Functional human Th17 clones with WT1-specific helper activity.

Th17 plays important roles in the pathogenesis of various inflammatory and autoimmune diseases. Although the importance of Th17 in tumor immunity has also been suggested, precise roles of tumor-associated antigen-specific Th17 still remain poorly understood, especially in humans. We previously identified WT1332, a 16-mer helper epitope derived from tumor-associated antigen Wilms' tumor gene 1 (WT1) product, and WT1332-specific Th1 clones were established. In the present study, WT1-specific Th17 clones were established by the stimulation of peripheral blood mononuclear cells with the WT1332 helper peptide under human Th17-polarizing conditions. The WT1-specific Th17 clone exhibited the helper function for proliferation of conventional CD4(+) T cells in the antigenic stimulation-specific manner. This is the first report of establishment of functional Th17 clones with both antigen (WT1332) specificity and antigen-specific helper activity. Th17 clones established here and the method to establish antigen-specific Th17 clones should be a useful tool to further analyze the roles of human Th17 in tumor immunity.

CD22-antagonists with nanomolar potency: the synergistic effect of hydrophobic groups at C-2 and C-9 of sialic acid scaffold.

In earlier studies, we identified the C-9 amido derivative 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcα2-6GalOMP) and the C-9 amino derivative 2 (9-(4'-hydroxy-4-biphenyl)methylamino-9-deoxy-Neu5Gcα2-6GalOMP) have the most promising affinity for mouse CD22 and human CD22, respectively. Replacing the subterminal galactose residue (2-6Gal-OMP) of 1 with benzyl (5) or biphenylmethyl (6) as aglycone led to even higher potency for mCD22. In this study, both compounds showed improved potency and selectivity for CD22 (IC(50) 70 nM) and 712-fold more selective for CD22 than for MAG. The corresponding derivatives of 2, compounds 8 and 9, showed comparable activity to 2 but lower potency and selectivity than 5 and 6. Although compounds 5-9 are simple and small molecular weight antagonists, they showed much high potency and selectivity than the corresponding compounds having α 2-6Gal linkage. Both biological and computational docking simulation studies suggest that the 2-6Gal-OMP residues of 1 and 2 are not critical for binding process and could be replaced with hydrophobic non-carbohydrate moieties. The data presented herein has significant implications for the design and discovery of next-generation CD22-antagonists.

Potent small molecule mouse CD22-inhibitors: exploring the interaction of the residue at C-2 of sialic acid scaffold.

Our previous study revealed that compound 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcalpha2-6GalOMP) has the most promising affinity for mCD22. Replacing the subterminal galactose residue of 1 with benzyl or biphenylmethyl as aglycone led to 38- and 20-fold higher potency, respectively. This discovery represents a new direction in inhibitor design suitable for pharmaceutical development.

HLA-DPB1*05: 01-restricted WT1332-specific TCR-transduced CD4+ T lymphocytes display a helper activity for WT1-specific CTL induction and a cytotoxicity against leukemia cells.

Wilms tumor gene 1 (WT1) is overexpressed in various malignant neoplasms, and has been demonstrated as an attractive target for cancer immunotherapy. We previously reported the identification of a WT1 protein-derived, 16-mer helper peptide WT1332 that could elicit Th1-type CD4+ T-cell response and bind to multiple HLA class II molecules. In this study, we examined the feasibility of adoptive therapy using CD4+ T cells that were transduced an HLA-DPB1*05:01-restricted, WT1332-specific T-cell receptor (TCR). HLA-DPB1*05:01-restricted, WT1332-specific TCR-transduced CD4+ T cells were successfully generated using lentiviral vector and exhibited strong proliferative response and Th1-type cytokine production in response to WT1332 peptide, WT1 protein, or WT1-expressing tumor cell lysate. Furthermore, the WT1332-specific TCR-transduced CD4+ T cells lysed HLA-DPB1*05:01-positive, WT1-expressing human leukemia cells through granzyme B/perforin pathway. Furthermore, stimulation of peripheral blood mononuclear cells with both HLA-A*24:02-restricted cytotoxic T lymphocytes-epitope peptide (modified 9-mer WT1235 peptide, WT1235m) and WT1332 helper peptide in the presence of WT1332-specific TCR-transduced CD4+ T cells strikingly enhanced the induction of WT1235m-specific cytotoxic T lymphocytes. Thus, these results demonstrated the feasibility of immunotherapy based on adoptive transfer of WT1332-specific TCR-transduced CD4+ T cells for the treatment of leukemia.

Recognition of a natural WT1 epitope by a modified WT1 peptide-specific T-cell receptor.

Wilms' tumor gene WT1 is highly expressed in leukemia and in various types of solid tumors and exerts an oncogenic function. Thus, WT1 protein is a most promising tumor-associated antigen. We have been successfully performing WT1 vaccination with a 9-mer modified WT1(235) peptide, which has one amino acid substitution (M→Y) at position 2 of 9-mer natural WT1(235) peptide (235-243 a.a.), for close to 700 HLA-A*24:02-positive patients with leukemia or solid tumors. Although vaccination of modified WT1(235) peptide induced natural WT1(235) peptide-recognizing cytotoxic T-lymphocytes (CTLs) and exerted cytotoxic activity towards leukemia and solid tumor cells that expressed the natural WT1(235) peptide (epitope) but not the vaccinated modified WT1(235) peptide (epitope), the molecular basis has remained unclear. In this study, we established a modified WT1(235) peptide-specific CTL clone, we isolated T-cell receptor (TCR) genes from it and transduced the TCR genes into CD8(+) T-cells. The TCR-transduced CD8(+) T-cells produced interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in response to stimulation not only with the modified WT1(235) peptide but also with the natural WT1(235) peptide and lysed modified or natural WT1(235) peptide-pulsed target cells and endogenously WT1-expressing leukemia cells in a HLA-A*24:02-restriction manner. These results provided us, for the first time at molecular basis, with a proof-of-concept of modified WT1(235) peptide-based immunotherapy for natural WT1(235) peptide-expressing malignancies.

Design, synthesis, and structure-affinity relationships of novel series of sialosides as CD22-specific inhibitors.

Sialosides incorporating substituted amides or amines at 9-position of sialic acid moiety have been synthesized and evaluated as CD22 inhibitors. Several derivatives exhibited inhibitory potency in sub- to low micromolar range (e. g., 8o, 9d, 9g, and 9k showed IC 50 values 0.40, 0.47, 0.24, and 0.23 microM, respectively, for hCD22, while 8p, 8q, and 9f, showed IC 50 values 1.70, 2.90, and 4.10 microM, respectively, for mCD22). The most significant result was the strongly enhanced affinity of 9g and 9k containing 9-(2' or 4'-hydroxy-4-biphenyl) methylamino substituents (600-fold more potent for hCD22 than the corresponding 9-hydroxy derivative; 7a). Molecular modeling study was carried out to get some insights into the molecular basis of CD22 inhibition. To the best of our knowledge, this is the first systematic structure-affinity relationship study on inhibition of CD22.