Improving Circulation Half-Life of Therapeutic Candidate N-TIMP2 by Unfolded Peptide Extension

Abstract

Matrix Metalloproteinases (MMPs) are drivers of many diseases including cancer and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are human proteins that inhibit MMPs and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties of a drug candidate, such as complete MMP inhibition, low toxicity and immunogenicity, high tissue permeability and others. A major challenge with TIMPs, however, is their formulation and delivery, as these proteins are quickly cleared from the bloodstream due to their small size. In this study, we explore a new method for plasma half-life extension for the N-terminal domain of TIMP2 (N-TIMP2) through appending it with a long intrinsically unfolded tail containing a random combination of Pro, Ala, and Thr (PATylation). We design, produce and explore two PATylated N-TIMP2 constructs with a tail length of 100- and 200-amino acids (N-TIMP2-PAT₁₀₀ and N-TIMP2-PAT₂₀₀, respectively). We demonstrate that both PATylated N-TIMP2 constructs possess apparent higher molecular weights compared to the wild-type protein and retain high inhibitory activity against MMP-9. Furthermore, when injected into mice, N-TIMP2-PAT₂₀₀ exhibited a significant increase in plasma half-life compared to the non-PATylated variant, enhancing the therapeutic potential of the protein. Thus, we establish that PATylation could be successfully applied to TIMP-based therapeutics and offers distinct advantages as an approach for half-life extension, such as fully genetic encoding of the gene construct, mono-dispersion, and biodegradability. Furthermore, PATylation could be easily applied to N-TIMP2 variants engineered to possess high affinity and selectivity toward individual MMP family members, thus creating attractive candidates for drug development against MMP-related diseases.

Figure 1.. Preparation of N-TIMP2 tagged and PATylated constructs.

(A) Schematic representation of expressed constructs of N-TIMP2 (top) and PATylated constructs (bottom). The N-TIMP2 domain is shown in cyan, the internal FLAG-tag in red, c-myc tag in green and 6xHis tag in blue. (B) Ten model decoys of N-TIMP2-PAT₁₀₀ fused to a 100 amino acid PAT tail as modeled by AlfaFold. N-TIMP2 is shown in cyan, PAT fusion in orange, and FLAG-tag in red. The structures of N-TIMP2-PAT₂₀₀ looked generally similar to those of N-TIMP2-PAT₁₀₀.

Figure 2:. Characterization of N-TIMP2 and the PATylated variants.

(A) SDS-PAGE under non-reducing conditions of N-TIMP-2 and two preps of N-TIMP2-PAT₂₀₀ (left) and N-TIMP2-PAT₁₀₀ (right). (B) CD spectra of the three constructs: N-TIMP2 (blue), N-TIMP2-PAT₁₀₀ (orange) and N-TIMP2-PAT₂₀₀ (green). Each curve is an average of 5 runs. (C) Inhibitory activity of the N-TIMP2 (blue), N-TIMP2-PAT₁₀₀ (orange) and N-TIMP2-PAT₂₀₀ (green) against MMP-9. Fraction of MMP-9 activity is plotted vs. concentration of added N-TIMP2-based inhibitor. The data were fitted to eq. 1 to obtain ${\text{K}}_{\mathrm{i}}^{\mathrm{app}}$.

Figure 3.. Pharmacokinetic study in mice demonstrates significant extension of plasma half-life for N-TIMP2-PAT₂₀₀.

(A) A schematic diagram of the ELISA setup for the detection of recombinant N-TIMP2-based constructs from plasma. An anti-C-myc tag antibody on the surface of a MaxiSorp^™ plate is used for capturing the N-TIMP2 constructs from plasma. The protein is detected by a biotinylated anti-FLAG-tag antibody, which binds to HRP-conjugated streptavidin that in turn catalyzes the oxidation of the TMB substrate to the measurable blue colored product. The blue, green and red regions highlighted on the N-TIMP2 protein respectively represent the 6xHIS tag, C-myc tag and FLAG tag. (B) Schematic diagram of pharmacokinetic study design, in which each N-TIMP2 variant was tested using 6 mice grouped into two sub-cohorts of 3 mice each, which were bled at alternating time points following the initial baseline bleed and IP injection of N-TIMP proteins. (C) Elimination half-life of N-TIMP2 (black triangles versus N-TIMP2-PAT₂₀₀ (green circles). Plot of average ± SEM (n=3) of log10 N-TIMP2 concentration vs time, modeled with a 2-phase decay with least-squares nonlinear fit for half-life determination. Statistical significance assessed via a sum-of-squares F test where H0: K_slow N-TIMP2-PAT₂₀₀ = K_slow N-TIMP2; Ha: K_slow N-TIMP2-PAT₂₀₀ ≠ K_slow N-TIMP2. α=0.05, p<0.0001.