund's adjuvant. The anti-HER2 antibody response was detected by ELISA, immunoblotting and flow cytometry. All the four HER2 subdomains along with the full extracellular domain (fECD) were able to induce specific anti-HER2 antibodies. Although anti-HER2 subdomains antibodies could not react with eukaryotic recombinant fECD protein by ELISA, they were able to recognize this protein by immunoblotting under both reduced and non-reduced conditions. Furthermore, only the sera of mice immunized with fECD protein could recognize native HER2 on HER2 overexpressing tumor cells (>99%) by flow cytometry. Moreover, fECD immunized mice sera inhibited the proliferation of tumor cells by XTT assay. The prokaryotic recombinant proteins of HER2 extracellular subdomains are immunogenic, yet the induced specific antibodies do not react with the native HER2 protein due to the paucity of post-translation modifications and /or distortion of the native conformation of isolated HER2 extracellular subdomains which might be potentially effective for induction of cell mediated immune response against HER2.

Charge variant analysis of proposed biosimilar to Trastuzumab

Trastuzumab is a humanized monoclonal antibody (mAb) employed for the treatment of HER2 Positive Breast Cancer. A HER2 overexpressing tumor cell binds to Trastuzumab and attracts immune cells which lead to induction of Antibody Dependent Cellular Cytotoxicity (ADCC) by binding to Fc receptors (CD16a or FcγRIIIa) on an effector cell, such as natural killer (NK) cells.The most commonly expressed receptor on NK cell is CD16a which binds to the Fc portion of Trastuzumab. The ligand-independent HER2–HER3 dimerization is the most potent stimulator of downstream pathways for regulation of cell growth and survival. An attempt has been made in this study to understand the impact of charge heterogeneity on the binding kinetics and potency of the monoclonal antibody. Trastuzumab has a pI range of 8.7–8.9 and is composed of mixture of acidic and basic variants beside the main peak. Ion exchange chromatography was used to isolate the acidic, basic, and main peak fractions from in-house proposed biosimilar to Trastuzumab and their activities were compared to the Innovator Trastuzumab Herclon®. Data from the mass analysis confirmed the potential modifications in both acidic and basic variant. Binding activity studies performed using Surface Plasmon Resonance (SPR) revealed that acidic variants had lesser binding to HER2 in comparison to the basic variants. Both acidic and basic variant showed no significant changes in their binding to soluble CD16a receptors. In vitro assay studies using a breast cancer cell line (BT-474) confirmed the binding potency of acidic variant to be lesser than basic variant, along with reduced anti-proliferative activity for the acidic variant of Trastuzumab. Overall, these data has provided meaningful insights to the impact of antibody charge variants on in vitro potency and CD16 binding affinity of trastuzumab.

Epitope Mapping of Human HER2 Specific Mouse Monoclonal Antibodies Using Recombinant Extracellular Subdomains

Background:Human epidermal growth factor receptor 2 (HER2) is overexpressed in several human malignancies and numerous studies have indicated that it plays important roles in the development and maintenance of the malignant phenotype. Targeting of HER2 molecules with monoclonal antibodies (mAbs) is a promising therapeutic approach. However, anti-HER2 mAbs affect cancer cells differently, depending on the distinct epitopes which are the targets.Methods:Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues.Results:Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one reacted with subdomain I+II, 2 recognized either subdomain III or IV and 2 recognized subdomain III+IV. However, none of our mAbs recognized the subdomain II alone. The mAbs displayed either inhibitory or stimulatory effects on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implied better reactivity of the mAbs recognizing linear epitopes.Conclusions:Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, there seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs.