Promosome LLC, Scientific PresentationScientific Presentation

Expression Technologies for

Enhancing

Protein Production

Promosome’s technologies enable increased protein production by increasing translation efficiencies

1) initiation

2) elongation

3) termination

Background: Translation involves 3 stages

Translation initiation involves ribosomal recruitment and recognition of a start site

Ribosomal recruitment can occur at the m7G cap

via various eukaryotic initiation factors (eIFs)

Ribosomal recruitment can also occur at an internal ribosome entry site (IRES)

An initiation codon is then recognized and aribosomal complex forms at this site

Translation Enhancer Elements (TEEs)

Translational Enhancer Elements (TEEs)

TEEs are short nucleotide sequences that increase translation efficiency

Some function as ribosomal recruitment sites

Powerful synthetic translational enhancers have been generated by linking together five or more individual TEEs

Gtx TEE can dramatically boost protein synthesisin some cell lines without increasing mRNA levels

Activities of synthetic translational enhancers with 5 copies of individual TEEs in CHO-DG44 cells

TEEs can enhance production of recombinant antibodies

Expression of this human antibody was enhanced maximally by TEEs in the 5’ leader of the L chain cistron

In this example, secretion of L chain dimers was also observed when L chain expression was enhanced

≈6-fold increase

TEEs enhance expression of a hard-to-express mucin-Fc fusion protein

(Recopharma)

≈2-3-fold increase

RESCUE

RESCUE

Increases protein production by eliminating negative features in mRNAs that can decrease translation initiation

Primarily involves alterations in mRNA coding regions

- but does not involve altering codon utilization, minimizing potential alterations to protein secondary structure

Applicable to multiple cell types

RESCUE examples in CHO cells

CAT enzyme (CHO-DG44) IgG L and H chain (CHO-K1)

EPO (CHO-DG44)

RESCUE-modification of mucin-Fc fusion protein (Recopharma)

≈7-8 fold increase

Summary

TEEsShort sequences identified for ability to enhance protein expression in CHO cells

Enable more protein to by synthesized per unit mRNA

May avoid limitations associated with high mRNA levels and enable high expressing cell lines without multiple chromosomal copies

RESCUEA novel technology is based on our new ideas about translation initiation

Potential advantages include increased protein expression and improved cell physiology

This technology appears to be broadly applicable to many cell types

RESCUE is expected to be compatible with other technologies