A Conjugate Based on Anti-HER2 Diaffibody and Auristatin E Targets HER2-Positive Cancer Cells (original) (raw)
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Journal of controlled release : official journal of the Controlled Release Society, 2018
Patients with HER2-positive tumors often suffer resistance to therapy, warranting development of novel treatment modalities. Affibody molecules are small affinity proteins which can be engineered to bind to desired targets. They have in recent years been found to allow precise targeting of cancer specific molecular signatures such as the HER2 receptor. In this study, we have investigated the potential of an affibody molecule targeting HER2, Z, conjugated with the cytotoxic maytansine derivate MC-DM1, for targeted cancer therapy. Z was expressed as a monomer (Z), dimer ((Z)) and dimer with an albumin binding domain (ABD) for half-life extension ((Z)-ABD). All proteins had a unique C-terminal cysteine that could be used for efficient and site-specific conjugation with MC-DM1. The resulting affibody drug conjugates were potent cytotoxic molecules for human cells over-expressing HER2, with sub-nanomolar IC-values similar to trastuzumab emtansine, and did not affect cells with low HER2 e...
Cancers, 2020
The human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in a variety of cancers and therapies targeting HER2 are routinely used in the clinic. Recently, small engineered scaffold proteins, such as affibody molecules, have shown promise as carriers of cytotoxic drugs, and these drug conjugates may become complements or alternatives to the current HER2-targeting therapies. Here, we investigated if a monovalent HER2-binding affibody molecule, ZHER2:2891, fused with a plasma half-life extending albumin binding domain (ABD), may be used as carrier of the cytotoxic maytansine derivate mcDM1. We found that the resulting drug conjugate, ZHER2:2891-ABD-E3-mcDM1, had strong affinity for its cognate molecular targets: HER2 and serum albumin. ZHER2:2891-ABD-E3-mcDM1 displayed potent cytotoxic activity towards cells with high HER2 expression, with IC50 values ranging from 0.6 to 33 nM. In vivo, an unspecific increase in uptake in the liver, imparted by the hydrophobic mcD...
mAbs
Despite advances in medical care, cancer remains a major threat to human health. Antibody-drug conjugates (ADCs) are a promising targeted therapy to overcome adverse side effects to normal tissues. In this field, the current challenge is obtaining homogeneous preparations of conjugates, where a defined number of drugs are conjugated to specific antibody sites. Site-directed cysteine-based conjugation is commonly used to obtain homogeneous ADC, but it is a time-consuming and expensive approach due to the need for extensive antibody engineering to identify the optimal conjugation sites and reductionoxidation protocols are specific for each antibody. There is thus a need for ADC platforms that offer homogeneity and direct applicability to the already approved antibody therapeutics. Here we describe a novel approach to derive homogeneous ADCs with drug-to-antibody ratio of 2 from any human immunoglobulin 1 (IgG 1), using trastuzumab as a model. The method is based on the production of heavy chains (HC) and light chains (LC) in two recombinant HEK293 independent cultures, so the original amino acid sequence is not altered. Isolated LC was effectively conjugated to a single drug-linker (vcMMAE) construct and mixed to isolated HC dimers, in order to obtain a correctly folded ADC. The relevance of the work was validated in terms of ADC homogeneity (HIC-HPLC, MS), purity (SEC-HPLC), isolated antigen recognition (ELISA) and biological activity (HER2-positive breast cancer cells cytotoxicity assays).
Pharmaceutics, 2021
Affibody molecules hold great promise as carriers of cytotoxic drugs for cancer therapy due to their typically high affinity, easy production, and inherent control of the drug molecules’ loading and spatial arrangement. Here, the impact of increasing the drug load from one to three on the properties of an affibody drug conjugate targeting the human epidermal growth factor receptor 2 (HER2) was investigated. The affibody carrier was recombinantly expressed as a fusion to an albumin-binding domain (ABD) for plasma half-life extension. One or three cysteine amino acids were placed at the C-terminus to which cytotoxic mcDM1 molecules were conjugated. The resulting drug conjugates, ZHER2–ABD–mcDM1 and ZHER2–ABD–mcDM13, were characterized in vitro, and their biodistribution in mice carrying HER2-overexpressing SKOV3 xenografts was determined. Increasing the drug load from one to three led to a decrease in affinity for HER2, but a significantly more potent cytotoxic effect on SKOV3 cells w...
Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a novel HER2 ADC, we selected an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. We modified the PBD dimer to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrated in vivo efficacy in models of HER2-positive and HER2-low cancers and was well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study in patients with HER2-positive metastatic breast cancer showed early signs of anti-tumor activity with limited toxicity. However, delayed dermal, ocular and pulmonary toxicities developed. The delayed onset, as well as non-resolva...
Separations
Antibody-drug conjugates (ADCs) are promising state-of-the-art biopharmaceutical drugs for selective drug-delivery applications and the treatment of diseases such as cancer. The idea behind the ADC technology is remarkable as it combines the highly selective targeting capacity of monoclonal antibodies with the cancer-killing ability of potent cytotoxic agents. The continuous development of improved ADCs requires systematic studies on the nature and effects of warhead modification. Recently, we focused on the hydrophilic modification of monomethyl auristatin E (MMAE), the most widely used cytotoxic agent in current clinical trial ADCs. Herein, we report on the use of micellar electrokinetic chromatography (MEKC) for studying the hydrophobic character of modified MMAE derivatives. Our data reveal a connection between the hydrophobicity of the modified warheads as free molecules and their cytotoxic activity. In addition, MMAE-trastuzumab ADCs were constructed and evaluated in prelimina...
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2014
Affibody molecules are small (7 kDa) nonimmunoglobulin scaffold proteins with favorable tumor-targeting properties. Studies concerning the influence of chelators on biodistribution of (99m)Tc-labeled Affibody molecules demonstrated that the variant with a C-terminal glycyl-glycyl-glycyl-cysteine peptide-based chelator (designated ZHER2:V2) has the best biodistribution profile in vivo and the lowest renal retention of radioactivity. The aim of this study was to evaluate (188)Re-ZHER2:V2 as a potential candidate for radionuclide therapy of human epidermal growth factor receptor type 2 (HER2)-expressing tumors. ZHER2:V2 was labeled with (188)Re using a gluconate-containing kit. Targeting of HER2-overexpressing SKOV-3 ovarian carcinoma xenografts in nude mice was studied for a dosimetry assessment. Binding of (188)Re-ZHER2:V2 to living SKOV-3 cells was demonstrated to be specific, with an affinity of 6.4 ± 0.4 pM. The biodistribution study showed a rapid blood clearance (1.4 ± 0.1 perce...
Pharmaceutics, 2021
Human epidermal growth factor receptor 2 (HER2) is a clinically validated target for breast cancer therapy. Previously, a drug-fused HER2-targeting affinity protein construct successfully extended the survival of mice bearing HER2-expressing xenografts. The aim of this study was to evaluate the influence of the number and positioning of the protein domains in the drug conjugate. Seven HER2-targeting affibody-based constructs, including one or two affibody molecules (Z) with or without an albumin-binding domain (ABD), namely Z, Z-ABD, ABD-Z, Z-Z, Z-Z-ABD, Z-ABD-Z, and ABD-Z-Z, were evaluated on their effects on cell growth, in vivo targeting, and biodistribution. The biodistribution study demonstrated that the monomeric constructs had longer blood retention and lower hepatic uptake than the dimeric ones. A dimeric construct, specifically ABD-Z-Z, could stimulate the proliferation of HER2 expressing SKOV-3 cells in vitro and the growth of tumors in vivo, whereas the monomeric construc...
Journal of Nuclear Medicine, 2009
The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced 99m Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. Methods: A C-terminal cysteine was introduced into the Affibody molecule Z HER2:342 to enable sitespecific labeling with 99m Tc. Two recombinant variants, His 6 -Z HER2:342 -Cys (dissociation constant [K D ], 29 pM) and Z HER2:2395 -Cys, lacking a His tag (K D , 27 pM), were labeled with 99m Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and g-camera imaging studies were performed in mice bearing HER2-expressing xenografts. Results: 99m Tc-His 6 -Z HER2:342 -Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. 99m Tc-Z HER2:2395 -Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 6 2.5 (mean 6 SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 6 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 6 24 and 121 6 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical g-camera 1 h after injection of 99m Tc-Z HER2:2395 -Cys. Conclusion: The Affibody molecule 99m Tc-Z HER2:2395 -Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.
European Journal of Nuclear Medicine and Molecular Imaging, 2010
Purpose Affibody molecules are a novel class of tumourtargeting proteins, which combine small size (7 kDa) and picomolar affinities. The Affibody molecule Z HER2:342 has been suggested for imaging of HER2 expression in order to select patients for trastuzumab therapy. When optimizing chelators for 99m Tc-labelling, we have found that synthetic Z HER2:342 conjugated with mercaptoacetyl-glycyl-glycylglycyl (maGGG) and mercaptoacetyl-glycyl-seryl-glycyl (maGSG) chelators provides relatively low renal uptake of radioactivity and could be suitable for therapy. Methods maGGG-Z HER2:342 and maGSG-Z HER2:342 were labelled with 186 Re and their biodistribution was studied in normal mice. Dosimetric evaluation and tumour targeting to HER2-overexpressed xenografts (SKOV-3) by 186 Re-maGSG-Z HER2:342 were studied.