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Swelling of polymeric hydrogels is sensitive to their cross-link densities. Here, we exploit this principle to prepare self-rupturing gels which are based on a commonly-used, nontoxic, and inexpensive polyelectrolyte, poly(acrylic acid), and are prepared through a simple and low-cost polymerization-based technique. The self-rupture of these covalently cross-linked gels is achieved by preparing them to have highly nonuniform cross-link densities. This heterogeneity in cross-linking leads to highly nonuniform swelling, which generates stresses that are high enough to induce gel rupture. The time required for this rupture to occur depends on the difference in the cross-link densities between the adjoining gel regions, gel size, order in which the variably cross-linked gel portions are synthesized, and on the ambient pH and ionic strength. Furthermore, when these self-rupturing gels are prepared to have liquid-filled (capsule-like) morphologies, they can act as timed/delayed release devices. The self-rupture of these capsules provides a burst payload release after a preprogrammed delay, which is on the timescale of days and can be easily tuned by varying the rupture time, i.e., by varying either the cross-link nonuniformity or the pH and ionic strength of the release media.

Wild-type Wilms tumor gene WT1 is highly expressed not only in hematopoietic malignancies, including leukemia and myelodysplastic syndromes (MDS), but also in various kinds of solid tumors. Human cytotoxic T lymphocytes (CTLs) which could specifically lyse WT1-expressing tumor cells with HLA class I restriction were generated in vitro. We have also demonstrated that mice immunized with the WT1 peptide or WT1 cDNA rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to normal organs which physiologically expressed WT1 in prophylactic and therapeutic models. Furthermore, we and others detected IgM and IgG WT1 antibodies in the patients with hematopoietic malignancies, indicating that WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing WT1- specific cellular immune responses were elicited in the patients. CD8 WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of the findings mentioned above, we performed a phase I clinical trial of WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that WT1 peptide cancer vaccine had efficacy in the clinical setting, because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of TAA-derived cancer vaccine may be enhanced by combination with stronger adjuvants, helper peptide, or conventional treatments such as molecular-target-based drugs.

Wild-type Wilms' tumor gene WT1 is expressed at high levels not only in most of acute myelocytic, acute lymphocytic, and chronic myelocytic leukemia, but also in various types of solid tumors including lung cancer. We tested the ability of the gene product (WT1) to serve as a target antigen for tumor -specific immunotherapy both in human in vitro system and mouse in vivo system. In the latter, we can evaluate the efficacy and the side effects of WT1 vaccination in vivo. In the human in vitro system, two WT1 peptides that contain HLA-A2.1 binding anchor motifs were determined to bind to HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from an HLA-A2.1-psitive donor were repeatedly stimulated in vitro with TAP-deficient T2 cells pulsed with each of these two peptides, and CD8-positive cytotoxic T lymphocytes (CTLs) that specifically lyse WT1-expressing, HLA-A2.1-positive tumor cells were induced. Other groups also have succeeded in generating CTLs which specifically lyse WT1-expressing leukemia cells, and which do not inhibit colony-formation of normal hematopoietic cells that express WT1 at physiological levels. In the mouse in vivo system, immunization of C57BL / 6 mice with one WT1 peptide with relatively high binding affinity for H-2D b molecules, which contain H-2D b binding anchor motifs, induced CTLs, which specifically lysed WT1-expressing tumor cells in an H-2D b -restricted manner. Furthermore, mice immunized with the WT1 peptide (peptide vaccination) or WT1 cDNA (DNA vaccination) rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to WT1-expressing normal organs by the induced CTLs. The WT1 protein has been identified as a novel tumor antigen and recent investigations provide a rationale for developing WT1-based adoptive T cell therapy and vaccination against various kinds of malignant neoplasms.

Major histocompatibility complex (MHC) class I molecules are cell-surface glycoproteins that bind peptides and present them to T cells. The formation of a peptide-MHC complex is the initial step in specific, T cell-mediated immune responses. But, unlike other receptor-ligand systems, peptides are essential for a stable conformation of the MHC proteins. To investigate the contribution of every amino acid of octapeptides to the stability and antigenic integrity of MHC proteins, complex octapeptide libraries with one defined amino acid and mixtures of 19 amino acids in the remaining seven positions were synthesized and tested for their capacity to stabilize the conformation of the mouse MHC class I molecule H-2Kb. Peptide transporter-deficient RMA-S cells were employed in this study. Amino acid preferences found for the eight sequence positions reveal constitutional, volumetric, and steric constraints that govern peptide selection by MHC molecules. The pattern of amino acid preferences indicates that the peptides behave as integral parts of the MHC proteins and follow rules established for the interrelationship of primary sequence and the conformation and stability of proteins in general.

We previously isolated from mouse spleen an octapeptide (LSPFPFDL) that in association with the class I major histocompatibility complex protein Ld is recognized by the antigen-specific receptor of an alloreactive CD8+ T-cell clone (2C). Guided by an assay dependent upon the same 2C T-cell receptor, we have now isolated from the same source another naturally occurring peptide. The second peptide (VAITRIEQLSPFPFDL) includes the entire octapeptide sequence and preliminary evidence suggests that it may be a natural precursor of the octapeptide. On finding extensive sequence homology between the 16-mer and part of human 2-oxoglutarate dehydrogenase, we determined the cDNA sequence of mouse 2-oxoglutarate dehydrogenase and found that the deduced amino acid sequence matches precisely the two naturally occurring peptides, indicating their origin by cellular processing of this ubiquitous self protein.

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.

Complex synthetic peptide libraries with defined amino acids in one or more positions of the H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes SIINFEKL and RGYVYQGL and mixtures of 19 amino acids in the remaining positions were used to analyze the structural requirements of peptide binding to MHC class I molecules and antigen recognition by CTLs. This approach provides means to assess semiquantitatively the contribution of every amino acid to the binding of peptides to major histocompatibility complex (MHC) molecules without biases introduced by naturally processed peptides. Primary and secondary anchor residues were defined for their major contribution to the binding efficiency of the peptides. In contrast to primary anchors, secondary anchor amino acids vary greatly in their side chains and position in the sequences. All amino acids in the octapeptide sequences were found to exhibit positive or negative influences on binding to the MHC molecules and on recognition of the resulting complexes by CTLs. Strong interdependence of the effects of the individual residues in the epitope sequences was demonstrated. CTL responses to peptide libraries were suppressed when residues were introduced; however, they were augmented when the critical residues for T cell recognition were fixed, suggesting a potential use of the peptide libraries for defining epitope sequences in general.