Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.

Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products. Eleven cysteine mutants were designed based on a structure model of the TSH-TSH receptor (TSHR) complex to create unique conjugation sites on both α and ß subunits for site-specific conjugation. Sequential screening of mutant expression level, oligomerization tendency, and conjugation efficiency resulted in the identification of the αG22C rhTSH mutant for stable expression and scale-up PEGylation. The introduced cysteine in the αG22C rhTSH mutant was partially blocked when isolated from conditioned media and could only be effectively PEGylated after mild reduction with cysteine. This produced a higher reaction yield, ~85%, for the monoPEGylated product. Although the mutation had no effect on receptor binding, PEGylation of αG22C rhTSH led to a PEG size-dependent decrease in receptor binding. Nevertheless, the 40 kDa PEG αG22C rhTSH showed a prolonged duration of action compared to rhTSH in a rat pharmacodynamics model. Reverse-phase HPLC and N-terminal sequencing experiments confirmed site-specific modification at the engineered Cys 22 position on the α-subunit. This work is another demonstration of successful PEGylation of a cysteine-knot protein by an engineered cysteine mutation.

Site-Specific Antibody Conjugation to Engineered Double Cysteine Residues.

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMABTM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy.

Engineered Fc-glycosylation switch to eliminate antibody effector function.

Antibodies mediate effector functions through Fcγ receptor (FcγR) interactions and complement activation, causing cytokine release, degranulation, phagocytosis, and cell death. They are often undesired for development of therapeutic antibodies where only antigen binding or neutralization would be ideal. Effector elimination has been successful with extensive mutagenesis, but these approaches can potentially lead to manufacturability and immunogenicity issues. By switching the native glycosylation site from position 297 to 298, we created alternative antibody glycosylation variants in the receptor interaction interface as a novel strategy to eliminate the effector functions. The engineered glycosylation site at Asn298 was confirmed by SDS-PAGE, mass spectrometry, and X-ray crystallography (PDB code 6X3I). The lead NNAS mutant (S298N/T299A/Y300S) shows no detectable binding to mouse or human FcγRs by surface plasmon resonance analyses. The effector functions of the mutant are completely eliminated when measured in antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. In vivo, the NNAS mutant made on an antibody against a human lymphocyte antigen does not deplete T cells or B cells in transgenic mice, in contrast to wild-type antibody. Structural study confirms the successful glycosylation switch to the engineered Asn298 site. The engineered glycosylation would clash with approaching FcγRs based on reported Fc-FcγR co-crystal structures. In addition, the NNAS mutants of multiple antibodies retain binding to antigens and neonatal Fc receptor, exhibit comparable purification yields and thermal stability, and display normal circulation half-life in mice and non-human primate. Our work provides a novel approach for generating therapeutic antibodies devoid of any ADCC and CDC activities with potentially lower immunogenicity.

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFß1 antibody.

Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFß1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFß1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFß1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFß1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A.

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.

Carbohydrate-mediated polyethylene glycol conjugation of TSH improves its pharmacological properties.

Thyrogen (thyrotropin alfa for injection), recombinant human TSH (rhTSH), has been successfully used to enhance diagnostic radioiodine scanning and thyroglobulin testing in the follow-up of patients with thyroid cancer and as an adjunctive treatment for radioiodine thyroid remnant ablation. However, the short half-life of rhTSH in the circulation requires a multidose regimen. We developed novel sialic acid-mediated and galactose-mediated conjugation chemistries for targeting polyethylene glycol (PEG) to the three N-linked glycosylation sites on the protein, to prolong plasma half-life by eliminating kidney filtration and potential carbohydrate-mediated clearance. Conjugates of different PEG sizes and copy numbers were screened for reaction yield, TSH receptor binding, and murine phamacokinetics/pharmacodynamics studies. The best performing of these products, a 40-kDa mono-PEGylated sialic acid-mediated conjugate, exhibited a 3.5-fold longer duration of action than rhTSH in rats, as a 5-fold lower affinity was more than compensated by a 23-fold extension of circulation half-life. Biochemical characterization confirmed conjugation through the sialic acids. Correlation of PEG distribution on the three N-linked glycosylation sites and the PEG effect on receptor binding supported the previously reported structure-function relationship of rhTSH glycosylation. This long-acting rhTSH has the potential to significantly improve patient convenience and provider flexibility while reducing potential side effects associated with a sudden elevation of serum TSH.

In vitro and in vivo evaluation of a non-carbohydrate targeting platform for lysosomal proteins.

Lysosomal storage diseases arise from a genetic loss-of-function defect in enzymes mediating key catabolic steps resulting in accumulation of substrate within the lysosome. Treatment of several of these disorders has been achieved by enzyme replacement therapy (ERT), in which a recombinant version of the defective enzyme is expressed in vitro and administered by infusion. However, in many cases the biodistribution of the administered protein does not match that of the accumulated substrate due to the glycosylation-mediated clearance of the enzymes from circulation, resulting in poor or absent substrate clearance from some tissues. To overcome this limitation, we have evaluated several peptide-based targeting motifs to redirect recombinant human alpha-galactosidase (rhalphaGal) to specific receptors. A reversible thiol-based PEGylation chemistry was developed to achieve multivalent peptide display with lysosomal release. In vitro, cell uptake was peptide dependent and independent of the normal mannose-6-phosphate receptor mediated pathway. Surprisingly, despite increased plasma half-life and decreased liver uptake, none of the peptide conjugates showed significantly altered biodistribution in alphaGal-knockout mice. This suggests that these peptide-based targeting motifs are unlikely to provide substantial therapeutic benefit likely due to the complexity of factors affecting PK and biodistribution.