L-Asparaginase (ASNase, EC 3.5.1.1) is a key component of the established combined chemotherapy used for the treatment of pediatric acute lymphoblastic leukemia, having significantly increased the percentage of complete remissions in patients since its introduction in 1970s. The benefit of ASNase treatment is supported by extensive clinical data, while resistance to asparaginase is correlated with poor prognosis. ASNase is an amidohydrolase which shows a prevalent asparaginolytic and a secondary glutaminolytic activity; its therapeutic benefit comes from the depletion of asparagine from the blood stream, on which leukemic cells depends, given their absent or compromised capability to express asparagine synthetase (EC 6.3.5.4) under stress conditions. The ASNase molecules currently used in the clinics are derived from either E. coli (EcAII) or E. chrysanthemi, with the first line drug being PEG-Asparaginase (Oncaspar ®). However, most of the available ASNase products lack optimal pharmaceutical features, in particular because of ASNase high toxicity, due to its untargeted activity; high immunogenicity, due to its bacterial origin and large size; short blood serum half-life, and poor efficacy in specific sub-classes of patients (high risk). The aim of this work was to address these limitations, in order to improve EcAII efficacy, and in particular its high toxicity, on one side, by targeting the drug onto leukemic cells, and its high immunogenicity, on the other side, by miniaturizing the drug. It is expected that tackling these two points should also help to increase the drug efficacy in the treatment of high-risk patients. The adopted strategy consisted in the design of a radically new, anti-CD19 Asparaginase-based Antibody Drug Conjugate (ADC), which was conceived by our research group after the successful engineering of a single domain antibody (sdAb) with asparagynolitic activity, obtained through the rational transfer of E. coli type II asparaginase catalytic residues onto a camelid sdAb backbone (sdASNase) (PATENT# E0115946). The addition of a targeting domain nanobody to the catalytic sdASNase lead to the new concept of Targeted Catalytic Nanobodies (TCANs).In particular, the molecule designed in this work (TCAN3) was composed by a newly selected anti-CD19 nanobody (targeting domain) and by the catalytic sdASNase nanobody (catalytic domain). In order to produce such molecule, several steps were followed. Firstly, the extracellular domain of CD19 was expressed as a C-terminal fusion with the human Fc fragment. For the selection of targeting nanobodies, the Phage Display technique was set up in house, but, due to obstacles encountered in both recombinant CD19 purification and classical Phage Display selection, the Yeast Two Hybrid system was chosen as an alternative strategy for the screening of anti-CD19 intracellular nanobodies. The collaboration with the group of Prof. Cattaneo (SNS, Pisa) resulted in the successful isolation of a nanobody, whose binding to CD19 was confirmed through ELISA tests. The TCAN3 was then assembled joining the selected anti-CD19 nanobody targeting domain to the available sdASNase nanobody through a linker. The TCAN3 was then expressed and purified, and its binding to CD19 was confirmed through ELISA tests. In the meantime, preliminary tests for co-localization studies of CD19 and lysosomes were set up. In conclusion, in this work, TCAN3, a promising targeted asparaginase-based molecule which could help in leukemia therapy, and especially in high-risk forms, was designed and expressed. TCANs, focusing for the first time on the specificity of the metabolic traits of a given tumor and coupling the correct catalytic activity to the appropriate target specificity,might potentially represent a novel general approach to tackle any type of cancer which shows sensitivity to a specific metabolite deprivation.

Asparaginase-based antibody Drug Conjugates

PESSINO, GRETA
2021-05-26T00:00:00+02:00

Abstract

L-Asparaginase (ASNase, EC 3.5.1.1) is a key component of the established combined chemotherapy used for the treatment of pediatric acute lymphoblastic leukemia, having significantly increased the percentage of complete remissions in patients since its introduction in 1970s. The benefit of ASNase treatment is supported by extensive clinical data, while resistance to asparaginase is correlated with poor prognosis. ASNase is an amidohydrolase which shows a prevalent asparaginolytic and a secondary glutaminolytic activity; its therapeutic benefit comes from the depletion of asparagine from the blood stream, on which leukemic cells depends, given their absent or compromised capability to express asparagine synthetase (EC 6.3.5.4) under stress conditions. The ASNase molecules currently used in the clinics are derived from either E. coli (EcAII) or E. chrysanthemi, with the first line drug being PEG-Asparaginase (Oncaspar ®). However, most of the available ASNase products lack optimal pharmaceutical features, in particular because of ASNase high toxicity, due to its untargeted activity; high immunogenicity, due to its bacterial origin and large size; short blood serum half-life, and poor efficacy in specific sub-classes of patients (high risk). The aim of this work was to address these limitations, in order to improve EcAII efficacy, and in particular its high toxicity, on one side, by targeting the drug onto leukemic cells, and its high immunogenicity, on the other side, by miniaturizing the drug. It is expected that tackling these two points should also help to increase the drug efficacy in the treatment of high-risk patients. The adopted strategy consisted in the design of a radically new, anti-CD19 Asparaginase-based Antibody Drug Conjugate (ADC), which was conceived by our research group after the successful engineering of a single domain antibody (sdAb) with asparagynolitic activity, obtained through the rational transfer of E. coli type II asparaginase catalytic residues onto a camelid sdAb backbone (sdASNase) (PATENT# E0115946). The addition of a targeting domain nanobody to the catalytic sdASNase lead to the new concept of Targeted Catalytic Nanobodies (TCANs).In particular, the molecule designed in this work (TCAN3) was composed by a newly selected anti-CD19 nanobody (targeting domain) and by the catalytic sdASNase nanobody (catalytic domain). In order to produce such molecule, several steps were followed. Firstly, the extracellular domain of CD19 was expressed as a C-terminal fusion with the human Fc fragment. For the selection of targeting nanobodies, the Phage Display technique was set up in house, but, due to obstacles encountered in both recombinant CD19 purification and classical Phage Display selection, the Yeast Two Hybrid system was chosen as an alternative strategy for the screening of anti-CD19 intracellular nanobodies. The collaboration with the group of Prof. Cattaneo (SNS, Pisa) resulted in the successful isolation of a nanobody, whose binding to CD19 was confirmed through ELISA tests. The TCAN3 was then assembled joining the selected anti-CD19 nanobody targeting domain to the available sdASNase nanobody through a linker. The TCAN3 was then expressed and purified, and its binding to CD19 was confirmed through ELISA tests. In the meantime, preliminary tests for co-localization studies of CD19 and lysosomes were set up. In conclusion, in this work, TCAN3, a promising targeted asparaginase-based molecule which could help in leukemia therapy, and especially in high-risk forms, was designed and expressed. TCANs, focusing for the first time on the specificity of the metabolic traits of a given tumor and coupling the correct catalytic activity to the appropriate target specificity,might potentially represent a novel general approach to tackle any type of cancer which shows sensitivity to a specific metabolite deprivation.
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Descrizione: Asparaginase-based Antibody Drug Conjugates
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11571/1435095
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