William Siders

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.

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.

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.

Impact of alemtuzumab treatment on the survival and function of human regulatory T cells in vitro

Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon‐β in relapsing–remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement‐dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab‐exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25hi T‐cell population was necessary for the suppressive activity of alemtuzumab‐exposed T cells. The mechanism of this suppression was found to be dependent on both cell–cell contact and interleukin‐2 consumption. These findings suggest that an alemtuzumab‐mediated increase in the proportion of Treg cells may play a role in promoting the long‐term efficacy of alemtuzumab in patients with multiple sclerosis.

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.

Enhancement of the anti-tumor activity of therapeutic monoclonal antibodies by CXCR4 antagonists

Abstract The interaction between CXCR4 on the surface of tumor cells and CXCL12 in the stroma is believed to contribute to tumor cell survival and protection against drug treatment. Inhibition of stromal survival signals by CXCR4 antagonists has been reported to render tumor cells more sensitive to chemotherapy, but little is known about potential synergy with monoclonal antibodies. In this study, administration of the small molecule CXCR4 antagonists plerixafor and GENZ-644494 was found to enhance the anti-tumor activity of the monoclonal antibodies alemtuzumab and rituximab in disseminated lymphoma models. The observed enhancement in therapeutic efficacy by CXCR4 antagonists appeared to involve several factors, including interference with the tumor-promoting signals delivered by CXCL12, disruption of the tumor/stroma interaction and mobilization of effector neutrophils capable of mediating antibody-dependent cell-mediated cytotoxicity. The involvement of neutrophils was further supported by the observed reversal in therapeutic benefit upon neutrophil depletion.

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.

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 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.

Anti-murine CD52 antibody treatment does not adversely affect the migratory ability of immune cells

OBJECTIVE: To evaluate the migratory properties of various immune cell populations following anti-muCD52 treatment in murine models of inflammation. BACKGROUND: Alemtuzumab is an anti-CD52 humanized monoclonal antibody that causes depletion of circulating lymphocytes followed by a distinctive pattern of repopulation. Alemtuzumab showed superior efficacy vs. subcutaneous interferon beta-1a in phase 3 trials in active relapsing-remitting multiple sclerosis patients who were treatment-naive (CARE-MS I) or had relapsed on prior therapy (CARE-MS II). An anti-muCD52 antibody was utilized to expand our understanding of anti-CD52 therapy in mouse models of immune cell migration. DESIGN/METHODS: Immune cell migration was evaluated using both in vitro and in vivo analysis. Lymphocyte migration in vitro was evaluated in transwell assays using CD4 T cells purified from anti-muCD52-treated mice. In vivo migration was assessed using an acute CNS inflammation model in which mice were injected intracerebroventricularly with lipopolysaccharide (LPS) following anti-muCD52 treatment. Similarly, migration of innate immune cell subsets was evaluated in peritonitis model of inflammation, with animals being injected intraperitoneally with thioglycollate 3 days following anti-muCD52 treatment. The numbers of CNS-infiltrating lymphocytes or peritoneum-infiltrating cells were measured by polychromatic flow cytometry. RESULTS: Immune cells from anti-muCD52-treated mice retained their ability to migrate. In vitro, CD4 T cells were able to migrate in response to SDF-1alpha as efficiently as cells from vehicle-treated animals. In vivo, lymphocytes in anti-muCD52-treated animals were able to migrate into the CNS following local induction of inflammation by LPS. Similarly, innate immune cell migration was not affected by anti-muCD52 treatment, as similar numbers and types of cells were observed in inflamed peritoneum of anti-muCD52- and vehicle-treated animals. CONCLUSIONS: These results indicate that following anti-muCD52 treatment, immune cells retain their ability to migrate. These findings suggest that anti-muCD52 treatment may not compromise immune surveillance, but further studies are required. Study Supported by: Genzyme, a Sanofi company Disclosure: Dr. Havari has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Turner has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Dodge has received personal compensation for activities with Genzyme Corp. as an employee. Mr. Treleaven has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Shihabuddin has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Roberts has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Kaplan has received personal compensation for activities with Genzyme Corp. as an employee. Dr. Siders has received personal compensation for activities with Genzyme, Corp. as an employee.