The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F

Woitok, Mira; Klose, Diana; Niesen, Judith; Richter, Wolfgang; Abbas, Muhammad Nadeem; Stein, Christoph; Fendel, Rolf; Bialon, Magdalena; Püttmann, Christiane; Fischer, Rainer; Barth, Stefan; Kolberg, Katharina

doi:10.1016/j.canlet.2016.08.003

Cited by 39 publications

(73 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The AURIF-based ADCs were generated as previously described 31. Briefly, the purified scFv-SNAP proteins were incubated with a twofold molar excess of BG-modified AURIF (Tube Pharmaceuticals GmbH, Vienna, Austria) for 2 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

Woitok¹,

Klose²,

Fiore³

et al. 2017

OTT

Self Cite

View full text Add to dashboard Cite

Antibody–drug conjugates (ADCs) can deliver toxins to specific targets such as tumor cells. They have shown promise in preclinical/clinical development but feature stoichiometrically undefined chemical linkages, and those based on full-size antibodies achieve only limited tumor penetration. SNAP-tag technology can overcome these challenges by conjugating benzylguanine-modified toxins to single-chain fragment variables (scFvs) with 1:1 stoichiometry while preserving antigen binding. Two (human and mouse) scFv-SNAP fusion proteins recognizing the epidermal growth factor receptor (EGFR) were expressed in HEK 293T cells. The purified fusion proteins were conjugated to auristatin F (AURIF). Binding activity was confirmed by flow cytometry/immunohistochemistry, and cytotoxic activity was confirmed by cell viability/apoptosis and cell cycle arrest assays, and a novel microtubule dynamics disassembly assay was performed. Both ADCs bound specifically to their target cells in vitro and ex vivo, indicating that the binding activity of the scFv-SNAP fusions was unaffected by conjugation to AURIF. Cytotoxic assays confirmed that the ADCs induced apoptosis and cell cycle arrest at nanomolar concentrations and microtubule disassembly. The SNAP-tag technology provides a platform for the development of novel ADCs with defined conjugation sites and stoichiometry. We achieved the stable and efficient linkage of AURIF to human or murine scFvs using the SNAP-tag technology, offering a strategy to improve the development of personalized medicines.

show abstract

Section: Methodsmentioning

confidence: 99%

Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

Woitok¹,

Klose²,

Fiore³

et al. 2017

OTT

Self Cite

View full text Add to dashboard Cite

show abstract

“…Commercially available and self-labeling SNAP-tag technology, in which a 182-residue polypeptide tag covalently links proteins of interest to ligands, 93 has also been used to generate homogeneous ADCs, including novel recombinant ADCs designed to kill breast cancer cells. 94 To aid novel ADC development, initiatives to create ADCs more efficiently have begun. Antibody derivatives including single-chain fragment variable (scFv) regions were recently used in ADC development instead of mAbs because they are easier to modify and produce.…”

Section: What's New For Adcs?mentioning

confidence: 99%

“…94,95 These scFv-based ADCs also achieve greater tissue/tumor penetration than ADCs based off full-length mAbs. 94,95 With the aim of improving ADC efficacy, new ADC technology adds a highly hydrophilic and polyvalent Fleximer polymer to overcome the limitations of direct drug-antibody conjugation by permitting high drug loading with a variety of payloads, without compromising the physicochemical and PK properties of the ADC. 96 Research suggests ADCs may be synergistic with immune checkpoint inhibitors, providing a strong rationale for exploring these combination strategies.…”

Section: What's New For Adcs?mentioning

confidence: 99%

Antibody–Drug Conjugates as Cancer Therapeutics: Past, Present, and Future

Vezina

Cotreau

Han

et al. 2017

The Journal of Clinical Pharma

View full text Add to dashboard Cite

Antibody-drug conjugates (ADCs) represent an innovative therapeutic approach that provides novel treatment options and hope for patients with cancer. By coupling monoclonal antibodies (mAbs) to cytotoxic small-molecule payloads with a plasma-stable linker, ADCs offer the potential for increased drug specificity and fewer off-target effects than systemic chemotherapy. As evidence for the potential of these therapies, many new ADCs are in various stages of clinical development. Because their structure poses unique challenges to pharmacokinetic and pharmacodynamic characterization, it is critical to recognize the differences between ADCs and conventional chemotherapy in the design of ADC clinical development strategies. Although some properties may be determined mainly by either the mAb or the small-molecule portion, the behavior of these agents is not always predictable. Furthermore, because the absorption, distribution, metabolism, and excretion (ADME) of ADCs are influenced by all 3 of its components (mAb, linker, and payload), it is important to characterize the intact molecule, any target-mediated catabolic clearance of the mAb, and the ADME properties of the small-molecule payload. Here we describe key issues in the clinical development of ADCs, including considerations for designing first-in-human studies for ADCs. We discuss some difficulties of ADC pharmacokinetic characterization and current approaches to overcoming these challenges. Finally, we consider all aspects of clinical pharmacology assessment required during drug development, using examples from the literature to illustrate the discussion.

show abstract

“…For example, in the case of cancerous ovarian cells, the SNAP fusion protein was able to eliminate EGFR+ cells with IC 50 values of 45-66 nmol/l and 40-90 nmol/l [77]. In the case of breast cancer cells, the recombinant fusion proteins were stable in human serum, were internalized rapidly, and demonstrated potent inhibitory and pro-apoptotic effects in vitro at 3-21 nmol/l [74]. …”

Section: Photoimmunotherapymentioning

confidence: 99%

“…The antibody fragment (scFv-425) that binds to EGFR was used as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy targeting skin, breast, pancreatic and ovarian cancer cells. The scFv-425-SNAP-fusion protein was conjugated to a range of BG-modified probes: a microtubule inhibitor auristatin F (AURIF) [74], a chlorin e6 photosensitizer in PDT [75], and the IR700 fluorophore in PIT [76,77]. All these probes were specifically targeted to the tumor and induced cytotoxicity.…”

Section: Photoimmunotherapymentioning

confidence: 99%

Human Antibody Fusion Proteins/Antibody Drug Conjugates in Breast and Ovarian Cancer

Padayachee

Biteghe

Malindi

et al. 2017

Transfus Med Hemother

Self Cite

View full text Add to dashboard Cite

Considerable research efforts have been dedicated to understanding ovarian and breast cancer mechanisms, but there has been little progress translating the research into effective clinical applications. Hence, personalized/precision medicine has emerged because of its potential to improve the accuracy of tumor targeting and minimize toxicity to normal tissue. Targeted therapy in both breast and ovarian cancer has focused on antibodies, antibody drug conjugates (ADCs), and very recently the introduction of human antibody fusion proteins. Small molecule inhibitors and monoclonal antibodies (mAbs) are used in conjunction with chemotherapeutic drugs as a form of treatment but problems arise from a board expression of the target antigen in healthy tissues. Also, insufficient tumor penetration due to tight binding affinity and macromolecular size of mAbs compromise the efficacy of these ADCs. A more targeted approach is thus needed, and ADCs were designed to meet this need. However, in ADCs the method of conjugation of drug to antibody is >1, altering the structure of the drug which leads to off-target effects. Random conjugation also causes the drug to affect the pharmokinetics and biodistribution of the antibody and may cause nonspecific binding and internalization. Recombinant therapeutic proteins achieve controlled conjugation reactions and combine cytotoxicity and targeting in one molecule. They can also be engineered to extend half-life, stability and mechanism of action, and offer novel delivery routes. SNAP-tag fusion proteins are an example of a theranostic recombinant protein as they provide a unique antibody format to conjugate a variety of benzyl guanine modified labels, e.g. fluorophores and photosensitizers in a 1:1 stoichiometry. On the one hand, SNAP tag fusions can be used to optically image tumors when conjugated to a fluorophore, and on the other hand the recombinant proteins can induce necrosis/apoptosis in the tumor when conjugated to a photosensitizer upon exposure to a changeable wavelength of light. The dual nature of SNAP-tag fusions as both a diagnostic and therapeutic tool reinforces its significant role in cancer treatment in an era of precision medicine.

show abstract

The efficient elimination of solid tumor cells by EGFR-specific and HER2-specific scFv-SNAP fusion proteins conjugated to benzylguanine-modified auristatin F

Cited by 39 publications

References 34 publications

Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

Comparison of a mouse and a novel human scFv-SNAP-auristatin F drug conjugate with potent activity against EGFR-overexpressing human solid tumor cells

Antibody–Drug Conjugates as Cancer Therapeutics: Past, Present, and Future

Human Antibody Fusion Proteins/Antibody Drug Conjugates in Breast and Ovarian Cancer

Contact Info

Product

Resources

About