BACKGROUND CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene (“gene editing”) — in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN) — is safe. METHODS We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (−1.81 cells per day) was significantly less than the decline in unmodified cells (−7.25 cells per day) (P = 0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.)

The facultative intracellular bacterium Listeria monocytogenes is being developed as a cancer vaccine platform because of its ability to induce potent innate and adaptive immunity. For successful clinical application, it is essential to develop a Listeria platform strain that is safe yet retains the potency of vaccines based on wild-type bacteria. Here, we report the development of a recombinant live-attenuated vaccine platform strain that retains the potency of the fully virulent pathogen, combined with a >1,000-fold reduction in toxicity, as compared with wild-type Listeria. By selectively deleting two virulence factors, ActA (⌬actA) and Internalin B (⌬inlB), the immunopotency of Listeria was maintained and its toxicity was diminished in vivo, largely by blocking the direct internalin B-mediated infection of nonphagocytic cells, such as hepatocytes, and the indirect ActA-mediated infection by cellto-cell spread from adjacent phagocytic cells. In contrast, infection of phagocytic cells was not affected, leaving intact the ability of Listeria to stimulate innate immunity and to induce antigenspecific cellular responses. Listeria ⌬actA͞⌬inlB-based vaccines were rapidly cleared from mice after immunization and induced potent and durable effector and memory T-cell responses with no measurable liver toxicity. Therapeutic vaccination of BALB͞c mice bearing murine CT26 colon tumor lung metastases or palpable s.c. tumors (>100 mm 3 ) with recombinant Listeria ⌬actA͞⌬inlB expressing an endogenous tumor antigen resulted in breaking of self-tolerance and long-term survival. We propose that recombinant Listeria ⌬actA͞⌬inlB expressing human tumor-associated antigens represents an attractive therapeutic strategy for further development and testing in human clinical trials.C ancer immunotherapy represents a promising treatment strategy that has produced some tantalizing clinical responses for a variety of malignant diseases. Although promising, there continues to be a strong need for a practical immunization strategy that can be routinely adopted to specific malignancies and that consistently yields durable and robust therapeutic antitumor responses.Progress in molecular and cellular immunology, combined with increasing understanding of pathogen physiology and hostpathogen interaction has facilitated the design and use of attenuated bacteria as conventional vaccine vectors. However, the practical utility of live attenuated vaccines relies on achieving a proper balance between the virulence͞toxicity and immunogenicity of the vaccine. The potency of a pathogen to elicit adaptive immunity is related in part to its ability to stimulate significant innate immunity through recognition of microbial pathogen-associated molecular patterns by Toll-like receptors. Microbial encounter with professional antigen-presenting cells (APC), such as dendritic cells, results in activation and maturation (1) as well as secretion of high levels of T helper-1-type cytokines (2). This interaction initiates adaptive immune responses and therefore link...

Purpose Listeria monocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-100 is a live-attenuated Lm strain (Lm ΔactA/ΔinlB). Uptake by phagocytes in the liver results in local inflammatory responses, and activation and recruitment of NK and T cells, in association with increased survival of mice bearing hepatic metastases. The Lm ΔactA/ΔinlB strain, engineered to express human mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces mesothelin-specific T cell responses against mesothelin-expressing murine tumors. These two Phase 1 studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers, respectively. Experimental Design A single intravenous injection of ANZ-100 was evaluated in a dose escalation study in subjects with liver metastases. Nine subjects received 1×106, 3×107, or 3×108 colony forming units [cfu]. CRS-207 was evaluated in a dose-escalation study in subjects with mesothelioma, lung, pancreatic or ovarian cancers. 17 subjects received up to 4 doses of 1×108, 3×108, 1×109, or 1×1010 cfu. Results A single infusion of ANZ-100 was well tolerated to the maximum planned dose. Adverse events included transient laboratory abnormalities and symptoms associated with cytokine release. Multiple infusions of CRS-207 were well tolerated up to 1×109 cfu, the determined maximum tolerated dose. Immune activation was observed for both ANZ-100 and CRS-207 as measured by serum cytokine/chemokine levels and NK cell activation. In the CRS-207 study, Listeriolysin O and mesothelin-specific T cell responses were detected and 37% of subjects lived ≥ 15 months. Conclusions ANZ-100 and CRS-207 administration was safe and resulted in immune activation.

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.Phosphatidylinositol (PI) 3-kinase has been shown to mediate signaling induced by numerous growth factors and tumor antigens. The intracellular levels of the phospholipid products of PI 3-kinase increase in response to stimulation with growth factors or after oncogenic transformation (for reviews, see references 10,11,33,76,80). PI 3-kinase signaling appears to be required for a number of mitogens during the G 1 -to-Sphase transition of the cell cycle (63). Recently, it was demonstrated that PI 3-kinase regulates cell survival in response to various apoptotic stimuli (21, 49).PI 3-kinase is a heterodimeric complex consisting of an 85-kDa regulatory subunit, p85, and a 110-kDa catalytic subunit, p110 (11, 33). The p85 subunit contains two Src homology 2 (SH2) domains, which bind to tyrosine-phosphorylated receptors after stimulation of cells with growth factors and in this manner recruit the p85-p110 complex to the cell membrane. The region between the two SH2 domains of p85, the iSH2 region, mediates the association with p110, and this interaction is required for the enzymatic activity of p110 (37). Based on this observation we generated a chimeric molecule, p110*, in which the iSH2 region of p85 was covalently linked to its binding site at the p110 N terminus by using a flexible hinge region (30). p110* is a constitutively active PI 3-kinase which can activate signaling pathways independent of growth factor stimulation.The generation of constitutively active PI 3-kinase molecules has greatly facilitated the analysis of signa...

We developed a new class of vaccines, based on killed but metabolically active (KBMA) bacteria, that simultaneously takes advantage of the potency of live vaccines and the safety of killed vaccines. We removed genes required for nucleotide excision repair (uvrAB), rendering microbial-based vaccines exquisitely sensitive to photochemical inactivation with psoralen and long-wavelength ultraviolet light. Colony formation of the nucleotide excision repair mutants was blocked by infrequent, randomly distributed psoralen crosslinks, but the bacterial population was able to express its genes, synthesize and secrete proteins. Using the intracellular pathogen Listeria monocytogenes as a model platform, recombinant psoralen-inactivated Lm DeltauvrAB vaccines induced potent CD4(+) and CD8(+) T-cell responses and protected mice against virus challenge in an infectious disease model and provided therapeutic benefit in a mouse cancer model. Microbial KBMA vaccines used either as a recombinant vaccine platform or as a modified form of the pathogen itself may have broad use for the treatment of infectious disease and cancer.

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory͞growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of nontoxic doses of recombinant IL-2 (20,000 and 40,000 IU͞dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-␥ production in these treated mice. Moreover, treatment with the combination of tumor lysatepulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14-and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.Dendritic cells (DC) are potent antigen-presenting cells that can elicit primary and boost secondary immune responses to foreign antigens (1, 2). In a variety of settings, these specialized antigenpresenting cells can induce both the generation and proliferation of specific cytotoxic T lymphocyte (CTL) and T helper cells via antigen presentation by major histocompatibility complex (MHC) class I and class II molecules, respectively. We (3-6) and others (7-9) have described the induction of MHC class I and class II specific T cell responses following stimulation with tumor antigen(s)-pulsed DC in vitro and in vivo. Because of these properties, much attention has been directed toward the use of DC in vaccine strategies for the treatment of cancer. In this regard, DC pulsed with tumor-associated antigen(s) in various forms, including as whole cell lysates (3-6), peptides (7, 8), proteins (10), RNA (11), or DNA (12, 13), has been studied for antitumor effects in experimental animals. In these models, immunization with tumor antigen(s) presented by DC has shown much promise in effectively priming the cellular immune response as well as in eliciting tumor regression in vivo. Of importance, host-derived CD8 ϩ and, to a lesser extent, CD4 ϩ T cells have been shown to play the central role in the antitumor effects mediated by DC-based vaccines (6).We have been studying approaches to enhance the therapeutic activity of murine and human DC, which involve...

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.

Intermittent therapy with IL-2 and ART produced a substantially greater increase in CD4 cells and was associated with a larger decrease in viral load than ART alone. Clinical end-point trials will be necessary to determine whether the enhanced viral suppression and CD4 cell increases associated with IL-2 therapy will translate into improved clinical outcomes. JAMA. 2000;284:183-189