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Tumor targeted delivery of cytotoxic payloads using affibody molecules and ABD-derived affinity proteins
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancer treatment cost billions of dollars every year, but the mortality rate is still high. An ideal treatment is the so-called “magic bullets” that recognize and kill tumor cells while leaving normal cells untouched. In recent years, some nonimmunoglobulin alternative scaffold affinity proteins, such as affibody molecules and ADAPTs, have emerged and been used to specifically recognize different tumor antigens. In this thesis, I studied the properties and anti-tumor activities of affibody and ADAPT fusion toxins and affibody drug conjugates. In the first two papers, I studied a panel of recombinant affitoxins (affibody toxin fusion proteins) consisting of an anti-HER2 affibody molecule (ZHER2), an albumin binding domain (ABD) and a truncated version of Pseudomonas Exotoxin A(PE38X8). The affitoxins demonstrated specific anti-tumor activity on HER2-overexpressing tumor cells in vitro. A biodistribution experiment showed that addition of an ABD increased the blood retention by 28-fold and a (HE)3 N-terminal purification tag decreased hepatic uptake of the affitoxin compared with a His6 tag. In paper III, I studied immunotoxins consisting of an anti-HER2 ABD-derived affinity protein (ADAPT), an ABD and a minimized and deimmunized version of Pseudomonas exotoxin A (PE25). These immunotoxins demonstrated potent and specific cytotoxicity toward HER2 overexpressing tumor cells in vitro similar to affitoxins. In paper IV, I produced a panel of affibody drug conjugates consisting of ZHER2, ABD and malemidocaproylmertansine (mc-DM1). The conjugates had selective toxic activity on HER2-overexpressing tumor cells in vitro comparable with the approved drug trastuzumab emtansine. The conjugate, ZHER2-ZHER2-ABD-mc-DM1 was found to prolong the life span of tumor bearing mice and delayed the growth ofxenografted SKOV-3 tumors. In conclusion, affibody molecules and ADAPTs are promising alternatives to antibodies for targeted tumor therapy.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2018. , p. 78
Series
TRITA-CBH-FOU ; 2018:26
Keywords [en]
Targeted tumor therapy, immunotoxins, ADCs, affibody molecule, ADAPT, pseudomonas exotoxin A, maytansinoid
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-228015ISBN: 978-91-7729-827-4 (print)OAI: oai:DiVA.org:kth-228015DiVA, id: diva2:1206290
Public defence
2018-06-14, Oskar Kleins Auditorium, Roslagstullsbacken 21, Albanova University Center,, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20180517

Available from: 2018-05-17 Created: 2018-05-16 Last updated: 2018-05-17Bibliographically approved
List of papers
1. Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin Z(HER2:2891)-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain
Open this publication in new window or tab >>Target-specific cytotoxic effects on HER2-expressing cells by the tripartite fusion toxin Z(HER2:2891)-ABD-PE38X8, including a targeting affibody molecule and a half-life extension domain
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2015 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 47, no 2, p. 601-609Article in journal (Refereed) Published
Abstract [en]

Development of cancer treatment regimens including immunotoxins is partly hampered by their immunogenicity. Recently, deimmunized versions of toxins have been described, potentially being better suited for translation to the clinic. In this study, a recombinant tripartite fusion toxin consisting of a deimmunized version of exotoxin A from Pseudomonas aeruginosa (PE38) genetically fused to an affibody molecule specifically interacting with the human epidermal growth factor receptor 2 (HER2), and also an albumin binding domain (ABD) for half-life extension, has been produced and characterized in terms of functionality of the three moieties. Biosensor based assays showed that the fusion toxin was able to interact with human and mouse serum albumin, but not with bovine serum albumin and that it interacted with HER2 (K-D=5 nM). Interestingly, a complex of the fusion toxin and human serum albumin also interacted with HER2 but with a somewhat weaker affinity (K-D=12 nM). The IC50-values of the fusion toxin ranged from 6 to 300 pM on SKOV-3, SKBR-3 and A549 cells and was lower for cells with higher surface densities of HER2. The fusion toxin was found specific for HER2 as shown by blocking available HER2 receptors with free affibody molecule before subjecting the cells to the toxin. Analysis of contact time showed that 10 min was sufficient to kill 50% of the cells. In conclusion, all three regions of the fusion toxin were found to be functional.

Keywords
affibody molecule, immunotoxin, albumin binding domain, PE38, HER2
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-171879 (URN)10.3892/ijo.2015.3027 (DOI)000357965700022 ()2-s2.0-84932646862 (Scopus ID)
Funder
Swedish Cancer Society
Note

QC 20150817

Available from: 2015-08-17 Created: 2015-08-10 Last updated: 2018-05-16Bibliographically approved
2. Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin
Open this publication in new window or tab >>Influence of molecular design on biodistribution and targeting properties of an Affibody-fused HER2-recognising anticancer toxin
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2016 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 49, no 3, p. 1185-1194Article in journal (Refereed) Published
Abstract [en]

Targeted delivery of toxins is a promising way to treat disseminated cancer. The use of monoclonal antibodies as targeting moiety has provided proof-of-principle for this approach. However, extravasation and tissue penetration rates of antibody-based immunotoxins are limited due to antibody bulkiness. The use of a novel class of targeting probes, Affibody molecules, provides smaller toxin-conjugated constructs, which may improve targeting. Earlier, we have demonstrated that affitoxins containing a HER2-targeting Affibody moiety and a deimmunized and truncated exotoxin A from Pseudomonas aeruginosa, PE38X8, provide highly selective toxicity to HER2-expressing cancer cells. To evaluate the influence of molecular design on targeting and biodistribution properties, a series of novel affitoxins were labelled with the residualizing radionuclide In-111. In this study, we have shown that the novel conjugates are more rapidly internalized compared with the parental affitoxin. The use of a (HE)(3) purification tag instead of a hexahistidine tag enabled significant (p<0.05) reduction of the hepatic uptake of the affitoxin in a murine model. Fusion of the affitoxin with an albumin-binding domain (ABD) caused appreciable extension of the residence time in circulation and several-fold reduction of the renal uptake. The best variant, In-111-(HE)(3)-Z(HER2)-ABD-PE38X8, demonstrated receptor-specific accumulation in HER2-expressing SKOV-3 xenografts. In conclusion, a careful molecular design of scaffold protein based anticancer targeted toxins can appreciably improve their biodistribution and targeting properties.

Keywords
Affibody molecule, immunotoxin, albumin binding domain, PE38, HER2, biodistribution, In-111
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-192970 (URN)10.3892/ijo.2016.3614 (DOI)000382447300034 ()27573289 (PubMedID)2-s2.0-84978531659 (Scopus ID)
Funder
Swedish Research CouncilSwedish Cancer Society
Note

QC 20160930

Available from: 2016-09-30 Created: 2016-09-23 Last updated: 2018-05-16Bibliographically approved
3. Potent and specific fusion toxins consisting of a HER2-binding ABD-derived affinity protein (ADAPT), fused to truncated versions of Pseudomonas exotoxin A
Open this publication in new window or tab >>Potent and specific fusion toxins consisting of a HER2-binding ABD-derived affinity protein (ADAPT), fused to truncated versions of Pseudomonas exotoxin A
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(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-228014 (URN)
Note

QC 20180517

Available from: 2018-05-16 Created: 2018-05-16 Last updated: 2018-05-17Bibliographically approved
4. Affibody-derived Drug Conjugates: Potent Cytotoxic Drugs ForTreatment Of HER2 Over-Expressing Tumors
Open this publication in new window or tab >>Affibody-derived Drug Conjugates: Potent Cytotoxic Drugs ForTreatment Of HER2 Over-Expressing Tumors
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences Engineering and Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-228013 (URN)
Note

QC 20180517

Available from: 2018-05-16 Created: 2018-05-16 Last updated: 2018-05-17Bibliographically approved

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