Studies on Ligand Directed Enzyme Prodrug Therapy and Production of Long Acting Protein Therapeutics for Targeted Cancer Treatment
Al-Mansoori, Layla
DOI:
10.33612/diss.131689831
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Publication date: 2020
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Al-Mansoori, L. (2020). Studies on Ligand Directed Enzyme Prodrug Therapy and Production of Long Acting Protein Therapeutics for Targeted Cancer Treatment. University of Groningen.
https://doi.org/10.33612/diss.131689831
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17. Di Matteo P, Mangia P, Tiziano E, Valentinis B, Porcellini S, Doglioni C, et al. Anti-metastatic activity of the tumor vascular targeting agent NGR-TNF. Clin Exp Metastasis. 2015;32(3):289-300.
18. Al-mansoori L, Bashraheel SS, Qahtani ADA, O’Connor CD, Elsinga P, Goda SK. In vitro studies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy. Oncotarget. 2020.
19. McCullough JL, Chabner BA, Bertino JR. Purification and properties of carboxypeptidase G 1. J Biol Chem. 1971;246(23):7207-13. In review
20. Fu K, Cheng Q, Liu Z, Chen Z, Wang Y, Ruan H, et al. Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells. PLoS One. 2014;9(8):e104426.
21. Jevsevar S, Kunstelj M, Porekar VG. PEGylation of therapeutic proteins. Biotechnol J. 2010;5(1):113-28.
22. Lawrence PB, Price JL. How PEGylation influences protein conformational stability. Curr Opin Chem Biol. 2016;34:88-94.
23. Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc. 2006;1(6):2876-90.
24. Veronese FM, Pasut G. PEGylation, successful approach to drug delivery. Drug Discov Today. 2005;10(21):1451-8.
25. Qi F, Hu C, Yu W, Hu T. Conjugation with Eight-Arm PEG Markedly Improves the In Vitro Activity and Prolongs the Blood Circulation of Staphylokinase. Bioconjugate chemistry. 2018;29(2):451-8.
26. Mu Q, Hu T, Yu J. Molecular insight into the steric shielding effect of PEG on the conjugated staphylokinase: biochemical characterization and molecular dynamics simulation. PLoS One. 2013;8(7):e68559.
27. Morgenstern J, Baumann P, Brunner C, Hubbuch J. Effect of PEG molecular weight and PEGylation degree on the physical stability of PEGylated lysozyme. International journal of pharmaceutics. 2017;519(1-2):408-17.
28. Wan X, Zhang J, Yu W, Shen L, Ji S, Hu T. Effect of protein immunogenicity and PEG size and branching on the anti-PEG immune response to PEGylated proteins. Process Biochemistry. 2017;52:18391.
Chapter (6)
General Discussion and Future Perspectives
After cardiovascular diseases cancer is the second leading cause of death worldwide with 14% of global deaths (1). Several factors are the underlying cause of the high prevalence of cancer cases such as environmental stress, dietary and genetic reasons. As a result, an increased number of mutations enhances carcinogenesis and the development of tumors. Various therapeutic approaches have been applied to treat cancer as chemotherapy, radiotherapy and surgery, which were found to be insufficient and accompanied with multiple undesirable severe side effects such as non-specific systemic toxicity and drug resistance (2). Hence, new therapeutic methods came into the field of cancer therapy introducing highly cytotoxic drugs targeted selectively to tumor cells thus lowering un-favored side effects.
Targeted cancer therapy requires a cancer specific marker with well-characterized ligands that bind specifically to this marker. The ligand is employed as a carrier for the cytotoxic molecule (drug) to the cancer cells via its selective binding to the marker. The efficiency of such a therapeutic complex relies on several factors as binding specificity, affinity, accessibility and physical properties (explained in chapter 2.).
Targeting cancer cells can proceed either directly by binding the cytotoxic drug with a cancer marker specific ligand, or could be indirect by binding the ligand to a therapeutic enzyme known to hydrolyze and activate the non-toxic drug “prodrug” (LDEPT). The second approach is considered to be safer and ensures the cytotoxic effect of the drug at the site of tumor (3).
Several cancer-specific ligands have been approved by FDA for clinical use. Some of these markers have been recently investigated for further improvement and higher efficiency including antigens such as Prostate specific membrane antigen “PSMA”, carcinoembryonic antigen “CEA and receptors as folate receptor, somatostatin receptor, integrins as αvβ3 and
clusters of differentiation molecules (CD13). All these markers were discussed in details in chapter 2.
The overall purpose of the work in this thesis is 1) to produce “biobetter” drugs to overcome some of the pitfalls of the antibody directed enzyme prodrug therapy (ADEPT) such as immunogenicity and 2) the production of the enzyme-carrier complex. We also introduced the ligand directed enzyme prodrug therapy LDEPT which achieves the aim of ADEPT as ligand-enzyme conjugates are easier and cheaper to produce than antibody-enzyme conjugates.
Two of the main components of the therapy are the enzyme (CPG2 in our case) and the carrier (antibody or ligand).
CPG2 is an exopeptidase used to detoxify the overdose of methotrexate, and in addition to activate prodrugs in ADEPT (4-6). Because of the bacterial “non-mammalian” origin of the enzyme this is accompanied with immunogenic reaction upon repeated administration in vivo, whereas CPG2 has a lower stability resulting in lower efficacy. We used two widely exploited methods to produce new variants: protein PEGylation and genetically fused with human serum albumin (HSA) (7, 8). We succeeded to produce the PEGylated and HSA conjugated forms of CPG2 (PEG-CPG2, HSA-CPG2) (Chapter 3). When the produced CPG2 isoforms were tested for their serum
General Discussion and Future Perspectives
After cardiovascular diseases cancer is the second leading cause of death worldwide with 14% of global deaths (1). Several factors are the underlying cause of the high prevalence of cancer cases such as environmental stress, dietary and genetic reasons. As a result, an increased number of mutations enhances carcinogenesis and the development of tumors. Various therapeutic approaches have been applied to treat cancer as chemotherapy, radiotherapy and surgery, which were found to be insufficient and accompanied with multiple undesirable severe side effects such as non-specific systemic toxicity and drug resistance (2). Hence, new therapeutic methods came into the field of cancer therapy introducing highly cytotoxic drugs targeted selectively to tumor cells thus lowering un-favored side effects.
Targeted cancer therapy requires a cancer specific marker with well-characterized ligands that bind specifically to this marker. The ligand is employed as a carrier for the cytotoxic molecule (drug) to the cancer cells via its selective binding to the marker. The efficiency of such a therapeutic complex relies on several factors as binding specificity, affinity, accessibility and physical properties (explained in chapter 2.).
Targeting cancer cells can proceed either directly by binding the cytotoxic drug with a cancer marker specific ligand, or could be indirect by binding the ligand to a therapeutic enzyme known to hydrolyze and activate the non-toxic drug “prodrug” (LDEPT). The second approach is considered to be safer and ensures the cytotoxic effect of the drug at the site of tumor (3).
Several cancer-specific ligands have been approved by FDA for clinical use. Some of these markers have been recently investigated for further improvement and higher efficiency including antigens such as Prostate specific membrane antigen “PSMA”, carcinoembryonic antigen “CEA and receptors as folate receptor, somatostatin receptor, integrins as αvβ3 and
clusters of differentiation molecules (CD13). All these markers were discussed in details in chapter 2.
The overall purpose of the work in this thesis is 1) to produce “biobetter” drugs to overcome some of the pitfalls of the antibody directed enzyme prodrug therapy (ADEPT) such as immunogenicity and 2) the production of the enzyme-carrier complex. We also introduced the ligand directed enzyme prodrug therapy LDEPT which achieves the aim of ADEPT as ligand-enzyme conjugates are easier and cheaper to produce than antibody-enzyme conjugates.
Two of the main components of the therapy are the enzyme (CPG2 in our case) and the carrier (antibody or ligand).
CPG2 is an exopeptidase used to detoxify the overdose of methotrexate, and in addition to activate prodrugs in ADEPT (4-6). Because of the bacterial “non-mammalian” origin of the enzyme this is accompanied with immunogenic reaction upon repeated administration in vivo, whereas CPG2 has a lower stability resulting in lower efficacy. We used two widely exploited methods to produce new variants: protein PEGylation and genetically fused with human serum albumin (HSA) (7, 8). We succeeded to produce the PEGylated and HSA conjugated forms of CPG2 (PEG-CPG2, HSA-CPG2) (Chapter 3). When the produced CPG2 isoforms were tested for their serum
stability and immunogenicity, they found to be more stable and with lower ex vivo immunogenicity compared with the free CPG2. Moreover, we found that PEGylation added another improvement by enhancing the catalytic activity of PEG-CPG2. Our resulting modified CPG2 proved to provide a “biobetter form” of CPG2 for further applications in LDEPT or drug detoxification. The other component of the therapeutic complex is the selection of proper ligand for the cancer specific marker. One of the promising cancer specific markers which has been extensively investigated recently is aminopeptidase N (APN, CD13). Several studies highlighted its importance in tumor metastasis and angiogenesis and blocking the enzyme could lead to lower cancer spreading (9-11). Furthermore, high APN expression on cancer cells has been proven to be a promising marker for imaging and targeting cancer cells (12, 13). To target APN, a small tripeptide discovered from the phage display libraries was found to have a selective high binding affinity to APN (14). Recent studies utilized this small peptide to carry the cytotoxic drug to the cancer cells (15-17). In our study, we produced a fusion protein composed of CPG2 with the APN specific peptide, cyclic asparagine-glycine-arginine (CNGRC) (Chapter 4). Production of this complex had several advantages such as the feasibility of production, small size of ligand (peptide) making it more accessible to tumor cells compared to antibodies. Moreover, it is the first conjugate of CPG2 with a peptide, since all previously conjugated CPG2 compounds were with full antibodies or its fragments. Two forms of the conjugates were generated namely 1) single fusion protein where the peptide CNGRC is linked to one end of the enzyme CPG2 via a linker consisting of two glycine residues (CNGRC-CPG2) and 2) double fusion protein with two
CNGRC groups linked to the enzyme CPG2 (CNGRC-CPG2-CNGRC), one at each terminus (carboxyl and amino). The resulting fusion proteins were examined for their stability, immunogenicity and CPG2 catalytic activity. The resulting fusion proteins showed higher stability and catalytic activity, lower ex vivo immunogenicity compared to the free CPG2. However, the double fusion protein exhibited remarkable enhancement in the stability and catalytic activity demonstrating structural changes induced by the fusion of CPG2 with two CNGRC groups which improved the resulting complex.
We investigated the binding capability of the resulting fusion proteins to cancer cells differentially expressing APN. The double fusion protein exhibited high binding affinity to cells expressing high levels of APN, consequently this affected the cytotoxicity of prodrug used with the double fusion protein which was significantly higher compared with free CPG2. Collectively, the results confirmed notable improvement in the therapeutic features of the produced double fusion protein in targeting high APN expressing cancer cells in vitro.
In the first part of the study we obtained a biobetter form of CPG2 by chemical conjugation with PEG and genetically fused with HSA producing PEG-CPG2 and HSA-CPG2. We then investigated the effect of PEGylation on the therapeutic features of the previously produced CPG2 fusion proteins (CNGRC-CPG2 and CNGRC-CPG2-CNGRC) (Chapter 5). The resulting PEGylated fusion proteins exhibited differential changes in their features compared with the non-PEGylated ones. The PEGylated single fusion protein showed better properties than the un-PEGylated CPG2 such as higher catalytic activity and stronger binding affinity to high APN expressing cells
stability and immunogenicity, they found to be more stable and with lower ex vivo immunogenicity compared with the free CPG2. Moreover, we found that PEGylation added another improvement by enhancing the catalytic activity of PEG-CPG2. Our resulting modified CPG2 proved to provide a “biobetter form” of CPG2 for further applications in LDEPT or drug detoxification. The other component of the therapeutic complex is the selection of proper ligand for the cancer specific marker. One of the promising cancer specific markers which has been extensively investigated recently is aminopeptidase N (APN, CD13). Several studies highlighted its importance in tumor metastasis and angiogenesis and blocking the enzyme could lead to lower cancer spreading (9-11). Furthermore, high APN expression on cancer cells has been proven to be a promising marker for imaging and targeting cancer cells (12, 13). To target APN, a small tripeptide discovered from the phage display libraries was found to have a selective high binding affinity to APN (14). Recent studies utilized this small peptide to carry the cytotoxic drug to the cancer cells (15-17). In our study, we produced a fusion protein composed of CPG2 with the APN specific peptide, cyclic asparagine-glycine-arginine (CNGRC) (Chapter 4). Production of this complex had several advantages such as the feasibility of production, small size of ligand (peptide) making it more accessible to tumor cells compared to antibodies. Moreover, it is the first conjugate of CPG2 with a peptide, since all previously conjugated CPG2 compounds were with full antibodies or its fragments. Two forms of the conjugates were generated namely 1) single fusion protein where the peptide CNGRC is linked to one end of the enzyme CPG2 via a linker consisting of two glycine residues (CNGRC-CPG2) and 2) double fusion protein with two
CNGRC groups linked to the enzyme CPG2 (CNGRC-CPG2-CNGRC), one at each terminus (carboxyl and amino). The resulting fusion proteins were examined for their stability, immunogenicity and CPG2 catalytic activity. The resulting fusion proteins showed higher stability and catalytic activity, lower ex vivo immunogenicity compared to the free CPG2. However, the double fusion protein exhibited remarkable enhancement in the stability and catalytic activity demonstrating structural changes induced by the fusion of CPG2 with two CNGRC groups which improved the resulting complex.
We investigated the binding capability of the resulting fusion proteins to cancer cells differentially expressing APN. The double fusion protein exhibited high binding affinity to cells expressing high levels of APN, consequently this affected the cytotoxicity of prodrug used with the double fusion protein which was significantly higher compared with free CPG2. Collectively, the results confirmed notable improvement in the therapeutic features of the produced double fusion protein in targeting high APN expressing cancer cells in vitro.
In the first part of the study we obtained a biobetter form of CPG2 by chemical conjugation with PEG and genetically fused with HSA producing PEG-CPG2 and HSA-CPG2. We then investigated the effect of PEGylation on the therapeutic features of the previously produced CPG2 fusion proteins (CNGRC-CPG2 and CNGRC-CPG2-CNGRC) (Chapter 5). The resulting PEGylated fusion proteins exhibited differential changes in their features compared with the non-PEGylated ones. The PEGylated single fusion protein showed better properties than the un-PEGylated CPG2 such as higher catalytic activity and stronger binding affinity to high APN expressing cells
and subsequently cytotoxicity with the prodrug, compared to the non-PEGylated one. However, with the double fusion protein the binding affinity following PEGylation was lowered thus affecting the cytotoxicity of the prodrug in association of PEGylated double fusion protein. Overall, the results provided another promising candidate being the PEGylated single fusion protein with improved features upon PEGylation.
Our work was carried in vitro and demonstrated promising results with some therapeutic complexes (double fusion protein “CNGRC-CPG2-CNGRC” and PEGylated single fusion protein “PEG CNGRC-CPG2”) which encourages future investigation in vivo to study their pharmacokinetics, stability, immunogenicity and cytotoxicity. A suitable animal model is the mouse model with a HT1080 tumor xenograft. After injection of HT1080 cells to mice for developing fibrosarcoma tumor xenografts, these tumor-bearing mice can be used to investigate in vivo antitumor activity of produced CPG2 fusion conjugates and their effect on tumor growth inhibition and angiogenesis. Moreover, positron emission tomography (PET) can be employed to investigate protein kinetics of the administered CPG2 fusion conjugates by labelling with a long-lived PET-radionuclide (89Zr).
Another option is PET-labelling of prodrugs which will be converted to very power cytotoxic compounds by glucarpidase. PET it is then possible to measure the regional distribution of a PET-labelled prodrug, for ADEPT or ADEPT as a function of time in living species non-invasively.
Using this method pathophysiological conditions can be compared to study the viability of different drugs in relation to each other and to the pro-drugs which are currently in use. Furthermore using PET the
pharmacokinetics of the PET-labelled prodrug can accurately be measured non-invasively and it can be assessed if the prodrug reach the target. Other important questions which can be answered using PET: What is the systemic uptake of a locally administered prodrug? What is the half live time of the pro-drug in the different tissues including the tumor.
Prodrugs which produce powerful cytotoxic compounds can be radiolabelled with either 11C or 18F as PET-radionuclide because.using these radionuclides
the physicochemical and biological properties are not or slightly affected. Currently, there are no PET-labelled prodrugs or PET- labelled glucarpidase for ADEPT available. PET-studies can be carried out both in appropriate animal models and in patients.
In addition, several well-characterized PET-biomarkers are available and used in the clinic to assess cancer treatment. These biomarkers can also be used to in vivo monitor ADEPT. Examples of the PET-biomarkers are [18F]FDG
(glucose consumption), [18F]FLT (cell proliferation), and [18]FET (amino acid
transport/protein synthesis). In case of a successful treatment, the metabolic activity of the tumor should decrease and therefore the PET-signal upon administration of such a PET-biomarker will decrease accordingly.
Antibody directed enzyme prodrug therapy could be implemented effectively for the targeted treatment of most common solid cancers, provided the cancer specific antibody is available to deliver the CPG2. Clinical studies with the CPG2 system have demonstrated the effectiveness of this therapy (18). The main stumbling block has been immunogenicity of the CPG2. The work
and subsequently cytotoxicity with the prodrug, compared to the non-PEGylated one. However, with the double fusion protein the binding affinity following PEGylation was lowered thus affecting the cytotoxicity of the prodrug in association of PEGylated double fusion protein. Overall, the results provided another promising candidate being the PEGylated single fusion protein with improved features upon PEGylation.
Our work was carried in vitro and demonstrated promising results with some therapeutic complexes (double fusion protein “CNGRC-CPG2-CNGRC” and PEGylated single fusion protein “PEG CNGRC-CPG2”) which encourages future investigation in vivo to study their pharmacokinetics, stability, immunogenicity and cytotoxicity. A suitable animal model is the mouse model with a HT1080 tumor xenograft. After injection of HT1080 cells to mice for developing fibrosarcoma tumor xenografts, these tumor-bearing mice can be used to investigate in vivo antitumor activity of produced CPG2 fusion conjugates and their effect on tumor growth inhibition and angiogenesis. Moreover, positron emission tomography (PET) can be employed to investigate protein kinetics of the administered CPG2 fusion conjugates by labelling with a long-lived PET-radionuclide (89Zr).
Another option is PET-labelling of prodrugs which will be converted to very power cytotoxic compounds by glucarpidase. PET it is then possible to measure the regional distribution of a PET-labelled prodrug, for ADEPT or ADEPT as a function of time in living species non-invasively.
Using this method pathophysiological conditions can be compared to study the viability of different drugs in relation to each other and to the pro-drugs which are currently in use. Furthermore using PET the
pharmacokinetics of the PET-labelled prodrug can accurately be measured non-invasively and it can be assessed if the prodrug reach the target. Other important questions which can be answered using PET: What is the systemic uptake of a locally administered prodrug? What is the half live time of the pro-drug in the different tissues including the tumor.
Prodrugs which produce powerful cytotoxic compounds can be radiolabelled with either 11C or 18F as PET-radionuclide because.using these radionuclides
the physicochemical and biological properties are not or slightly affected. Currently, there are no PET-labelled prodrugs or PET- labelled glucarpidase for ADEPT available. PET-studies can be carried out both in appropriate animal models and in patients.
In addition, several well-characterized PET-biomarkers are available and used in the clinic to assess cancer treatment. These biomarkers can also be used to in vivo monitor ADEPT. Examples of the PET-biomarkers are [18F]FDG
(glucose consumption), [18F]FLT (cell proliferation), and [18]FET (amino acid
transport/protein synthesis). In case of a successful treatment, the metabolic activity of the tumor should decrease and therefore the PET-signal upon administration of such a PET-biomarker will decrease accordingly.
Antibody directed enzyme prodrug therapy could be implemented effectively for the targeted treatment of most common solid cancers, provided the cancer specific antibody is available to deliver the CPG2. Clinical studies with the CPG2 system have demonstrated the effectiveness of this therapy (18). The main stumbling block has been immunogenicity of the CPG2. The work
presented in this thesis has successfully eliminated this problem through the production of long acting enzyme and also the production of novel conjugates with small peptides instead of the whole antibody as a carrier to direct the enzyme to the tumour
The “biobetter” molecules produced in this work will pave the way for more efficient and effective ADEPT and LDEPT as a single treatment or in combination with other protocol such as immunotherapy or radiotherapy for greater clinical benefit.
Summary of the work with medium-term future planning is shown in the below figure.
presented in this thesis has successfully eliminated this problem through the production of long acting enzyme and also the production of novel conjugates with small peptides instead of the whole antibody as a carrier to direct the enzyme to the tumour
The “biobetter” molecules produced in this work will pave the way for more efficient and effective ADEPT and LDEPT as a single treatment or in combination with other protocol such as immunotherapy or radiotherapy for greater clinical benefit.
Summary of the work with medium-term future planning is shown in the below figure. Ch apt er (6 ) G en er al Dis cu ss io n an d Fu tu re P er spe ct iv 181 Su mm ary of th e key resu lts of th e the sis and pos sible f utu re w ork. CPG2: Car box yp eptidase G2, CNGR C: Cy asparagine -g ly cine -ar gi nine ,APN: A m ino pept idase N, HSA: hu man s eru m albu m in, X: CNG RC, PEG: Pol ye thelen e gl yc
References:
1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69-90.
2. Xu G, McLeod HL. Strategies for enzyme/prodrug cancer therapy. Clin Cancer Res. 2001;7(11):3314-24.
3. Gerber DE. Targeted therapies: a new generation of cancer treatments. Am Fam Physician. 2008;77(3):311-9.
4. Bagshawe KD. Antibody-directed enzyme prodrug therapy (ADEPT) for cancer. Expert Rev Anticancer Ther. 2006;6(10):1421-31.
5. Sherwood RF, Melton RG, Alwan SM, Hughes P. Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Use of a novel triazine dye affinity method. Eur J Biochem. 1985;148(3):447-53.
6. Green JM. Glucarpidase to combat toxic levels of methotrexate in patients. Ther Clin Risk Manag. 2012;8:403-13.
7. Schellenberger V, Wang CW, Geething NC, Spink BJ, Campbell A, To W, et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nature biotechnology. 2009;27(12):1186-90.
8. Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi M, Valentini M, et al. Site-directed enzymatic PEGylation of the human granulocyte colony-stimulating factor. Febs j. 2009;276(22):6741-50.
9. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407(6801):249-57.
10. Amoscato AA, Sramkoski RM, Babcock GF, Alexander JW. Neutral surface aminopeptidase activity of human tumor cell lines. Biochimica et biophysica acta. 1990;1041(3):317-9.
11. Wickstrom M, Larsson R, Nygren P, Gullbo J. Aminopeptidase N (CD13) as a target for cancer chemotherapy. Cancer Sci. 2011;102(3):501-8.
12. Schreiber CL, Smith BD. Molecular Imaging of Aminopeptidase N in Cancer and Angiogenesis. Contrast Media Mol Imaging. 2018;2018:5315172.
13. Gai Y, Jiang Y, Long Y, Sun L, Liu Q, Qin C, et al. Evaluation of an Integrin alphavbeta3 and Aminopeptidase N Dual-Receptor Targeting Tracer for Breast Cancer Imaging. Molecular pharmaceutics. 2020;17(1):349-58.
14. Curnis F, Arrigoni G, Sacchi A, Fischetti L, Arap W, Pasqualini R, et al. Differential binding of drugs containing the NGR motif to CD13 isoforms in tumor vessels, epithelia, and myeloid cells. Cancer research. 2002;62(3):867-74.
15. Curnis F, Cattaneo A, Longhi R, Sacchi A, Gasparri AM, Pastorino F, et al. Critical role of flanking residues in NGR-to-isoDGR transition and CD13/integrin receptor switching. J Biol Chem. 2010;285(12):9114-23.
16. Gregorc V, Gaafar RM, Favaretto A, Grossi F, Jassem J, Polychronis A, et al. NGR-hTNF in combination with best investigator choice in previously treated malignant pleural mesothelioma (NGR015): a randomised, double-blind, placebo-controlled phase 3 trial. The Lancet Oncology. 2018;19(6):799-811.
17. Mohammadi-Farsani A, Habibi-Roudkenar M, Golkar M, Shokrgozar MA, Jahanian-Najafabadi A, KhanAhmad H, et al. A-NGR fusion protein induces apoptosis in human cancer cells. Excli j. 2018;17:590-7.
18. Francis RJ, Sharma SK, Springer C, Green AJ, Hope-Stone LD, Sena L, et al. A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours. British journal of cancer. 2002;87(6):600-7
