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Conditional activation of engineered affinity proteins by proteolytic processing is an interesting approach for a wide range of applications. We have generated an anti-idiotypic masking domain with specificity for the binding surface of an EGFR-targeting affibody molecule using an in-house developed staphylococcal display method. The masking domain could specifically abrogate EGFR-binding on cancer cells when fused to the EGFR-targeting affibody molecule via a linker comprising a protease cleavage site. EGFR-binding was restored by proteolytic cleavage of the linker region resulting in release of the masking domain. A saturation mutagenesis study provided detailed information on the interaction between the EGFR-targeting affibody molecule and the masking domain. Introducing an anti-idiotypic masking affibody domain is a viable approach for blocking EGFR-binding and al-lows for conditional activation by proteolytic processing. The results warrant further studies evaluating the therapeutic and diagnostic applicability both in vitro and in vivo., QC 20230307

Safety and efficacy of cancer-targeting treatments can be improved by conditional activation enabled by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumourigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease -dependent activation has the potential to increase tumour-selective targeting while decreasing exposure to healthy tissues, thus improving the safety profile for patients. Higher selectivity could also allow for adminis-tration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). We could show that binding to endogenous EGFR on cancer cells in vitro was restored following proteolytic removal of ZB05. In this study we evaluate a novel affibody-based pro -drug design, which incorporates a protease substrate sequence recognized by cancer-associated proteases and demonstrate the potential of this approach for selective tumour-targeting and shielded uptake in healthy tissues in vivo using tumour-bearing mice. This may widen the therapeutic index of cytotoxic EGFR-targeted thera-peutics by decreasing side effects, improving selectivity of drug delivery, and enabling the use of more potent cytotoxic drugs.

This review describes the principles for generation of affibody molecules using bacterial display on the Gram-negative Escherichia coli and the Gram-positive Staphylococcus carnosus, respectively. Affibody molecules are small and robust alternative scaffold proteins that have been explored for therapeutic, diagnostic, and biotechnological applications. They typically exhibit high-stability, affinity, and specificity with high modularity of functional domains. Due to the small size of the scaffold, affibody molecules are rapidly excreted through renal filtration and can efficiently extravasate from blood and penetrate tissues. Preclinical and clinical studies have demonstrated that affibody molecules are promising and safe complements to antibodies for in vivo diagnostic imaging and therapy. Sorting of affibody libraries displayed on bacteria using fluorescence-activated cell sorting is an effective and straightforward methodology and has been used successfully to generate novel affibody molecules with high affinity for a diverse range of molecular targets., Part of book ISBN 978-1-0716-3278-9 978-1-0716-3279-6 978-1-0716-3281-9QC 20230816

Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived pro-teins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naive affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recog-nition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide tech-nology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity., QC 20230523

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we studied a novel drug conjugate, Z(HER3)-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin-binding domain for in vivo half-life extension. Z(HER3)-ABD-mcDM1 showed a strong affinity to the extracellular domain of HER3 (K-D 6 nM), and an even stronger affinity (K-D 0.2 nM) to the HER3-overexpressing pancreatic carcinoma cell line, BxPC-3. The drug conjugate showed a potent cytotoxic effect on BxPC-3 cells with an IC50 value of 7 nM. Evaluation of a radiolabeled version, [Tc-99m]Tc-Z(HER3)-ABD-mcDM1, showed a relatively high rate of internalization, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could target BxPC-3 (pancreatic carcinoma) and DU145 (prostate carcinoma) xenografts in mice, with an uptake peaking at 6.3 +/- 0.4% IA/g at 6 h post-injection for the BxPC-3 xenografts. The general biodistribution showed uptake in the liver, lung, salivary gland, stomach, and small intestine, organs known to express murine ErbB3 naturally. The results from the study show that Z(HER3)-ABD-mcDM1 is a highly potent and selective drug conjugate with the ability to specifically target HER3 overexpressing cells. Further pre-clinical and clinical development is discussed., Sara S. Rinne and Wen Yin share first authorship

QC 20221031

QC 20220119

HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt-chelator complex on the biodistribution of anti-HER3 affibody molecule (HE)(3)-Z(HER3) and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE)(3)-Z(HER3)-X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [Co-57]Co (surrogate for Co-55). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [Co-57]Co-(HE)(3)-Z(HER3)-DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA 3 h pi, [Co-57]Co-(HE)(3)-Z(HER3)-DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [Co-57]Co-chelator complex influenced the uptake in tumors and normal tissue. [Co-57]Co-(HE)(3)-Z(HER3)-DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [Co-57]Co-(HE)(3)-Z(HER3)-DOTA improved imaging contrast compared to early-time-point imaging with [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA.

QC 20201215

QC 20201023

Upregulation of the human epidermal growth factor receptor type 3 (HER3) is a common mechanism to bypass HER-targeted cancer therapy. Affibody-based molecular imaging has the potential for detecting and monitoring HER3 expression during treatment. In this study, we compared the imaging properties of newly generated Ga-68-labeled anti-HER3 affibody molecules (HE)(3)-Z(HER3)-DOTA and (HE)(3)-Z(HER3)-DOTAGA with previously reported [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. We hypothesized that increasing the negative charge of the gallium-68/chelator complex would reduce hepatic uptake, which could lead to improved contrast of anti-HER3 affibody-based PET-imaging of HER3 expression. (HE)(3)-Z(HER3)-X (X = DOTA, DOTAGA) were produced and labeled with gallium-68. Binding of the new conjugates was specific in HER3 expressing BxPC-3 and DU145 human cancer cells. Biodistribution and in vivo specificity was studied in BxPC-3 xenograft bearing Balb/c nu/nu mice 3 h pi. DOTA- and DOTAGA-containing conjugates had significantly higher concentration in blood than [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA. Presence of the negatively charged Ga-68-DOTAGA complex reduced the unspecific hepatic uptake, but did not improve overall biodistribution of the conjugate. [Ga-68]Ga-(HE)(3)-Z(HER3)-DOTAGA and [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA had similar tumor-to-liver ratios, but [Ga-68]Ga-(HE)(3)-Z(HER3)-NODAGA had the highest tumor uptake and tumor-to-blood ratio among the tested conjugates. In conclusion, [Ga-68] Ga-(HE)(3)-Z(HER3)-NODAGA remains the favorable variant for PET imaging of HER3 expression.

QC 20191202

QC 20191203

QC 20190814

Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD 035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified., The first two authors contributed equally to this work.

Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

QC 20181217

Cancer-directed therapies targeting membrane-bound receptors are often limited by systemic toxicity. The epidermal growth factor receptor (EGFR) is a receptor commonly overexpressed in cancer but its abundance in healthy tissues, particularly in skin, often results treatment-induced toxicities. Efficacy and safety of EGFR-targeting drugs could be dramatically improved by increasing the selectivity for tumours. The tumour microenvironment differs significantly from normal tissues which can be exploited to achieve local conditional drug activation and selective tumour-targeting. Proteases are often upregulated in cancer to promote tumour growth, invasion, and metastasis. We have developed an affibody-based prodrug with conditional targeting of EGFR conferred by an anti-idiotypic affibody masking domain (ZB05). Binding to EGFR is restored following removal of ZB05 by cancer-associated proteases. We show that cleavage by proteases is necessary for EGFR-mediated targeting of cancer cells and delivery of cytotoxic drugs in vitro. These results warrant further evaluation of the potential pharmacokinetic advantages of an EGFR-targeting affibody prodrug in vivo compared to non-masked EGFR-targeting variants., QC 20220315

Safety and efficacy of cancer-targeting treatments can be improved by conditional activation conferred by the distinct milieu of the tumour microenvironment. Proteases are intricately involved in tumorigenesis and commonly dysregulated with elevated expression and activity. Design of prodrug molecules with protease-dependent activation has the potential to increase tumor-selective targeting, while decreasing the exposure to healthy tissues, thus improving safety, allowing for administration of higher doses or use of more aggressive treatment options, leading to higher therapeutic efficacy. We have previously performed in vitro characterizations of an affibody-based prodrug approach for protease-mediated targeting of EGFR. In this study we demonstrate the potential for selective tumor-targeting and shielded uptake in healthy tissues in vivo using tumor-bearing mice for an EGFR-targeting affibody prodrug., QC 20220315

Systemic toxicity is a significant challenge faced when developing therapeutic agents for cancer. High endogenous expression of cancer biomarkers limits the efficacy and safety of targeted therapies. The epidermal growth factor receptor (EGFR) is commonly overexpressed in certain cancers making it an interesting target for directed cancer therapy, but the potential is limited by the presence in healthy tissues. Conditional activation of targeted drugs can be achieved by utilizing the unique microenvironment of tumours. Proteases are important for tumorigenesis and are often upregulated in cancer. The design of a therapeutic agent that is selectively activated by tumor-associated proteases can improve the efficacy and safety by lowering systemic exposure. We have generated an anti-idiotypic masking domain with specificity for the binding surface of an EGFR-targeting affibody molecule using staphylococcal display. We found that the masking domain abrogates EGFR-binding in a proof-of-concept prodrug construct and that protease-mediated removal of the masking domain restores binding to EGFR. These results suggest that introducing an anti-idiotypic masking affibody domain is a viable approach for blocking EGFR-binding and warrants further studies evaluating the therapeutic applicability both in vitro and in vivo., QC 20220316

Increasing evidence suggests that therapy targeting the human epidermal growth factor receptor 3 (HER3) could be a viable route for targeted cancer therapy. Here, we have studied a novel drug conjugate, ZHER3-ABD-mcDM1, consisting of a HER3-targeting affibody molecule, coupled to the cytotoxic tubulin polymerization inhibitor DM1, and an albumin- binding domain for in vivo half-life extension. ZHER3-ABD-mcDM1 showed strong affinity to the extracellular domain of HER3 (KD 6 nM), and an even stronger affinity (KD 0.2 nM) to the HER3-overexpressing pancreatic cancer cell line, BxPC3. The drug conjugate showed a potent cytotoxic effect on BxPC3 cells with an IC50 value of 7 nM. Further in vitro evaluation of a radiolabeled version [99mTc]Tc-ZHER3-ABD-mcDM1, showed that it had a relatively high rate of internalization into HER3-expressing BxPC-3 as well as DU145 (prostate cancer) cells, with a 27% internalized fraction after 8 h. Further in vivo evaluation showed that it could specifically target BxPC-3 and DU145-derived xenografts in mice, with an uptake peaking at 6.3 ± 0.4 %ID/g at 6 h post-injection for the BxPC-3-derived xenografts. The general biodistribution showed uptake in liver, lung, salivary gland, stomach, and small intestine, organs known to express HER3 naturally. The results from the study show that ZHER3-ABD- mcDM1 is a highly potent and specific drug conjugate, that may be further developed towards HER3-targeted cancer therapy., QC 20220225

The outcome of clinical trials evaluating drugs targeting the human epidermal growth factor receptor 3 (HER3) has been poor, with primary concerns related to lack of efficacy. The development of novel modalities that have the potential to improve the efficacy of HER3-targeting drugs is therefore warranted. Here, we have investigated the in vitro and in vivo properties of affibody-based drug conjugates targeting HER3. The HER3- targeting affibody molecule ZHER3 was fused in a mono- and bivalent format to an albumin binding domain (ABD) for in vivo half-life extension and was coupled to the cytotoxic drug DM1 via a non-cleavable maleimidocaproyl (mc) linker. The bivalent drug conjugate, ZHER3-ABD-ZHER3-mcDM1, demonstrated 10-fold higher toxicity to the HER3-expressing human cell line BxPC3 compared to the monovalent drug conjugate ZHER3-ABD-mcDM1. In vivo, a moderate uptake was recorded for [99mTc]Tc-labeled ZHER3-ABD-ZHER3-mcDM1 in BxPC3-derived tumors (3.5 ± 0.3%IA/g) at 24 h after injection, and clearance was predominately renal-mediated. Treatment of mice with implanted BxPC3 pancreatic human tumors showed that a combination of ZHER3-ABD-ZHER3-mcDM1 and its non-toxic analog ZHER3-ABD-ZHER3 was superior to a treatment scheme including only ZHER3-ABD- ZHER3, providing tumor growth inhibition and longer median survival of the mice (90 d) in comparison to the monotherapy (68 d) and vehicle control (49 d). In conclusion, the bivalent drug conjugate ZHER3-ABD-ZHER3-mcDM1 produced the strongest cytotoxic effect on HER3-expressing BxPC3 cells compared to previously investigated constructs, including non-toxic ZHER3-ABD-ZHER3 and the monovalent drug conjugate ZHER3-ABD- mcDM1, without apparent toxicity in vivo., QC 20230824