Immunization with a poly (lactide co-glycolide) encapsulated plasmid DNA expressing antigenic regions of HPV 16 and 18 results in an increase in the precursor frequency of T cells that respond to epitopes from HPV 16, 18, 6 and 11.

A phase II trial was conducted in subjects with human papillomavirus (HPV) associated high-grade cervical dysplasia testing the safety and efficacy of a microparticle encapsulated pDNA vaccine. Amolimogene expresses T cell epitopes from E6 and E7 proteins of HPV types 16 and 18. An analysis was performed on a subset of HLA-A2+ subjects to test whether CD8+ T cells specific to HPV 16, 18, 6 and 11 were increased in response to amolimogene immunization. Of the 21 subjects receiving amolimogene, 11 had elevated CD8+ T cell responses to HPV 16 and/or 18 peptides and seven of these also had increases to corresponding HPV 6 and/or 11 peptides. In addition, T cells primed and expanded in vitro with an HPV 18 peptide demonstrated cross-reactivity to the corresponding HPV 11 peptide. These data demonstrate that treatment with amolimogene elicits T cell responses to HPV 16, 18, 6 and 11.

Consecutive low doses of cyclophosphamide preferentially target Tregs and potentiate T cell responses induced by DNA PLG microparticle immunization.

Cyclophosphamide in combination with immunotherapeutic approaches preferentially impinges on T(reg) activity and allows for robust generation of T cell effectors. Reduced dosages of cyclophosphamide are necessary to restrict its cytotoxic effects to the negative regulatory cell populations while sparing effector lymphocytes. We investigated cyclophosphamide dosing in combination with ZYC300, a PLG-encapsulated plasmid DNA vaccine which encodes the cytochrome P450 family member, CYP1B1, a known human tumor-associated antigen. In mice, three consecutive, low doses of cyclophosphamide comprised a superior regimen in enhancing the magnitude, diversity of epitopes, and avidity to individual epitopes of specific T cell responses when compared to regimens that used either a single low or a single high dose. Consecutive low doses of cyclophosphamide predominantly targeted T(regs) while sparing overall T lymphocyte counts. Thus, we report the synergistic activity of pharmacologic T(reg) depletion with cyclophosphamide on quantitatively and qualitatively increasing T cell responses to a known human tumor-associated antigen.

In vivo electroporation enhances the potency of poly-lactide co-glycolide (PLG) plasmid DNA immunization.

Immunization with plasmid DNA that has been encapsulated in poly lactide-co-glycolide (PLG) microparticles targets the plasmid DNA to antigen presenting cells and elicits immune responses to the encoded antigen(s). Application of a series of electrical pulses (EPT) immediately following unformulated DNA injection enhances expression of the encoded antigen and increases immune responses. The combination of using EPT before or after PLG-encapsulated plasmid DNA immunization was tested to determine if enhanced immune responses would be generated. The results show that the combination lead to both enhanced expression of antigen and more robust T cell responses, even if EPT was applied prior to immunization. The data also demonstrate that recruitment of phagocytes to the injection site was markedly enhanced by EPT, and this resulted in an increase of the antigen expression levels in these cells. Co-administration of microparticles and EPT also effected localized necrosis of muscle fibers, caused persistent Th-1-modulated cytokine production, and lead to the release of two endogenous adjuvants, uric acid and HMGB1. In all, we describe that increased immunogenicity observed with the combination of PLG-encapsulated plasmid DNA microparticle with EPT was caused by an increase in the recruitment of antigen presenting cells which mediated a more robust T cell response than observed with immunization alone.

Targeting cytochrome P450 CYP1B1 with a therapeutic cancer vaccine.

The role that the immune system plays in limiting tumor formation and growth is becoming increasingly clear and passive immunotherapeutic approaches, such as the use of monoclonal antibodies, are now being successfully applied in clinical practice. Active immunization against tumors, however, has not yet been shown to have the same level of clinical efficacy. Two important reasons for this lack of efficacy have to do with the antigens being targeted, as well as the immunization approaches that have been tested. This review will highlight some of the requirements thought to be important for the successful development of an active immunization approach, with a focus on the ongoing development efforts for a novel agent targeting the cytochrome P450 family member, CYP1B1.

The Smu tandem repeat region is critical for Ig isotype switching in the absence of Msh2.

Deficiencies of the Msh2 protein or the Smu tandem repeat (SmuTR) sequences each reduce isotype switching in mice by about 2- to 3-fold. We find that switching in mice deficient for both Msh2 and SmuTR is nearly ablated. We propose that the SmuTR provides closely spaced cleavage sites that can undergo switch recombination independent of Msh2, whereas cleavages in sequences flanking the SmuTR require Msh2 processing to allow recombinational joining. We also find that changes in Smu sequences alter the focus of switch junctions within Sgamma sequences, indicating that sequences of switch regions act together in the choice of switch recombination junctions. These findings help to explain the conservation of tandemly repeated switch regions associated with heavy chain constant genes in species capable of switching.

Repeated immunization with plasmid DNA formulated in poly(lactide-co-glycolide) microparticles is well tolerated and stimulates durable T cell responses to the tumor-associated antigen cytochrome P450 1B1.

Injection of microparticle-encapsulated DNA elicits immune responses to plasmid-encoded antigens in mice and humans. Cytochrome P450 CYP1B1 (CYP1B1) is a member of the CYP1 P450 enzyme family that is overexpressed in a variety of solid tumors. The work described herein was performed to study the kinetics of stimulating T cell responsiveness with an encapsulated DNA encoding CYP1B1 and provides support for the clinical development of this formulation. Immunization of HLA-A2/Kb transgenic mice with human CYP1B1 encoding plasmid DNA formulated in poly(lactide-co-glycolide) (PLG) microparticles elicits CD8+ T cells that respond to human CYP1B1-positive target cells. The duration of the immune response, the effect on the immune response of multiple injections, and the safety of repeated injections were studied. These results show that the PLG-encapsulated DNA therapeutic elicits durable immune responses to CYP1B1, the responses are dependent on repeat immunization, and that the formulation is well tolerated.