Polymer nanoparticles for immunotherapy from encapsulated tumor-associated antigens and whole tumor cells.

Encapsulation of tumor-associated antigens (TAA) in polymer nanoparticles is a promising approach to increasing the efficiency of antigen (Ag) delivery for antitumor vaccines. We optimized a polymer preparation method to deliver both defined tumor-associated proteins and the complex mixtures of tumor Ags present in tumors. Tumor Ags were encapsulated in a biodegradable, 50:50 poly(D,L-lactide co-glycolide) copolymer (PLGA) by emulsification and solvent extraction. Two particular Ags were studied, gp100 (a melanoma-associated antigen) and ovalbumin (OVA), as well as mixtures of proteins and lysates of tumor cells. The efficiency of encapsulation was measured by protein assays of dissolved nanoparticles. Ag stability after release from nanoparticles was verified by SDS-acrylamide gel electrophoresis and Western blot analysis. Molecular weight and protein loading interact to define the encapsulation efficiency and release rate of nanoparticles formulated from 50:50 PLGA. A midrange molecular weight polymer had more desirable release properties at 100 mg/mL than at 50 mg/mL protein loading, indicating the need for optimization of nanoparticle formulation for preparations with different particle loadings. Mixtures of proteins derived from cell lysates were reliably encapsulated into nanoparticles, which released the spectrum of proteins contained in lysates. Antigenic proteins were co-encapsulated with cell lysate and released from nanoparticles; these Ags retained their antigenicity and functioned better than soluble Ags when tested in in vitro assays of T cell cytokine formation and in vivo tumor vaccination challenge.

Photoactivated 8-methoxypsoralen treatment causes a peptide-dependent increase in antigen display by transformed lymphocytes.

Ex vivo exposure of malignant human T cells to photoactivated 8-methoxypsoralen (8-MOPa), followed by their i.v. return, appears to vaccinate patients against tumor-associated antigens of cutaneous T cell lymphoma in a procedure termed photopheresis. The molecular basis of this Food and Drug Administration-approved therapy, administered in 100 centers worldwide, is unclear. Most of the attention to the mechanism of action of the drug has focused on its capacity to form covalent cross-links with pyrimidine bases of DNA, thereby inhibiting cellular proliferation. Because immunologic factors appear to be important in the clinical response and could potentially serve as a model for immunotherapy of other malignancies, we explored the possibility that 8-MOP-treated cells display increased quantities of antigenic peptides at their cell surface. In this work, human B-lymphoblastoid tissue culture lines were exposed to 8-MOPa and expression of cell surface class I major histocompatibility complex proteins assessed, since CD8 T cells recognize antigenic moieties in the context of class I molecules. A peak 200-300% increase in MHC class I expression in 8-MOPa-treated cells occurred at 20 hr. 8-MOPa was far more effective in inducing this increase in class I MHC than other modalities, including mitomycin C, gamma-irradiation, ultraviolet B or heat or cold shock. This increase in surface class I MHC molecules appears to be driven by the degradation of cytoplasmic proteins into small peptides, followed by the transport of these peptides to MHC class I molecules in the endoplasmic reticulum. The data suggest that 8-MOPa treatment may augment the immunogenicity of tumor and/or antigen-presenting cells by enhancing processing and transport of class I MHC antigenic peptides.

Variation in genomic Alu repeat density as a basis for rapid construction of low resolution physical maps of human chromosomes.

Human DNA restriction fragments containing high numbers of Alu repeat sequences can be preferentially detected in the presence of other human DNA restriction fragments in DNA from human: rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave mammalian DNA infrequently. This ability to lower the observed human DNA complexity allowed us to develop an approach to order rapidly somatic hybrid cell lines retaining overlapping human genomic domains. The ordering process also generates a relative physical map of the human fragments detected with Alu probe DNA. This process can generate physical mapping information for human genomic domains as large as an entire chromosome (100,000 kb). The strategy is demonstrated by ordering Alu-detected NotI fragments in a panel of mouse: human hybrid cells that span the entire long arm of human chromosome 17.