Investigation of the mechanism of action of alemtuzumab in a human CD52 transgenic mouse model.

Alemtuzumab is a humanized monoclonal antibody against CD52, an antigen found on the surface of normal and malignant lymphocytes. It is approved for the treatment of B-cell chronic lymphocytic leukaemia and is undergoing Phase III clinical trials for the treatment of multiple sclerosis. The exact mechanism by which alemtuzumab mediates its biological effects in vivo is not clearly defined and mechanism of action studies have been hampered by the lack of cross-reactivity between human and mouse CD52. To address this issue, a transgenic mouse expressing human CD52 (hCD52) was created. Transgenic mice did not display any phenotypic abnormalities and were able to mount normal immune responses. The tissue distribution of hCD52 and the level of expression by various immune cell populations were comparable to those seen in humans. Treatment with alemtuzumab replicated the transient increase in serum cytokines and depletion of peripheral blood lymphocytes observed in humans. Lymphocyte depletion was not as profound in lymphoid organs, providing a possible explanation for the relatively low incidence of infection in alemtuzumab-treated patients. Interestingly, both lymphocyte depletion and cytokine induction by alemtuzumab were largely independent of complement and appeared to be mediated by neutrophils and natural killer cells because removal of these populations with antibodies to Gr-1 or asialo-GM-1, respectively, strongly inhibited the activity of alemtuzumab whereas removal of complement by treatment with cobra venom factor had no impact. The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab.

Reduction of inflammation and preservation of neurological function by anti-CD52 therapy in murine experimental autoimmune encephalomyelitis.

Alemtuzumab, a monoclonal antibody directed against human CD52, is used in the treatment of MS. To characterize the impact of anti-CD52 administration, a monoclonal antibody to mouse CD52 (anti-muCD52) was generated and evaluated in EAE mouse models of MS. A single course of anti-muCD52 provided a therapeutic benefit accompanied by a reduction in the frequency of autoreactive T lymphocytes and production of pro-inflammatory cytokines. Examination of the CNS revealed a decrease in infiltrating lymphocytes, demyelination and axonal loss. Electrophysiological assessment showed preservation of axonal conductance in the spinal cord. These findings suggest that anti-CD52 therapy may help preserve CNS integrity.

Immune status following alemtuzumab treatment in human CD52 transgenic mice.

Alemtuzumab is a monoclonal antibody against the CD52 antigen present at high levels on the surface of lymphocytes. While treatment of multiple sclerosis patients with alemtuzumab results in marked depletion of lymphocytes from the circulation, it has not been associated with a high incidence of serious infections. In a human CD52 transgenic mouse, alemtuzumab treatment showed minimal impact on the number and function of innate immune cells. A transient decrease in primary adaptive immune responses was observed post-alemtuzumab but there was little effect on memory responses. These results potentially help explain the level of immunocompetence observed in alemtuzumab-treated MS patients.

Involvement of neutrophils and natural killer cells in the anti-tumor activity of alemtuzumab in xenograft tumor models.

Alemtuzumab is a recombinant humanized IgG1 monoclonal antibody directed against CD52, an antigen expressed on the surface of normal and malignant B and T lymphocytes. Alemtuzumab is approved for the treatment of B-cell chronic lymphocytic leukemia (B-CLL), but the exact mechanism by which the antibody depletes malignant lymphocytes in vivo is not clearly defined. To address this issue, the anti-tumor activity of alemtuzumab was studied in disseminated and subcutaneous xenograft tumor models. The density of CD52 target antigen on the surface of tumor cells appeared to correlate with the anti-tumor activity of alemtuzumab. Deglycosylation of alemtuzumab resulted in a loss of cytotoxicity in vitro and was found to abolish anti-tumor activity in vivo. Individual inactivation of effector mechanisms in tumor-bearing mice indicated that the protective activity of alemtuzumab in vivo was primarily dependent on ADCC mediated by neutrophils and to a lesser extent NK cells. Increasing the number of circulating neutrophils by treatment with G-CSF enhanced the anti-tumor activity of the antibody, thus providing further evidence for the involvement of neutrophils as effector cells in the activity of alemtuzumab.

Induction of antitumor immunity by semi-allogeneic and fully allogeneic electrofusion products of tumor cells and dendritic cells.

Immunization with the electrofusion product of tumor cells and dendritic cells (DCs) is a promising approach to cancer immunotherapy. Production of electrofusion vaccines currently requires the acquisition of tumor material and must be tailored to each individual. Alternative vaccine configurations were explored in this study. Results indicated that fusion vaccines with fully syngeneic, semi-allogeneic or fully allogeneic components, were all effective in inducing specific, long-lasting antitumor immunity. This previously undescribed activity of a fully allogeneic fusion product introduces the possibility of using defined allogeneic tumor and DC lines to simplify vaccine manufacturing.

Cytotoxic T lymphocyte responses to transgene product, not adeno-associated viral capsid protein, limit transgene expression in mice.

The use of adeno-associated viral (AAV) vectors for gene replacement therapy is currently being explored in several clinical indications. However, reports have suggested that input capsid proteins from AAV-2 vector particles may result in the stimulation of cytotoxic T lymphocyte (CTL) responses that can result in a loss of transduced cells. To explore the impact of anti-AAV CTLs on AAV-mediated transgene expression, both immunocompetent C57BL=6 mice and B cell-deficient muMT mice were immunized against the AAV2 capsid protein (Cap) and were injected intravenously with an AAV-2 vector encoding alpha-galactosidase (alpha-Gal). C57BL=6 mice, which developed both CTL and neutralizing antibody responses against Cap, failed to show any detectable alpha-Gal expression. In contrast, serum alpha-Gal levels comparable to those of naive mice were observed in muMT mice despite the presence of robust CTL activity against Cap, indicating that preexisting Cap-specific CTLs did not have any effect on the magnitude and duration of transgene expression. The same strategy was used to assess the impact of CTLs against the alpha-Gal transgene product on AAV-mediated gene delivery and persistence of transgene expression. Preimmunization of muMT mice with an Ad=alpha-Gal vector induced a robust CTL response to alpha-Gal. When these mice were injected with AAV2=alpha-Gal vector, initial levels of alpha-Gal expression were reduced by more than 1 log and became undetectable by 2 weeks postinjection. Overall, our results indicate that CTLs against the transgene product as opposed to AAV capsid protein are more likely to interfere with AAV transgene expression.

Induction of specific antitumor immunity in the mouse with the electrofusion product of tumor cells and dendritic cells.

Dendritic cells (DCs) are potent antigen-presenting cells capable of inducing primary T-cell responses. Several immunotherapy treatment strategies involve manipulation of DCs, both in vivo and ex vivo, to promote the immunogenic presentation of tumor-associated antigens. In this study, an electrofusion protocol was developed to induce fusion between tumor cells and allogeneic bone marrow-derived DCs. Preimmunization with irradiated electrofusion product was found to provide partial to complete protection from tumor challenge in the murine Renca renal cell carcinoma model and the B16 and M3 melanoma models. Vaccinated survivors developed specific immunological memory and were able to reject a subsequent rechallenge with the same tumor cells but not a syngeneic unrelated tumor line. Antitumor protection in the B16 model was accompanied by the development of a polyclonal cytotoxic T-lymphocyte response against defined melanoma-associated antigens. The therapeutic potential of this type of approach was suggested by the ability of a Renca-DC electrofusion product to induce tumor rejection in a substantial percentage of hosts (60%) bearing pre-established tumor cells. These results indicate that treatment with electrofused tumor cells and allogeneic DCs is capable of inducing a potent antitumor response and could conceivably be applied to a wide range of cancer indications for which tumor-associated antigens have not been identified.

Tumor treatment with complexes of cationic lipid and noncoding plasmid DNA results in the induction of cytotoxic T cells and systemic tumor elimination.

We have demonstrated recently that treatment of established peritoneal mesothelial tumors with complexes composed of cationic lipid and noncoding plasmid DNA (pNull) results in the inhibition of tumor growth accompanied by the induction of a tumor-specific cellular immune response. In this study, treatment of mice bearing intraperitoneal (i.p.) M3 melanoma tumors with i.p. injections of lipid/pNull complex was found to inhibit tumor growth and induce the development of a cytolytic response against several M3 melanoma-associated antigens. Depletion of CD8(+) T cells, as opposed to natural killer (NK) or CD4(+) T cells, essentially abrogated the therapeutic effect of lipid+pNull complex, thus supporting the involvement of cytotoxic CD8(+) T cells in the antitumor response. The antitumor effect of lipid/pNull complex was maximal following delivery into a tumor-bearing compartment. For example, i.p. delivery of complex was more effective than intravenous (i.v.) or subcutaneous (s.c.) treatment of i.p. M3 tumors. In addition, i.v. injection of complex displayed therapeutic activity against lung metastases caused by i.v. injection of tumor cells, and intratumoral injection of complex into solid s.c. tumors caused regression in most animals. Importantly, the immune response induced by local treatment of tumors with complex also offered systemic protection against tumor cells at distal sites, as illustrated by the eradication of both peritoneal tumors and lung metastases in mice treated with complex delivered i.p. Treatment with lipid/pNull complex, therefore, represents an attractive immune-based treatment modality that could potentially be applied to many tumor types.