Recombinant human Tissue Inhibitor of Metalloprotease −2; a novel anti‐cancer therapeutic
Authors: Chowdhury Ananda , Wei Beiyang , Yamada Yuji , Castro Nadia , Salomon David , Schneider Joel P. , Stetler‐Stevenson William G. 
Published: November 2023

INTRODUCTION

Tissue inhibitor of Metalloprotease‐2 (TIMP‐2), a soluble endogenous ~22 kDa metalloprotease inhibitor, exhibits anti‐cancer properties by metalloprotease inhibition in tumor microenvironment, direct receptor‐mediated growth inhibition, increased chemo –sensitivity and decreased cell motility. Herein we report a complete production‐characterization process, biophysical analysis, anti‐MMP activity and anti‐proliferative activity of rhTIMP‐2 in cell lines and mouse models of breast and lung cancer. Further we report the development and characterization of rhTIMP‐2 extended release peptide hydrogels and desferoxamine(des) labelled rhTIMP‐2 for dosage form and PET scan probe development respectively.

METHODS

Human TIMP‐2 cDNA with 6xHistidine epitope tag was codon optimized, transfected to HEK 293F cells and grown in optimized conditions up to 5L scale in wave bags. rhTIMP‐2 was captured by Ni2+ IMAC, polished by preparative RP‐HPLC and formulated in pure MQ water. rhTIMP‐2 was analyzed by SDS‐PAGE, Western Blotting, ESI‐TOF mass spec and CD spectroscopy. rhTIMP‐2 was also studied by 2D HSQC NMR and by Isothermal titration calorimetry for binding to integrin alpha‐3: beta‐1 receptor ectodomain both at 37°C. 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. In‐vivo rhTIMP‐2 was administered i.p. to athymic nude mice bearing breast cancer tumors against HBSS controls.

RESULTS

Codon optimization and stable clonal selection resulted in about 7‐fold increase in rhTIMP‐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 greater than 90% (96.21% recovery) and 99% (81.75% recovery) percentage purities respectively. Purified rTIMP‐2 exhibited single band and peak in SDS‐PAGE gel and by ESI‐TOF mass spec.; and was immuno‐reactive against anti‐TIMP‐2 and poly‐histidine antibodies. Circular Dichorism spectra indicated high degree of β‐pleated sheet in rhTIMP‐2 2° structure. 2D NMR of rhTIMP‐2 His6 in natural abundance indicated a well folded monodisperse rhTIMP‐2 with ~10–15% population of alternate rhTIMP‐2 species. All production batches of the rTIMP‐2 were negative in MAP testing with Endotoxin levels at 3.702 EU/ml (<10 EU/ml acceptable in cGMP). TIMP‐2 effectively displayed dose dependent inhibition of MMP‐2 (type IV collagenase) activity with an IC50 of < 50nM; with no MMP‐2 inhibitory observed in the Ala+TIMP‐2 (N′ terminal Ala appended TIMP‐2 with no MMP inhibitory activity). In‐vitro pre‐treatment with 50 nM rTIMP‐2 in gelatin coated plates suppressed rhEGF induced growth of A549 (lung cancer) and JygM(A) (triple negative breast cancer cell lines) to statistically significant levels. Athymic nude mice were injected with JygM(A) breast cancer cell lines under the mammary fat pads. Mice with palpable tumors were randomized and treated with 3 ug rhTIMP‐2/day/mouse intra‐peritonealy up to a period of 35 days that lead to ~30% reduction of tumor volume compared to the HBSS (vehicle control). We have also evaluated the effects of encapsulation and delivery of rhTIMP‐2 from cationic peptide hydrogel–which shows extended release to up to 28 days with little or no loss of its biologic activity. Similarly, little or no loss of biological activity was observed upon desferoxamine(des) conjugation to rhTIMP‐2. Zn89 attachment to the des subunit has been completed and the Zn89‐rhTIMP‐2 is being used for uptake, distribution and metabolism studies.

SUMMARY AND FUTURE DIRECTIONS

Conclusively we have developed a robust, scalable bioprocess method for rhTIMP‐2 (and analogs) production, characterization to support the availability and analysis through pre‐clinical development. In‐vitro and in‐vivo results shows reduction in cell proliferation and tumor volume in breast, lung cancer cell lines and tumors. Extended release dosage forms and Zn89 labelled TIMP‐2 are now being used to characterize the PK‐PD and distribution of the biologic. With all supportive data, reagents and in‐depth biophysical analysis we present a comprehensive report of rhTIMP‐2 as a novel biologic therapeutic for cancer treatment and we plan to move forward towards continued pre‐clinical/clinical development and protein engineering.

Support or Funding Information

This work is supported by center for cancer research, intramural research program, NCI/NIH research grants ZIA BC011204 & ZIA SC 009179 to WGSS