12 research outputs found
Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye
To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8
Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye
To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8
Investigations into the Cytotoxic and Mutagenic Effects of DNA Adducts and DNA Structures on DNA Replication
Byproducts of normal cellular processes and environmental toxicants are capable of interacting with cellular DNA, producing a variety of chemical modifications. In addition, when duplex DNA becomes single-stranded, certain DNA sequences are capable of adopting non-B structures potentially causing genomic instability. To avoid activation of cellular checkpoints due to stalled replication forks, cells are equipped with translesion synthesis (TLS) polymerases capable of bypassing DNA lesions arising from these endogenous and exogenous sources. TLS polymerases are able to carry out lesion bypass because they lack the proofreading activity of replicative polymerases and possess larger active sites. In some cases lesion bypass carried out by these polymerases is accurate and efficient, while in other cases, TLS introduce mutations into the genome. Investigations into the cytotoxic and mutagenic effects DNA lesions and non-B structures have on DNA replication are needed to understand their role in cancer, aging and disease. In these studies, comprehensive experiments were conducted to examine the effect regioisomeric alkylated thymidine lesions have on DNA replication; specifically the role TLS polymerases play in processing these lesions. Produced as a result from exposure to tobacco smoke, alkylated thymidines were shown to be poorly repaired in cells and thus, likely to contribute the mutations detected in lung cancer patients. Using novel LC-MS/MS methods, alkylated thymidines were shown to be both blocking and highly mutagenic to most DNA polymerases in vitro, primarily introducing T&rarrC and T&rarrA mutations. On the other hand, endogenously produced N2-alkyl-2'-deoxyguanosine lesions were shown to be accurately and efficiently bypassed through the combination of two TLS polymerases; pol &kappa or pol &iota inserts the correct dCMP opposite the lesion, and pol &zeta extends past the lesion. In addition, cells deficient in pol &kappa and pol &iota showed elevated levels of G&rarrA and G&rarrT mutations, which were attributed to TLS carried out by pol &eta. Taking advantage of the competitive replication and adduct bypass (CRAB) assay, we also investigated the effect non-B, G-quadruplex (G4) structures have on DNA replication. The experiments revealed that two G4 sequences located in the promoter regions of the oncogene c-Kit and the proto-oncogene c-Myb were capable of blocking DNA replication. Additionally, E. coli TLS pols II and IV, along with E. coli DNA helicase RecD, were shown to be involved in resolving the c-Kit-G4 sequence
Replication across Regioisomeric Ethylated Thymidine Lesions by Purified DNA Polymerases
Causal
links exist between smoking cigarettes and cancer development.
Some genotoxic agents in cigarette smoke are capable of alkylating
nucleobases in DNA, and higher levels of ethylated DNA lesions were
observed in smokers than in nonsmokers. In this study, we examined
comprehensively how the regioisomeric <i>O</i><sup>2</sup>-, <i>N</i>3-, and <i>O</i><sup>4</sup>-ethylthymidine
(<i>O</i><sup>2</sup>-, <i>N</i>3-, and <i>O</i><sup>4</sup>-EtdT, respectively) perturb DNA replication
mediated by purified human DNA polymerases (hPols) η, κ,
and ι, yeast DNA polymerase ζ (yPol ζ), and the
exonuclease-free Klenow fragment (Kf<sup>–</sup>) of <i>Escherichia coli</i> DNA polymerase I. Our results showed that
hPol η and Kf<sup>–</sup> could bypass all three lesions
and generate full-length replication products, whereas hPol Îą
stalled after inserting a single nucleotide opposite the lesions.
Bypass conducted by hPol κ and yPol ζ differed markedly
among the three lesions. Consistent with its known ability to efficiently
bypass the minor groove <i>N</i><sup>2</sup>-substituted
2′-deoxyguanosine lesions, hPol κ was able to bypass <i>O</i><sup>2</sup>-EtdT, though it experienced great difficulty
in bypassing <i>N</i>3-EtdT and <i>O</i><sup>4</sup>-EtdT. yPol ζ was only modestly blocked by <i>O</i><sup>4</sup>-EtdT, but the polymerase was strongly hindered by <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT. LC–MS/MS
analysis of the replication products revealed that DNA synthesis opposite <i>O</i><sup>4</sup>-EtdT was highly error-prone, with dGMP being
preferentially inserted, while the presence of <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT in template DNA directed
substantial frequencies of misincorporation of dGMP and, for hPol
ι and Kf<sup>–</sup>, dTMP. Thus, our results suggested
that <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT
may also contribute to the AT → TA and AT → GC mutations
observed in cells and tissues of animals exposed to ethylating agents
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The roles of DNA polymerases Îş and Îą in the error-free bypass of N2-carboxyalkyl-2'-deoxyguanosine lesions in mammalian cells.
To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N(2)-modified 2'-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) and N(2)-carboxymethyl-2'-deoxyguanosine (N(2)-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N(2)-CEdG lesions. In contrast, no mutation was found after the N(2)-CEdG- and N(2)-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N(2)-CEdG and N(2)-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately
<i>In-Vitro</i> Replication Studies on <i>O</i><sup>2</sup>‑Methylthymidine and <i>O</i><sup>4</sup>‑Methylthymidine
<i>O</i><sup>2</sup>- and <i>O</i><sup>4</sup>-methylthymidine (<i>O</i><sup>2</sup>-MdT
and <i>O</i><sup>4</sup>-MdT) can be induced in tissues
of laboratory
animals exposed with <i>N</i>-methyl-<i>N</i>-nitrosourea,
a known carcinogen. These two <i>O</i>-methylated DNA adducts
have been shown to be poorly repaired and may contribute to the mutations
arising from exposure to DNA methylating agents. Here, <i>in
vitro</i> replication studies with duplex DNA substrates containing
site-specifically incorporated <i>O</i><sup>2</sup>-MdT
and <i>O</i><sup>4</sup>-MdT showed that both lesions blocked
DNA synthesis mediated by three different DNA polymerases, including
the exonuclease-free Klenow fragment of <i>Escherichia coli</i> DNA polymerase I (Kf<sup>–</sup>), human DNA polymerase κ
(pol Îş), and <i>Saccharomyces cerevisiae</i> DNA polymerase
η (pol η). Results from steady-state kinetic measurements
and LC-MS/MS analysis of primer extension products revealed that Kf<sup>–</sup> and pol η preferentially incorporated the correct
nucleotide (dAMP) opposite <i>O</i><sup>2</sup>-MdT, while <i>O</i><sup>4</sup>-MdT primarily directed dGMP misincorporation.
While steady-state kinetic experiments showed that pol Îş-mediated
nucleotide insertion opposite <i>O</i><sup>2</sup>-MdT and <i>O</i><sup>4</sup>-MdT is highly promiscuous, LC-MS/MS analysis
of primer extension products demonstrated that pol Îş favorably
incorporated the incorrect dGMP opposite both lesions. Our results
underscored the limitation of the steady-state kinetic assay in determining
how DNA lesions compromise DNA replication <i>in vitro</i>. In addition, the results from our study revealed that, if left
unrepaired, <i>O</i>-methylated thymidine lesions may constitute
important sources of nucleobase substitutions emanating from exposure
to alkylating agents
From proof of concept to the routine use of an automated and robust multi-dimensional liquid chromatography mass spectrometry workflow applied for the charge variant characterization of therapeutic antibodies
The identification and quantification of post-translational modifications (PTMs) is a crucial step required during the development of therapeutic proteins. In particular, the characterization of charge variants separated by cation exchange chromatography (CEX) is a tedious process commonly performed with an off-line manual fraction collection followed by peptide mapping. To improve the efficiency of this time-consuming approach, a generic on-line multi-dimensional LC/MS approach was developed for the characterization of various monoclonal antibody (mAb) isotypes and a bi-specific antibody (BsAb). Fractions collected from 1D CEX analysis were consecutively reduced on a 2D reversed phase liquid chromatography (RPLC) column (polyphenyl), digested within 1–2 min using a 3D immobilized trypsin cartridge, and finally the obtained peptides were separated on another 4D RPLC column (C18), and simultaneously identified with a Q Exactive™ mass spectrometer. 2D RPLC columns and 3D trypsin cartridges from different suppliers were compared, as well as the effects of reducing agents. The effect of 2D and 4D RPLC column temperature, and 2D RPLC column mass load were also systematically studied. Under optimal conditions, the multi-dimensional LC/MS system described in this paper is a robust tool for the on-line digestion of proteins and shows high repeatability. Similar levels of oxidation and deamidation were measured using the off-line and on-line approaches for the same stressed samples. The lower amounts of deamidation and isomerization measured at some asparagine and aspartic acid residues by the on-line approach compared to the manual off-line procedure suggest reduced artifacts using the on-line methodology. The multi-dimensional LC/MS described here enables fast, on-line, automated characterization of therapeutic antibodies without the need for off-line fraction collection and sample pre-treatment (manual approach). The entire workflow can be completed within less than one day, compared to weeks with the manual off-line procedure
Chemical Structure and Properties of Interstrand Cross-Links Formed by Reaction of Guanine Residues with Abasic Sites in Duplex DNA
A new
type of interstrand cross-link resulting from the reaction of
a DNA abasic site with a guanine residue on the opposing strand of
the double helix was recently identified, but the chemical connectivity
of the cross-link was not rigorously established. The work described
here was designed to characterize the chemical structure and properties
of dG–AP cross-links generated in duplex DNA. The approach involved
characterization of the nucleoside cross-link “remnant”
released by enzymatic digestion of DNA duplexes containing the dG–AP
cross-link. We first carried out a chemical synthesis and complete
spectroscopic structure determination of the putative cross-link remnant <b>9b</b> composed of a 2-deoxyribose adduct attached to the exocyclic <i>N</i><sup>2</sup>-amino group of dG. A reduced analogue of the
cross-link remnant was also prepared (<b>11b</b>). Liquid chromatography–tandem
mass spectrometric (LC-MS/MS) analysis revealed that the retention
times and mass spectral properties of synthetic standards <b>9b</b> and <b>11b</b> matched those of the authentic cross-link remnants
released by enzymatic digestion of duplexes containing the native
and reduced dG–AP cross-link, respectively. These results establish
the chemical connectivity of the dG–AP cross-link released
from duplex DNA and provide a foundation for detection of this lesion
in biological samples. The dG–AP cross-link in duplex DNA was
remarkably stable, decomposing with a half-life of 22 days at pH 7
and 23 °C. The intrinsic chemical stability of the dG–AP
cross-link suggests that this lesion in duplex DNA may have the power
to block DNA-processing enzymes involved in transcription and replication
Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate <i>in Vivo</i> Deamidation of Therapeutic Antibodies
Deamidation
of therapeutic antibodies may result in decreased drug
activity and undesirable changes in pharmacokinetics and immunogenicity.
Therefore, it is necessary to monitor the deamidation levels [during
storage] and after <i>in vivo</i> administration. Because
of the complexity of <i>in vivo</i> samples, immuno-affinity
capture is widely used for specific enrichment of the target antibody
prior to LC–MS. However, the conventional use of bead-based
methods requires large sample volumes and extensive processing steps.
Furthermore, with automation difficulties and extended sample preparation
time, bead-based approaches may increase artificial deamidation. To
overcome these challenges, we developed an automated platform to perform
tip-based affinity capture of antibodies from complex matrixes with
rapid digestion and peptide elution into 96-well microtiter plates
followed by LC–MS analysis. Detailed analyses showed that the
new method presents high repeatability and reproducibility with both
intra and inter assay CVs < 8%. Using the automated platform, we
successfully quantified the levels of deamidation of a humanized monoclonal
antibody in cynomolgus monkeys over a time period of 12 weeks after
administration. Moreover, we found that deamidation kinetics between <i>in vivo</i> samples and samples stressed <i>in vitro</i> at neutral pH were consistent, suggesting that the <i>in vitro</i> stress test may be used as a method to predict the liability to
deamidation of therapeutic antibodies <i>in vivo</i>