Pre‐clinical Development of rhTIMP‐2 as a Novel Anti‐Cancer Therapeutic
Authors: Chowdhury Ananda , Wei Beiyang , Yamada Yuji , Schneider Joel P. , Stetler‐Stevenson William G. 
Published: November 2023

BACKGROUND

Tissue Inhibitor of Metalloprotease ‐2 (TIMP‐2), is a 21.75 kDa protein that has unique anti‐cancer properties involving direct Metalloprotease (MMP) inhibition, as well as anti‐angiogenic agent and direct anti‐proliferative effects on tumor cells. TIMP‐2 is a promising biologic candidate for further pre‐clinical studies. Herein we report bioprocess development, analytical characterization, in‐vitro analysis of tumor cell growth and formulation development of recombinant human TIMP‐2.

METHODS

TIMP‐2‐His6 cDNA was codon optimized using a proprietary algorithm (NEO BioLab, Cambridge, MA), the cDNA synthesized, transfected to HEK 293F cells and grown in optimized conditions up to 1L scale. rTIMP‐2 was captured by Ni2+ IMAC, polished by preparative RP‐HPLC and formulated in pure MQ water. rTIMP‐2‐His6 protein was analyzed by SDS‐PAGE, Western Blotting and ESI‐TOF mass spec and circular dichorism analysis. All production batches were Endotoxin and MAP tested. In‐vitro MMP‐2 inhibitory and A549 cancer cell growth inhibitory assay was done by colorimetric plate‐based methods. rTIMP‐2 was formulated in peptide hydrogels and release and efficacy was studied in‐vitro.

RESULTS

Codon optimization and stable selection resulted in about 7‐fold increase in rTIMP‐2 His6 expression compared to identical culture with WT rTIMP‐2 cDNA transfected HEK‐293F cells. Purification by IMAC Ni2+ chromatography and RP‐HPLC yielded TIMP‐2‐His6 with 96.21% and 81.75% recovery, respectively. Purified rTIMP‐2‐His6 exhibited single band SDS‐PAGE gel and by single peak in ESI‐TOF mass spec.; and was immuno‐reactive against anti‐TIMP‐2 and anti‐poly‐histidine antibodies. Circular Dichorism spectra indicated high degree of β‐pleated sheet in rTIMP‐2 2° structure. All production batches of the rTIMP‐2‐His6 were negative in MAP testing with Endotoxin levels at 3.702 EU/ml. TIMP‐2‐His6 effectively displayed dose dependent inhibition of MMP‐2 (type IV collagenase) activity with an IC50 of ~10nM; with no MMP‐2 inhibitory observed with the Ala+TIMP‐2‐His6 derivative (N‐terminal Ala appended TIMP‐2‐His6 with no MMP inhibitory activity). A549 lung cancer cell lines demonstrated 1.5 fold‐increased proliferation with 100‐ng/ml rEGF treatment. Pre‐treatment with 50 nM rTIMP‐2 suppressed EGF‐induced growth of A549 cells in‐vitro by ~90%. rTIMP‐2 packaged into anionic peptide hydrogel AcVES 3 (net charge: −5) and cationic HLT‐2 (net charge: +5) demonstrated extended release over a period of 30 days. Released rTIMP‐2 exhibited preserved MMP‐2 inhibitory activity.

SUMMARY AND FUTURE DIRECTIONS

We have developed a robust and scalable bioprocess method for rTIMP‐2‐His6 production and performed initial analytical characterization of this recombinant protein. Efficacy of the rTIMP‐2 was investigated in‐vitro for MMP‐2 inhibitory and independent effects. Further in‐depth dosing, PK‐PD, safety‐efficacy and ADME studies will soon be initiated. We plan to study bioavailability and distribution of Zr89‐labeled rTIMP‐2‐His6 by MRI imaging and continued protein engineering and formulation development of rTIMP‐2.

Support or Funding Information

This work was funded by the NCI Center for Cancer Research Intramural Research Program Grant # ZIA SC009179‐2.