University of Groningen
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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Citation for published version (APA):
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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Chapter (7) Summary
185
Summary:
For the last two decades, a significant progress has been made in the area of targeted cancer therapy thereby improving the efficacy of cancer treatment. Targeted cancer therapy has been the focus of most anti-cancer drugs developed recently to treat malignancies. The advance of knowledge in several related fields as genetics, cell biology and mitogenic pathways in addition to individual variation has played a crucial role in the advance of these treatments and to the development and design of tailored therapeutic protocols.
In Chapter 2 we highlighted recent updates utilizing cancer cell specific ligands in targeting tumor cells. A variety of ligands conjugated with a cytotoxic drug for delivery to the cancer cell site were reported leading to selective cancer cell death. Moreover, we discussed the criteria for proper selection of ligands, drugs and linkers to form the optimal therapeutic complex. The utilization of some cancer markers is discussed in this chapter demonstrating recent manipulations applied to form an advanced efficient ligand-drug or ligand-protein “enzyme” conjugates resulting in better cancer treatment with low or no side effects.
Antibody directed enzyme prodrug therapy (ADEPT) is a promising system designed to target malignancies where the driver, (antibody) is conjugated to the therapeutic enzyme. In our study is we selected carboxypeptidase G2 “CPG2”. Several other enzymes have been employed in targeted cancer therapy as β-lactamase and cytosine deaminase, however CPG2 is the only one that reached clinical trials. Thus, in our work we used CPG2 to produce
Chapter (7) Summary
186
long acting derivatives of the CPG2. We developed a novel ligand-CPG2 conjugates to enhance the therapeutic properties of CPG2 and the produced conjugates.
Instead of antibody conjugated CPG2 as in ADEPT, in Chapter 3 we designed and successfully generated a ligand “peptide”-CPG2 conjugates. The peptide conjugated to CPG2 is cyclic Cysteine-Asparagine-Glycine Arginine-Cysteine (CNGRC), which is known to selectively bind aminopeptidase N (CD13) which is highly expressed on tumor cells. APN is required by metastatic cancer cells stimulating angiogenesis. However, the conjugated peptide was linked to CPG2 with G-G linker in two ways yielding two forms either a single fusion protein where the peptide is linked to one terminus of CPG2 (CNGRC-CPG2), or double fusion protein where the peptide is constructed to bind both carboxyl and amino termini of CPG2 (CNGRC-CPG2-CNGRC). The resulting conjugates were tested for their catalytic activity and binding affinity to the targeted APN on cancer cells. The double fusion with CNGRC was found to enhance catalytic activity of CPG2 and binding affinity to cancer cell lines with high APN expression, and significantly lowered Ex-vivo immunotoxic effect. Moreover, the cytotoxic effect of the prodrug was found to be higher in association of the double fusion protein compared to the single fusion protein. Upon structural analysis using circular dichroism (CD) spectroscopy we found a notable impact of double fusion with CNGRC on the secondary structural composition (α-helix and β-sheet) of the resulting CPG2 complex, which we propose to contribute to the altered previously mentioned properties.
Summary:
For the last two decades, a significant progress has been made in the area of targeted cancer therapy thereby improving the efficacy of cancer treatment. Targeted cancer therapy has been the focus of most anti-cancer drugs developed recently to treat malignancies. The advance of knowledge in several related fields as genetics, cell biology and mitogenic pathways in addition to individual variation has played a crucial role in the advance of these treatments and to the development and design of tailored therapeutic protocols.
In Chapter 2 we highlighted recent updates utilizing cancer cell specific ligands in targeting tumor cells. A variety of ligands conjugated with a cytotoxic drug for delivery to the cancer cell site were reported leading to selective cancer cell death. Moreover, we discussed the criteria for proper selection of ligands, drugs and linkers to form the optimal therapeutic complex. The utilization of some cancer markers is discussed in this chapter demonstrating recent manipulations applied to form an advanced efficient ligand-drug or ligand-protein “enzyme” conjugates resulting in better cancer treatment with low or no side effects.
Antibody directed enzyme prodrug therapy (ADEPT) is a promising system designed to target malignancies where the driver, (antibody) is conjugated to the therapeutic enzyme. In our study is we selected carboxypeptidase G2 “CPG2”. Several other enzymes have been employed in targeted cancer therapy as β-lactamase and cytosine deaminase, however CPG2 is the only one that reached clinical trials. Thus, in our work we used CPG2 to produce
long acting derivatives of the CPG2. We developed a novel ligand-CPG2 conjugates to enhance the therapeutic properties of CPG2 and the produced conjugates.
Instead of antibody conjugated CPG2 as in ADEPT, in Chapter 3 we designed and successfully generated a ligand “peptide”-CPG2 conjugates. The peptide conjugated to CPG2 is cyclic Cysteine-Asparagine-Glycine Arginine-Cysteine (CNGRC), which is known to selectively bind aminopeptidase N (CD13) which is highly expressed on tumor cells. APN is required by metastatic cancer cells stimulating angiogenesis. However, the conjugated peptide was linked to CPG2 with G-G linker in two ways yielding two forms either a single fusion protein where the peptide is linked to one terminus of CPG2 (CNGRC-CPG2), or double fusion protein where the peptide is constructed to bind both carboxyl and amino termini of CPG2 (CNGRC-CPG2-CNGRC). The resulting conjugates were tested for their catalytic activity and binding affinity to the targeted APN on cancer cells. The double fusion with CNGRC was found to enhance catalytic activity of CPG2 and binding affinity to cancer cell lines with high APN expression, and significantly lowered Ex-vivo immunotoxic effect. Moreover, the cytotoxic effect of the prodrug was found to be higher in association of the double fusion protein compared to the single fusion protein. Upon structural analysis using circular dichroism (CD) spectroscopy we found a notable impact of double fusion with CNGRC on the secondary structural composition (α-helix and β-sheet) of the resulting CPG2 complex, which we propose to contribute to the altered previously mentioned properties.
Chapter (7) Summary
187
Since CPG2 is of non-mammalian origin and utilized as a therapeutic enzyme to be used in-vivo, several drawbacks were found when exploited in clinical trials such as immunotoxicity and its low stability against serum protease affecting its half-life. In Chapter 4 we explored two methods (PEGylation and gene fusion with human serum albumin “HSA”) broadly used to enhance the stability of therapeutic molecules as proteins and drugs. We succeeded to design and produce PEGylated-CPG2 and HSA fused CPG2 (HSA-CPG2). The two variants of CPG2 were purified and investigated for their stability and ex-vivo immunotoxicity. The catalytic activity of CPG2 was preserved following PEGylation and HSA conjugation. In vitro studies demonstrated an enhanced serum stability when PEGylated or HSA conjugated were compared to the free CPG2. Moreover, a significant decrease in the immunogenicity on peripheral blood mononuclear cells “PBMCs” has been achieved. It can be concluded that the “biobetter” CPG2 variants with improved therapeutic properties could overcome the immunogenicity problem which is one of the obstacles in using the ADEPT strategy for cancer treatment.
These results encouraged us to produce PEGylated form of the previously investigated CNGRC-CPG2 fusion proteins. In Chapter 5 we performed the PEGylation of the single fused and double fused CPG2 to produce PEG CNGRC-CPG2 and PEG CNGRC-CPG2-CNGRC.
The results have shown that the catalytic activity of the PEGylated single fusion protein, PEG CNGRC-CPG2 is much higher than the catalytic activity of the non-PEGylated single fusion protein (CNGRC-CPG2). However, the catalytic activity of the PEGylated double fused CPG2 was reduced significantly. The same results were obtained regarding the binding of the
Chapter (7) Summary
188
PEGylated products. We found that the PEGylated double fused CPG2 had less binding capability to the APN on cancer cells than the free fused CPG2 and the PEGylated single fused CPG2. As a result of the improved catalytic activity of the PEGylated single fusion protein, the cytotoxic effect of prodrug in combination of the PEGylated single fusion protein was significantly higher compared with the non-PEGylated one. However, the PEGylated double fusion protein resulted in lower cell death (lower cytotoxicity) in combination with the prodrug in comparison with the free fused protein. In summary, we successfully produced new long acting glucarpidase derivatives. We also replaced the antibody needed to carry out ADEPT by a small peptide and demonstrated the efficacy of the novel conjugates in ligand directed enzyme prodrug therapy (LDEPT) protocol. We then embarked on the production of long acting of these conjugates to minimize the immunogenicity of this treatment.
By overcoming some pitfalls, the novel protein conjugates and the long acting derivatives produced in this work will contribute significantly to the further development of ADEPT and LDEPT. Future investigations for stability, specificity and immunotoxicity of the produced therapeutic conjugates have to be carried in vivo, to provide other effective options for targeted cancer therapy. Moreover prodrugs could be PET-labelled to investigate drug biodistribution and cytotoxic effect on cancer cells exclusively, besides exploiting PET-biomarkers to inspect in-vivo therapeutic efficacy of LDEPT.
Since CPG2 is of non-mammalian origin and utilized as a therapeutic enzyme to be used in-vivo, several drawbacks were found when exploited in clinical trials such as immunotoxicity and its low stability against serum protease affecting its half-life. In Chapter 4 we explored two methods (PEGylation and gene fusion with human serum albumin “HSA”) broadly used to enhance the stability of therapeutic molecules as proteins and drugs. We succeeded to design and produce PEGylated-CPG2 and HSA fused CPG2 (HSA-CPG2). The two variants of CPG2 were purified and investigated for their stability and ex-vivo immunotoxicity. The catalytic activity of CPG2 was preserved following PEGylation and HSA conjugation. In vitro studies demonstrated an enhanced serum stability when PEGylated or HSA conjugated were compared to the free CPG2. Moreover, a significant decrease in the immunogenicity on peripheral blood mononuclear cells “PBMCs” has been achieved. It can be concluded that the “biobetter” CPG2 variants with improved therapeutic properties could overcome the immunogenicity problem which is one of the obstacles in using the ADEPT strategy for cancer treatment.
These results encouraged us to produce PEGylated form of the previously investigated CNGRC-CPG2 fusion proteins. In Chapter 5 we performed the PEGylation of the single fused and double fused CPG2 to produce PEG CNGRC-CPG2 and PEG CNGRC-CPG2-CNGRC.
The results have shown that the catalytic activity of the PEGylated single fusion protein, PEG CNGRC-CPG2 is much higher than the catalytic activity of the non-PEGylated single fusion protein (CNGRC-CPG2). However, the catalytic activity of the PEGylated double fused CPG2 was reduced significantly. The same results were obtained regarding the binding of the
PEGylated products. We found that the PEGylated double fused CPG2 had less binding capability to the APN on cancer cells than the free fused CPG2 and the PEGylated single fused CPG2. As a result of the improved catalytic activity of the PEGylated single fusion protein, the cytotoxic effect of prodrug in combination of the PEGylated single fusion protein was significantly higher compared with the non-PEGylated one. However, the PEGylated double fusion protein resulted in lower cell death (lower cytotoxicity) in combination with the prodrug in comparison with the free fused protein. In summary, we successfully produced new long acting glucarpidase derivatives. We also replaced the antibody needed to carry out ADEPT by a small peptide and demonstrated the efficacy of the novel conjugates in ligand directed enzyme prodrug therapy (LDEPT) protocol. We then embarked on the production of long acting of these conjugates to minimize the immunogenicity of this treatment.
By overcoming some pitfalls, the novel protein conjugates and the long acting derivatives produced in this work will contribute significantly to the further development of ADEPT and LDEPT. Future investigations for stability, specificity and immunotoxicity of the produced therapeutic conjugates have to be carried in vivo, to provide other effective options for targeted cancer therapy. Moreover prodrugs could be PET-labelled to investigate drug biodistribution and cytotoxic effect on cancer cells exclusively, besides exploiting PET-biomarkers to inspect in-vivo therapeutic efficacy of LDEPT.
