Anti-CEA fusion proteins(Cancer Research Technology Ltd.)

Protein vaccines may be a useful strategy for cancer immunotherapy because recombinant tumor antigen proteins can be produced on a large scale at relatively low cost and have been shown to be safe for clinical application. However, protein vaccines have historically exhibited poor immunogenicity; thus, an improved strategy is needed for successful induction of immune responses. TAT peptide is a protein transduction domain composed of an 11-amino acid peptide (TAT(47-57): YGRKKRRQRRR). The positive charge of this peptide allows protein antigen fused with it to improve cell penetration. Poly(I:C) is a synthetic double-stranded RNA that is negatively charged and favors interaction with the cationic TAT peptide. Poly(I:C) has been reported on adjuvant role in tumor vaccine through promotion of immune responses. Therefore, we demonstrated that vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) can induce anti-tumor immunity in a murine colorectal tumor model. Splenocytes from mice vaccinated with a mixture of TAT-CEA fusion protein and poly(I:C) effectively induced CEA-specific IFN-γ-producing T cells and showed cytotoxic activity specific for MC-38-cea2 tumor cells expressing CEA. Vaccine with a mixture of TAT-CEA fusion protein and poly(I:C) delayed tumor growth in MC-38-cea-2 tumor-bearing mice. Depletion of CD8(+) T cells and NK cells reversed the inhibition of tumor growth in an MC-38-cea2-bearing mice, indicating that CD8(+) T cells and NK cells are responsible for anti-tumor immunity by vaccine with a mixture of TAT-CEA fusion protein and poly(I:C). Taken together, these results suggest that poly(I:C) could be used as a potent adjuvant to induce the anti-tumor immunity of a TAT-CEA fusion protein vaccine in a murine colorectal tumor model.

Protein pharmacokinetic modulation is becoming an important tool in the development of biotherapeutics. Proteins can be chemically or recombinantly modified to alter their half-lives and bioavailability to suit particular applications as well as improve side effect profiles. The most successful and clinically used approach to date is chemical conjugation with poly(ethylene glycol) polymers (PEGylation). Here, therapeutic protein half-life can be increased significantly while retaining biological function, reducing immunogenicity and cross-reaction. Naturally occurring alternatives to such synthetic polymers could have major advantages such as lower side effects due to biodegradability and metabolism. Polysialic acid (PSA) has been investigated as a pharmacokinetic modulatory biopolymer with many successful examples in preclinical and clinical development. Single-chain Fvs (scFvs) are a choice antibody format for human therapeutic antibody discovery. Because of their small size, they are rapidly eliminated from the circulation and often are rebuilt into larger proteins for drug development and a longer half-life. Here we show that chemical polysialylation can increase the half-life of an antiplacental alkaline (PLAP) and anticarcinoembryonic antigen (CEA) scFv (F1 and MFE-23, respectively) 3.4-4.9-fold, resulting in a 10.6-15.2-fold increase in blood exposure. Amine-directed coupling of the MFE-23 scFv reduced its immunoreactivity 20-fold which was resolved by site-specific polysialylation through an engineered C-terminal thiol residue. The site-specifically polysialylated MFE-23 scFv demonstrated up to 30-fold improved tumor uptake while displaying favorable tumor:normal tissue specificity. This suggests that engineering antibody fragments for site-specific polysialylation could be a useful approach to increase the half-life for a variety of therapeutic applications.