A novel Fc-engineered monoclonal antibody to CD37 with enhanced ADCC and high proapoptotic activity for treatment of B-cell malignancies.

The tetraspanin CD37 is widely expressed in B-cell malignancies and represents an attractive target for immunotherapy with mAbs. We have chimerized a high-affinity mouse Ab to CD37 and engineered the CH2 domain for improved binding to human Fcγ receptors. The resulting mAb 37.1 showed high intrinsic proapoptotic activity on malignant B cells accompanied by homotypic aggregation. Furthermore, the Ab-mediated high Ab-dependent cell-mediated cytotoxicity (ADCC) on lymphoma and primary CLL cells. mAb 37.1 strongly depleted normal B cells as well as spiked B-lymphoma cells in blood samples from healthy donors as well as malignant B cells in blood from CLL patients. In all assays, mAb 37.1 was superior to rituximab in terms of potency and maximal cell lysis. A single dose of mAb CD37.1 administered to human CD37-transgenic mice resulted in a reversible, dose-dependent reduction of peripheral B cells. In a Ramos mouse model of human B-cell lymphoma, administration of mAb 37.1 strongly suppressed tumor growth. Finally, a surrogate Fc-engineered Ab to macaque CD37, with in vitro proapoptotic and ADCC activities very similar to those of mAb 37.1, induced dose-dependent, reversible B-cell depletion in cynomolgus monkeys. In conclusion, the remarkable preclinical pharmacodynamic and antitumor effects of mAb 37.1 warrant clinical development for B-cell malignancies.

In vivo plasmid electroporation induces tumor antigen-specific CD8+ T-cell responses and delays tumor growth in a syngeneic mouse melanoma model.

Plasmid DNA-based molecular cancer vaccines generally suffer from suboptimal immunogenicity. One of the key limitations is insufficient level of gene expression, which was surmounted in our approach by using the novel technique of in vivo plasmid electroporation-enhanced vaccination (electrovaccination). Electrovaccination with plasmids encoding the full-length autologous melanocyte antigen tyrosinase-related protein-2 induced limited melanocyte destruction in a subset of mice. Despite examples of vitiligo, vaccinated mice were not protected from a subsequent challenge of B16F10M melanoma cells. Novel constructs were then designed and submitted to a functional screen. Best performance was obtained when the relevant H-2K(b)-restricted epitope SVYDFFVWL was placed into a context of sequences of the HLA-Cw3 molecule. After animals were electrovaccinated using this construct, direct enzyme-linked immunospot analysis of peripheral blood mononuclear cells indicated that very high numbers of T cells recognizing the specific tyrosinase-related protein-2 epitope were generated. CD8+ T cells isolated from the spleen also displayed a high degree of antigen-specific reactivity and vigorously reacted toward unmodified B16F10M cells. In vivo protective effects of this construct were demonstrated in mice using two different models; outgrowth of s.c. implanted B16F10M tumor cells was significantly delayed, and vaccinated mice developed no or only very few tumor nodules in an i.v. lung metastasis model. Thus, improved antigen vectors delivered by highly effective gene transfer methods may form the basis for future human applications.

Activation requirements of circulating antigen-specific human CD8(+) memory T cells probed with insect cell-based artificial antigen-presenting cells.

We sought to define the molecular setup of an antigen-presenting cell that elicits antigen-specific T cell responses in vitro using insect cells that were infected with recombinant baculoviruses. Expression of single-chain HLA was complemented step-by-step with costimulatory molecules, including CD54 and CD80, by co-infection with the relevant viruses. Role of CD8 was assessed by introducing hybrid class I molecules where the alpha-3 domain of the HLA heavy chain molecule was replaced by its murine K(b) counterpart. Circulating T cells that respond to the EBV-derived HLA-A2-restricted peptide GLGCTLVAML were previously shown to bear hallmarks of memory cells. We found that the HLA+peptide complex alone displayed on the surface of insect cells was sufficient to elicit IFN-gamma secretion from these freshly isolated CD8(+) T cells in ELISpot assays. Binding of CD8 was absolutely required, but coexpression of costimulatory molecules resulted only in minimal increase in the number of spots. Tumor antigen-specific CTL clones also reacted in a strictly antigen-specific manner, but required CD54 for quantitative responses. The amount of IFN-gamma produced by the individual reactive T cells was evaluated as spot size, and was also influenced by the costimulatory molecules: CD54 increased also the response magnitude of cultured CTL lines, while CD80 enhanced cytokine release from freshly isolated CD8(+) T cells. Understanding the stimulatory requirements of functionally competent effector/memory T cells and their exact enumeration will be helpful for increasing the efficacy of vaccines.