| 1. |
- Altai, Mohamed, et al.
(författare)
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Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs
- 2018
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Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Dahlsson Leitao, Charles
(författare)
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Affibody-mediated targeting of HER-family receptors for cancer imaging and therapy
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are remarkable molecules with diverse and specialized functions playing essential roles in most biological processes. One such function is protecting us from diseases by the action of antibodies in our immune system that can recognize and mediate the destruction of invading pathogens by binding to foreign epitopes found on non-self proteins. The concept of utilizing specific protein-protein interactions to achieve a therapeutic effect has for several decades been a cornerstone for the development of cancer-directed treatments. While antibodies have formed a basis for the development of such drugs, other protein alternatives may be engineered to complement current antibody-based treatments, and may even prove to possess superior features. This thesis focuses on the engineering of affibody molecules, a small alternative scaffold protein, for design and development of novel cancer-targeting therapeutic and diagnostic drugs. There are many different strategies that have been investigated for inhibiting cancer progression and tumour growth with perhaps one of the most straightforward involving disruption of dysregulated growth-promoting signalling pathways. Members of the human epidermal growth factor receptor (HER) family is prominently expressed in various cancer types and have been shown to be intricately involved in tumorigenesis. One of the members (HER3) often becomes upregulated in cancer and have been shown to mediate acquired resistance to targeted therapies by the mechanism of ligand-induced activation. We have designed five novel affibody-based HER3-targeting molecules able to prevent ligand-binding and consequently activation of HER3. We investigated the targeting properties and biodistribution profiles of these molecules in vivo and subsequently evaluated the anti-tumour efficacy for the most promising variants in direct comparison to a HER3-targeting antibody with a similar inhibitory mechanism. We observed a large influence of design on both the biodistribution properties and the in vivo efficacy of different affibody molecules. Moreover, we demonstrated that two of the affibody-formats were equally effective as the antibody in inhibiting tumour growth and prolonging survival of mice bearing HER3-positive xenografts. The effectiveness of cancer treatments depends on efficient diagnostic approaches that can reliably stratify patients based on these targetable biomarkers, which is possible using radionuclide molecular imaging. We have performed a direct comparison of the diagnostic potential for visualizing HER3-expressing tumours of affibody- and antibody-based imaging probes. We concluded that affibody molecules provide superior imaging quality with higher diagnostic potential and enable early visualization of HER3-expression in tumours. Another member of the HER family that is of interest for cancer therapy is HER1 (or EGFR) but due to substantial expression in healthy tissues, targeted therapies may lead to severe side-effects. One possible solution to this is taking advantage of the distinct milieu of the tumour microenvironment to design EGFR-targeting drugs that become conditionally activated at the tumour site, but not in normal tissues, with the aim of drastically reducing systemic toxicity. We have generated an affibody molecule with anti-idiotypic binding specificity for a previously generated EGFR-binding affibody molecule, which we used to construct an affibody-based prodrug. We were able to show that, in a proof-of-concept format, this anti-idiotypic masking domain was able to block the binding to EGFR until removed by protease-mediated cleavage. We subsequently developed and characterized a more refined version of this prodrug, which we call a pro-affibody, and could show that activation by cancer-associated proteases confers binding to EGFR-expressing cancer cells and enables conditional cytotoxic payload delivery in vitro. The pro-affibody was further evaluated in vivo using tumour-bearing mice to investigate the feasibility for masked uptake in healthy tissues while retaining binding-activity in tumours. We observed a substantial reduction in EGFR-specific liver uptake compared to a control construct without a masking domain, and a strong indication of protease-mediated EGFR-binding in tumours. In conclusion, the experimental work presented in this thesis provides a rationale for designing effective affibody-based cancer therapeutics and diagnostics with different targeting strategies and demonstrates the potential of such drugs from preclinical in vivo data.
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| 3. |
- Dahlsson Leitao, Charles, et al.
(författare)
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Molecular Design of HER3-Targeting Affibody Molecules : Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled Tracers
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 20:5
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Tidskriftsartikel (refereegranskat)abstract
- Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)3-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)3-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)3-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)3-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)3-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.
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| 4. |
- Garousi, Javad, et al.
(författare)
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Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts
- 2019
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE3-ZCAIX:2, was labeled with In-111 and characterized in vitro. Tumor-targeting properties of [In-111]In-DOTA-HE3-ZCAIX:2 were compared head-to-head with properties of the parental variant, [(99)mTc]Tc(CO)(3)-HE3-ZCAIX:2, and the most promising antibody fragment-based tracer, [In-111]In-DTPA-G250(Fab')(2), in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the (99)mTc-labeled parental variant, [In-111]In-DOTA-HE3-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [In-111]In-DOTA-HE3-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [In-111]In-G250(Fab']2. In conclusion, [In-111]In-DOTA-HE3-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.
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| 5. |
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| 6. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Bacterial Cell Display for Selection of Affibody Molecules
- 2023
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Ingår i: Genotype Phenotype Coupling. - : Springer Nature. ; , s. 99-112
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 7. |
- Leitao, Charles Dahlsson, et al.
(författare)
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Conditionally activated affibody-based prodrug targeting EGFR demonstrates improved tumor selectivity
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 8. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Conditionally activated affibody-based prodrug targeting EGFR demonstrates improved tumour selectivity
- 2023
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 357, s. 185-195
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting
- 2023
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 655, s. 75-81
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Leitao, Charles Dahlsson, et al.
(författare)
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EGFR-targeting affibody-based prodrug activated by cancer-associated proteases
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 11. |
- Leitao, Charles Dahlsson, et al.
(författare)
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Evaluating the Therapeutic Efficacy of Mono- and Bivalent Affibody-Based Fusion Proteins Targeting HER3 in a Pancreatic Cancer Xenograft Model
- 2020
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Ingår i: Pharmaceutics. - : MDPI. - 1999-4923. ; 12:6
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Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor 3 (HER3) has been increasingly scrutinized as a potential drug target since the elucidation of its role in mediating tumor growth and acquired therapy resistance. Affibody molecules are so-called scaffold proteins with favorable biophysical properties, such as a small size for improved tissue penetration and extravasation, thermal and chemical stability, and a high tolerance to modifications. Additionally, affibody molecules are efficiently produced in prokaryotic hosts or by chemical peptide synthesis. We have previously evaluated the biodistribution profiles of five mono- and bivalent anti-HER3 affibody molecules (designated as 3) fused to an albumin-binding domain (designated as A), 3A, 33A, 3A3, A33, and A3, that inhibit ligand-dependent phosphorylation. In the present study, we examined the therapeutic efficacy of the three most promising variants, 3A, 33A, and 3A3, in a direct comparison with the HER3-targeting monoclonal antibody seribantumab (MM-121) in a preclinical BxPC-3 pancreatic cancer model. Xenografted mice were treated with either an affibody construct or MM-121 and the tumor growth was compared to a vehicle group. Receptor occupancy was estimated by positron emission tomography/computed tomography (PET/CT) imaging using a HER3-targeting affibody imaging agent [Ga-68]Ga-(HE)(3)-Z(08698)-NODAGA. The affibody molecules could inhibit ligand-dependent phosphorylation and cell proliferation in vitro and demonstrated tumor growth inhibition in vivo comparable to that of MM-121. PET/CT imaging showed full receptor occupancy for all tested drug candidates. Treatment with 3A and 3A3 affibody constructs was more efficient than with 33A and similar to the anti-HER3 antibody seribantumab, showing that the molecular design of affibody-based therapeutics targeting HER3 in terms of the relative position of functional domains and valency has an impact on therapeutic effect.
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| 12. |
- Mestre Borras, Anna, 1994-, et al.
(författare)
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Generation of an anti-idiotypic affibody-based masking domain for conditional activation of EGFR-targeting
- 2023
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 73, s. 9-18
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 13. |
- Mestre Borras, Anna, et al.
(författare)
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Generation of an anti-idiotypic masking domain for the construction of an affibody-based EGFR-targeting prodrug
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 14. |
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| 15. |
- Orlova, Anna, 1960-, et al.
(författare)
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Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab
- 2018
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Ingår i: Molecular Pharmaceutics. - : AMER CHEMICAL SOC. - 1543-8384 .- 1543-8392. ; 15:8, s. 3394-3403
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 16. |
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| 17. |
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| 18. |
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| 19. |
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| 20. |
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| 21. |
- Rinne, Sara S., et al.
(författare)
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Benefit of Later-Time-Point PET Imaging of HER3 Expression Using Optimized Radiocobalt-Labeled Affibody Molecules
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:6
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 22. |
- Rinne, Sara S., et al.
(författare)
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HER3 PET Imaging : 68Ga-Labeled Affibody Molecules Provide Superior HER3 Contrast to 89Zr-Labeled Antibody and Antibody-Fragment-Based Tracers
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:19, s. 4791-4791
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Tidskriftsartikel (refereegranskat)abstract
- HER3 (human epidermal growth factor receptor type 3) is a challenging target for diag- nostic radionuclide molecular imaging due to the relatively modest overexpression in tumors and substantial expression in healthy organs. In this study, we compared four HER3-targeting PET tracers based on different types of targeting molecules in a preclinical model: the 89Zr-labeled therapeutic antibody seribantumab, a seribantumab-derived F(ab)2-fragment labeled with 89Zr and 68Ga, and the 68Ga-labeled affibody molecule [68Ga]Ga-ZHER3. The novel conjugates were radiolabeled and characterized in vitro using HER3-expressing BxPC-3 and DU145 human cancer cells. Biodistribu- tion was studied using Balb/c nu/nu mice bearing BxPC-3 xenografts. HER3-negative RAMOS xenografts were used to demonstrate binding specificity in vivo. Autoradiography was conducted on the excised tumors. nanoPET/CT imaging was performed. New conjugates specifically bound to HER3 in vitro and in vivo. [68Ga]Ga-DFO-seribantumab-F(ab’)2 was considered unsuitable for imaging due to the low stability and high uptake in normal organs. The highest tumor-to-non-tumor contrast with [89Zr]Zr-DFO-seribantumab and [89Zr]Zr-DFO-seribantumab-F(ab’)2 was achieved at 96 h and 48 h pi, respectively. Despite lower tumor uptake, [68Ga]Ga-ZHER3 provided the best imaging contrast due to the fastest clearance from blood and normal organs. The results of our study suggest that affibody-based tracers are more suitable for PET imaging of HER3 expression than antibody- and antibody-fragment-based tracers.
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| 23. |
- Rinne, Sara S., et al.
(författare)
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Increase in negative charge of Ga-68/chelator complex reduces unspecific hepatic uptake but does not improve imaging properties of HER3-targeting affibody molecules
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 24. |
- Rinne, Sara S., et al.
(författare)
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Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:4
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Tidskriftsartikel (refereegranskat)abstract
- Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)(3)-Z(HER3:08698)-DOTAGA affibody molecule with non-residualizing [I-125]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA was compared side-by-side with [In-111]In-(HE)(3)-Z(HER3:08698)-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24h pi showed faster clearance of the [I-125]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 +/- 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [I-125]I-PIB label. In conclusion, the use of non-residualizing [I-125]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.
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