CONSTRUCTION AND EVALUATION OF A VECTOR FOR USE IN A POLYNUCLEOTIDE VACCINE

Abstract

Polynucleotide vaccines (PNV) consist of plasmid DNA expression vectors that are generally administered by intramuscular injection and result in expression of a target protein. This expressed protein can be processed and presented to the immune system as an antigenic target. This type of immunization has been evaluated in numerous infectious disease model systems and has elicited immune responses including neutralizing antibodies, T lymphocyte proliferative responses, and T lymphocyte cytotoxic responses. The development of techniques which identify tumor associated antigens have extended the possible application of polynucleotide vaccination to anti-tumor immunomodulatory therapies. Current vectors being used for PNV strategies have design characteristics which pose potential difficulties for application of this new vaccine technique to anti-tumor therapies. Vectors with open reading frames, other than the desired one, may encode foreign polypeptides which have the potential to mask the immune response to subdominant tumor associated antigens (TAA). Successful anti-tumor immunotherapies will likely have to be individualized and directed against multiple TAAs which will require a vector with special cloning site characteristics. In light of these concerns, among others, a novel vector (pITL) was designed for particular application to anti-tumor PNV strategies. Standard molecular biology techniques were used to construct pITL. The vector pITL contains a portion of the RANTES (Regulated Upon Activation, Normal T Cell Expressed and Secreted) promoter which was found to be maximally functional in human skeletal muscle, a portion of the Col El origin of replication, the SupF gene as a selection marker, a stuffer region and a directional cloning site which can accept target sequences directly derived from individual tumor specimens by reverse transcriptase polymerase chain reaction (rtPCR) techniques, a 3' splice sequence, and poly A addition signal sequence. The constructed vector contains all the necessary elements to express the messenger RNA (mRNA) of a target antigen in a eukarotic cell. Advantages of pITL over existing PNV vectors, in addition to the cloning site, include; the absence of significant non-target sequence open reading frames, its limited size, and limited extraneous DNA. Polynucleotide anti-tumor vaccines made with this vector may consist of various plasmids each containing particular target sequences from an individual's tumor thus, generating a polyclonal, individualized anti-tumor immunotherapy. The functional capability of pITL, as an expression vector, was tested by inserting the coding sequence of a green fluorescent protein into the vector at the cloning site, in a manner analogous to potential tumor target antigens. This product was transfected into primary human skeletal muscle cells in vitro. Examination of these cells demonstrated the production of the green fluorescent protein thereby confirming appropriate vector function, ie. protein expression.