Isolation and molecular characterization of novel glucarpidases:Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment

<div><p>Repeated cycles of antibody-directed enzyme pro-drug therapy (ADEPT) and the use of glucarpidase in the detoxification of cytotoxic methotrexate (MTX) are highly desirable during cancer therapy but are hampered by the induced human antibody response to glucarpidase. Novel variants of glucarpidase (formal name: carboxypeptidase G2, CPG2) with epitopes not recognized by the immune system are likely to allow repeated cycles of ADEPT for effective cancer therapy. Towards this aim, over two thousand soil samples were collected and screened for folate hydrolyzing bacteria using folate as the sole carbon source. The work led to the isolation and the characterization of three new glucarpidase producing strains, which were designated as: <i>Pseudomonas lubricans</i> strain SF168, <i>Stenotrophomonas</i> sp SA and <i>Xenophilus azovorans</i> SN213. The <i>CPG2</i> genes of <i>Xenophilus azovorans</i> SN213 (named <i>Xen CPG2</i>) and <i>Stenotrophomonas sp</i> SA (named <i>Sten CPG2</i>) were cloned and molecularly characterized. Both Xen CPG2 and Sten CPG2 share very close amino acid sequences (99%); we therefore, focused on the study of Xen CPG2. Finally, we demonstrated that a polyclonal antibody raised against our new CPG2, Xen CPG2, does not react with the CPG2 from <i>Pseudomonas sp</i>. strain RS-16 (Ps CPG2) that are currently in clinical use. The two enzymes, therefore could potentially be used consecutively in the ADEPT protocol to minimize the effect of the human antibody response that hampers current treatment with Ps CPG2. The identified novel CPG2 in this study will, therefore, pave the way for safer antibody directed enzyme pro-drug therapy for cancer treatment.</p></div

Correction:Isolation and molecular characterization of novel glucarpidases: Enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment

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Studies on vascular response to full superantigens and superantigen derived peptides:Possible production of novel superantigen variants with less vasodilation effect for tolerable cancer immunotherapy

Superantigens (SAgs) are a class of antigens that cause non-specific activation of T-cells resulting in polyclonal T cell activation and massive cytokine release and causing symptoms similar to sepsis, e.g. hypotension and subsequent hyporeactivity. We investigated the direct effect of SAgs on vascular tone using two recombinant SAgs, SEA and SPEA. The roles of Nitric Oxide (NO) and potentially hyperpolarization, which is dependent on the K + channel activation, were also explored. The data show that SEA and SPEA have direct vasodilatory effects that were in part NO-dependent, but completely dependent on activation of K + channels. Our work also identified the functional regions of one of the superantigens, SPEA, that are involved in causing the vasodilation and possible hypotension. A series of 20 overlapping peptides, spanning the entire sequence of SPEA, were designed and synthesized. The vascular response of each peptide was measured, and the active peptides were identified. Our results implicate the regions, (61–100), (101–140) and (181–220) which cause the vasodilation and possible hypotension effects of SPEA. The data also shows that the peptide 181–220 exert the highest vasodilation effect. This work therefore, demonstrates the direct effect of SAgs on vascular tone and identify the active region causing this vasodilation. We propose that these three peptides could be effective novel antihypertensive drugs. We also overexpressed, in E.coli, four superantigens from codon optimized genes

Production of "biobetter" variants of glucarpidase with enhanced enzyme activity

Glucarpidase, also known as carboxypeptidase G(2), is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. K-m, V-max and K-cat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase

Production of "biobetter" glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment

Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, `biobetter' glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy

CD spectra and high voltage of Xen CPG2 and Ps CPG2.

<p>A) Combined CD spectra data of Xen CPG2 and Ps CPG2 in molar ellipticity relative the wavelength in far UV region, the spectra obtained by dragging their spectral data over each other, where smooth 0 is molar ellipticity of Xen CPG2 and smooth 1 is for CD spectra of Ps CPG2. All Spectral data are corrected for the baseline buffer, B) represents the combined High voltage (HV) for both enzymes where average 0 is Xen CPG2 and average 1 is Ps CPG2. Also the table shows the calculated protein secondary structure of Xen CPG2 and Ps CPG2 by CDNN deconvolution analysis using their CD spectral data.</p

Carboxypeptidase G2 activity is Zn^²⁺ dependent.

<p>MTX substrate solution and pure CPG2 in the presence of Zn²⁺ (star), control reaction of MTX substrate solution and buffer (filled triangle) and MTX substrate solution and pure CPG2 in the presence of Zn²⁺ and 10 mM EDTA (square) are shown. New isolated CPG2 is Zn²⁺ dependent.</p

Amino acids sequence alignment of glucarpidase genes.

<p>the uppermost sequence encodes the glucarpidase from <i>Pseudomonas sp</i>. Strain RS-16 (12), the lower sequences encode the new glucarpidases isolated from <i>Xenophilus azovorans SN213</i> and <i>Stenotrophomonas sp SA</i>, respectively (this work). Amino acid differences between the sequences are highlighted in red. Amino acids in blue are involved in the active site of the enzyme where two zinc ions and a bridging water molecule binds (30).</p

Antibody detection of newly isolated CPG2 relative to Ps CPG2.

<p><b>A.</b> Dot blot using anti His tag antibody and using anti Xen CPG2 antibody where 1, 2, and 3 are pure protein (Xen CPG2 and Ps CPG2) at concentrations (0.05, 0.1, and 0.2 mg/mL). <b>B.</b> Dot blot at different concentration of anti Xen CPG2 antibody where 1, 2, and 3 are blotting at dilutions 1:20 000, 1:10 000 and 1:3000 in blocking buffer. <b>C.</b> SDS-PAGE and Western blot analysis of the pure protein (Xen CPG2 and Ps CPG2) where M is PageRuler™ Unstained Protein Ladder (10–200 kDa), lanes 1, 2, 3 are 0.25, 0.1, and 0.05 mg/mL of Xen CPG2 and lanes 4, 5, 6 are the same series of protein concentrations of Ps CPG2.</p

MS of the protonated DAMPA is shown at 313.1m/z.

<p>a) DAMPA H+ peak is the product of folate hydrolysis by the isolated strain. b and c) P1, P2 are DMPA H+ produced by recombinant CPG2s, new and Ps CPG2 respectively and F1, F2 are intact folate isolated from the media of both recombinant enzymes.</p