We constructed two megabase-sized YACs containing large contiguous fragments of the human heavy and kappa (kappa) light chain immunoglobulin (Ig) loci in nearly germline configuration, including approximately 66 VH and 32 V kappa genes. We introduced these YACs into Ig-inactivated mice and observed human antibody production which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and repertoire. Diverse Ig gene usage together with somatic hypermutation enables the mice to generate high affinity fully human antibodies to multiple antigens, including human proteins. Our results underscore the importance of the large Ig fragments with multiple V genes for restoration of a normal humoral immune response. These mice are likely to be a valuable tool for the generation of therapeutic antibodies.

We describe a novel retroviral packaging system in which high titer amphotropic retrovirus was produced without the need to generate stable producer clones. kat expression vectors, which produce high levels of retroviral vector transcripts and retroviral packaging functions, were transfected into 293 cells followed by virus harvest 48 hours posttransfection. Viral titers as high as 3.8 proviral copies/cell/mL of frozen supernatant in 3T3 cells were obtained, 10- to 50-fold greater than transient viral titers reported using 3T3-based retroviral packaging lines. Cocultivation of primary human CD8+ T lymphocytes after transient transfection of 293 cells with kat plasmids resulted in transduction efficiencies of 10% to 40%, 5- to 10-fold greater compared to cocultivation with a high titer PA317 producer clone and significantly greater than previously reported results for transduction of primary human T lymphocytes with retroviral vectors. Virus produced using the kat system was shown to be free of detectable replication competent retrovirus by an extended provirus mobilization assay, demonstrating that this system is as safe as currently available stable packaging lines. The kat virus production system should be of general use for the rapid production of high titer viral supernatants, as well as for high-efficiency transduction hematopoietic cell types refractory to retroviral transduction.

We have developed an immunotherapeutic approach with potential application in the treatment of viral and malignant disease. We show that primary CD8+ T cells isolated from peripheral blood can be genetically modified by retroviral transduction to express high levels of universal (major histocompatibility complex-unrestricted) chimeric T- cell receptors specific for human immunodeficiency virus (HIV) antigens. Two classes of HIV-specific URs in which the antigen-binding domain is comprised of either CD4 or a single-chain antibody are capable of activating a number of T-cell effector functions in response to target cells, including cytolysis, in a highly sensitive and specific manner. Importantly, we have addressed a number of issues which, although particularly relevant to the clinical application of this approach in the treatment of HIV infection, may also impact on the potential of UR immunotherapy for other disease targets. The UR immunotherapeutic system is particularly suited for evaluation in the clinical setting.