SummaryImmunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fce, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fce appeared as a 70 000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fce binds to IgE receptors FceRI and FceRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fce to FceRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fce to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fce -but not IgE -induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fce caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.

Treatment of human epidermal growth factor receptor 2 (HER2/neu)-expressing breast cancer patients with a monoclonal antibody (mAb) directed against HER2/neu improves the outcome of chemotherapy. In cases in which remission is observed, antibody-dependent cell-mediated cytotoxicity (ADCC) seems to be one of the main mechanisms of anti-HER2/neu mAb action, implicating Fcγ receptors (FcγRs) in this tumoricidal activity. In vitro and in vivo studies have revealed that anti-HER2/neu-mediated ADCC is mainly accomplished by polymorphonuclear granulocytes (PMN). C5a, a cleavage product of the complement component C5, modulates FcγR expression via upregulation of activating and downregulation of inhibitory FcγRs. C5a also recruits PMNs to sites of inflammation and increases PMN survival. To enhance the recruitment and activation of C5a receptor-bearing cells into the tumor microenvironment, we developed antibody fusion proteins composed of a human IgG3 anti-HER2/neu antibody genetically fused to C5a [anti-HER2/neu IgG3-(C5a)] or to its derivative, C5a desArg [anti-HER2/neu IgG3-(C5a desArg )]. Both fusion proteins were expressed, properly assembled, and secreted by murine myeloma cells, and displayed chemotactic activity on human PMN. Under comparable conditions, anti-HER2/neu IgG3-(C5a desArg ) increased the survival of PMN more efficiently than anti-HER2/neu IgG3-(C5a) or C5a desArg . Surprisingly, incubation of the fusion proteins with breast cancer cells that overexpress HER2/neu (SK-BR-3) induced cell death at a dose at which the anti-HER2/neu IgG3 antibody was innocuous. In the presence of human peripheral blood leukocytes as effector cells, both fusion proteins induced tumor cell death more efficiently than anti-HER2/neu IgG3. These data suggest that anti-HER2/neu IgG3-(C5a) and anti-HER2/neu IgG3-(C5a desArg ) fusion proteins possess novel properties that could be useful in cancer immunotherapy. Mol Cancer Ther; 9(8); 2175-85. ©2010 AACR.

Supplementary Figure 1 from Decreased Survival of Human Breast Cancer Cells Expressing HER2/<i>neu</i> on <i>In vitro</i> Incubation with an Anti-HER2/<i>neu</i> Antibody Fused to C5a or C5a_desArg

<div>Abstract<p>Treatment of human epidermal growth factor receptor 2 (HER2/<i>neu</i>)-expressing breast cancer patients with a monoclonal antibody (mAb) directed against HER2/<i>neu</i> improves the outcome of chemotherapy. In cases in which remission is observed, antibody-dependent cell-mediated cytotoxicity (ADCC) seems to be one of the main mechanisms of anti-HER2/<i>neu</i> mAb action, implicating Fcγ receptors (FcγRs) in this tumoricidal activity. <i>In vitro</i> and <i>in vivo</i> studies have revealed that anti-HER2/<i>neu</i>-mediated ADCC is mainly accomplished by polymorphonuclear granulocytes (PMN). C5a, a cleavage product of the complement component C5, modulates FcγR expression via upregulation of activating and downregulation of inhibitory FcγRs. C5a also recruits PMNs to sites of inflammation and increases PMN survival. To enhance the recruitment and activation of C5a receptor–bearing cells into the tumor microenvironment, we developed antibody fusion proteins composed of a human IgG3 anti-HER2/<i>neu</i> antibody genetically fused to C5a [anti-HER2/<i>neu</i> IgG3-(C5a)] or to its derivative, C5a_desArg [anti-HER2/<i>neu</i> IgG3-(C5a_desArg)]. Both fusion proteins were expressed, properly assembled, and secreted by murine myeloma cells, and displayed chemotactic activity on human PMN. Under comparable conditions, anti-HER2/<i>neu</i> IgG3-(C5a_desArg) increased the survival of PMN more efficiently than anti-HER2/<i>neu</i> IgG3-(C5a) or C5a_desArg. Surprisingly, incubation of the fusion proteins with breast cancer cells that overexpress HER2/<i>neu</i> (SK-BR-3) induced cell death at a dose at which the anti-HER2/<i>neu</i> IgG3 antibody was innocuous. In the presence of human peripheral blood leukocytes as effector cells, both fusion proteins induced tumor cell death more efficiently than anti-HER2/<i>neu</i> IgG3. These data suggest that anti-HER2/<i>neu</i> IgG3-(C5a) and anti-HER2/<i>neu</i> IgG3-(C5a_desArg) fusion proteins possess novel properties that could be useful in cancer immunotherapy. Mol Cancer Ther; 9(8); 2175–85. ©2010 AACR.</p></div>

<div>Abstract<p>Treatment of human epidermal growth factor receptor 2 (HER2/<i>neu</i>)-expressing breast cancer patients with a monoclonal antibody (mAb) directed against HER2/<i>neu</i> improves the outcome of chemotherapy. In cases in which remission is observed, antibody-dependent cell-mediated cytotoxicity (ADCC) seems to be one of the main mechanisms of anti-HER2/<i>neu</i> mAb action, implicating Fcγ receptors (FcγRs) in this tumoricidal activity. <i>In vitro</i> and <i>in vivo</i> studies have revealed that anti-HER2/<i>neu</i>-mediated ADCC is mainly accomplished by polymorphonuclear granulocytes (PMN). C5a, a cleavage product of the complement component C5, modulates FcγR expression via upregulation of activating and downregulation of inhibitory FcγRs. C5a also recruits PMNs to sites of inflammation and increases PMN survival. To enhance the recruitment and activation of C5a receptor–bearing cells into the tumor microenvironment, we developed antibody fusion proteins composed of a human IgG3 anti-HER2/<i>neu</i> antibody genetically fused to C5a [anti-HER2/<i>neu</i> IgG3-(C5a)] or to its derivative, C5a_desArg [anti-HER2/<i>neu</i> IgG3-(C5a_desArg)]. Both fusion proteins were expressed, properly assembled, and secreted by murine myeloma cells, and displayed chemotactic activity on human PMN. Under comparable conditions, anti-HER2/<i>neu</i> IgG3-(C5a_desArg) increased the survival of PMN more efficiently than anti-HER2/<i>neu</i> IgG3-(C5a) or C5a_desArg. Surprisingly, incubation of the fusion proteins with breast cancer cells that overexpress HER2/<i>neu</i> (SK-BR-3) induced cell death at a dose at which the anti-HER2/<i>neu</i> IgG3 antibody was innocuous. In the presence of human peripheral blood leukocytes as effector cells, both fusion proteins induced tumor cell death more efficiently than anti-HER2/<i>neu</i> IgG3. These data suggest that anti-HER2/<i>neu</i> IgG3-(C5a) and anti-HER2/<i>neu</i> IgG3-(C5a_desArg) fusion proteins possess novel properties that could be useful in cancer immunotherapy. Mol Cancer Ther; 9(8); 2175–85. ©2010 AACR.</p></div>