Chimeric IgA antibodies against HLA class II effectively trigger lymphoma cell killing by Michael Dechant, Gestur Vidarsson, Bernhard Stockmeyer, Roland Repp, Martin J. Glennie, Martin Gramatzki, Jan G. J. van de Winkel, and Thomas Valerius Blood Volume 100(13):4574-4580 December 15, 2002 ©2002 by American Society of Hematology

Sandwich ELISA for antibody quantification Sandwich ELISA for antibody quantification.ELISA plates were coated with polyclonal antibodies against human IgA or human IgG. Sandwich ELISA for antibody quantification.ELISA plates were coated with polyclonal antibodies against human IgA or human IgG. After chimeric antibodies were added in serial dilutions, peroxidase-labeled antihuman κ antibody was used for detection. Polyclonal human IgA or rituxan served as standards for IgAs or IgGs, starting from 50 or 5 μg/mL, respectively. This assay also demonstrated the assembly of light and heavy chains of the chimeric antibodies. Data from 1 of 4 experiments with similar results are shown for IgG1, IgA1, and IgA2. Michael Dechant et al. Blood 2002;100:4574-4580 ©2002 by American Society of Hematology

Western blots of chimeric antibodies Western blots of chimeric antibodies.Antibodies were separated by SDS-PAGE with gradient gels (4%-15%) under nonreducing (A) or reducing (B) conditions. Western blots of chimeric antibodies.Antibodies were separated by SDS-PAGE with gradient gels (4%-15%) under nonreducing (A) or reducing (B) conditions. After transfer onto nitrocellulose, immunoblots were incubated with peroxidase-labeled polyclonal goat antihuman κ–light chain antibody and goat antihuman IgG or rabbit antihuman IgA, respectively. Blots were developed with enhanced chemoluminescent reaction reagent (ECL). Thus, the correct size of all isotypes of chimeric HLA class II antibody F3.3 was demonstrated. Michael Dechant et al. Blood 2002;100:4574-4580 ©2002 by American Society of Hematology

Antigen binding of chimeric antibodies Antigen binding of chimeric antibodies.Saturating concentrations of chimeric IgA1, IgA2, and IgG1 isotypes of HLA class II antibody F3.3 demonstrated similar binding to HLA class II–transfected L66 cells, as measured by indirect immunofluorescence (panel A;... Antigen binding of chimeric antibodies.Saturating concentrations of chimeric IgA1, IgA2, and IgG1 isotypes of HLA class II antibody F3.3 demonstrated similar binding to HLA class II–transfected L66 cells, as measured by indirect immunofluorescence (panel A; 1 of 2 experiments with similar results is shown). In order to estimate antibody affinity, dilutions of chimeric antibodies were investigated for antigen binding, and antibody concentrations at half-maximal binding were determined (panel B; mean ± SEM for each of 3 experiments is displayed). Thus, IgG1, IgA2, and IgA1 demonstrated half-maximal binding at 1.8 μg/mL (X, Y) and 3.5 μg/mL (Z), respectively. FITC-labeled antihuman kappa–light chain antibody was used for staining. Michael Dechant et al. Blood 2002;100:4574-4580 ©2002 by American Society of Hematology

Killing of lymphoma cells by chimeric antibodies Killing of lymphoma cells by chimeric antibodies.Lysis of ARH-77 lymphoma cells was measured in 3-hour 51Cr release assays, using 1 μg/mL of the indicated chimeric isotypes of HLA class II antibody F3.3. Killing of lymphoma cells by chimeric antibodies.Lysis of ARH-77 lymphoma cells was measured in 3-hour 51Cr release assays, using 1 μg/mL of the indicated chimeric isotypes of HLA class II antibody F3.3. Unseparated blood (whole blood) served as effector source, which was then fractionated into polymorphonuclear (PMN) or mononuclear (MNC) effector cells or into complement-containing plasma to identify effector mechanisms of chimeric antibodies. Isolated PMNs or MNCs were used at an effector-to-target-cell ratio of 40:1. Data are presented as mean percentage specific lysis ± SEM from at least 4 independent experiments. Significant (P < .05) antibody-mediated lysis is marked by an asterisk (*). Michael Dechant et al. Blood 2002;100:4574-4580 ©2002 by American Society of Hematology

Influence of E/T ratio and antibody concentration on IgA-mediated ADCC Influence of E/T ratio and antibody concentration on IgA-mediated ADCC.IgA1-, IgA2-, or IgG1-mediated killing of Raji Burkitt lymphoma cells was investigated using different E/T ratios (A) or antibody concentrations (B). Influence of E/T ratio and antibody concentration on IgA-mediated ADCC.IgA1-, IgA2-, or IgG1-mediated killing of Raji Burkitt lymphoma cells was investigated using different E/T ratios (A) or antibody concentrations (B). Antibody concentration in panel A was 1 μg/mL; E/T ratio in panel B was 40:1. Data are presented as mean percentage specific lysis ± SEM observed with isolated PMNs from at least 3 different donors. Significant killing (P < .05) is indicated by an asterisk (*). Michael Dechant et al. Blood 2002;100:4574-4580 ©2002 by American Society of Hematology

Role of Fc receptors in IgA-mediated killing Role of Fc receptors in IgA-mediated killing.Fc receptor–blocking antibodies My43 (FcαRI), 197 (FcγRI), AT10 F(ab′) (FcγRII), or 3G8 F(ab′)2 (FcγRIII) were used to identify which Fc receptor triggered ARH-77 killing by chimeric IgA1 or IgA2 versions of HLA ... Role of Fc receptors in IgA-mediated killing.Fc receptor–blocking antibodies My43 (FcαRI), 197 (FcγRI), AT10 F(ab′) (FcγRII), or 3G8 F(ab′)2 (FcγRIII) were used to identify which Fc receptor triggered ARH-77 killing by chimeric IgA1 or IgA2 versions of HLA class II antibody F3.3. PMNs served as effector cells. All Fc receptor antibodies were used at 10 μg/mL. As demonstrated, IgA-mediated lysis was completely inhibited by FcαRI antibody, but not by antibodies directed against Fcγ receptors. Data are presented as mean percentage specific lysis ± SEM from experiments with 4 different donors. Significant inhibition (P < .05) is indicated by an asterisk (*). Michael Dechant et al. Blood 2002;100:4574-4580 ©2002 by American Society of Hematology

Killing of primary B-CLL cells by chimeric HLA class II antibodies Killing of primary B-CLL cells by chimeric HLA class II antibodies.Chimeric IgG1, IgA1, or IgA2 versions of HLA class II antibody F3.3 were compared in ADCC against 51Cr-labeled, freshly isolated B-CLL cells. Killing of primary B-CLL cells by chimeric HLA class II antibodies.Chimeric IgG1, IgA1, or IgA2 versions of HLA class II antibody F3.3 were compared in ADCC against 51Cr-labeled, freshly isolated B-CLL cells. As effector source, we used unseparated blood from healthy donors or from G-CSF–primed patients (G-CSF). Both donor groups mediated significant killing (indicated by *) with all 3 antibody isotypes. Notably, only IgA1- and IgA2-mediated lysis, not IgG1-mediated lysis, was significantly enhanced during G-CSF therapy. Killing without antibody or without effectors was consistently low. Data are presented as mean percentage specific lysis ± SEM from experiments with at least 4 different donors from each group. Significant (P < .05) differences between healthy donors and G-CSF–primed patients are indicated by #. Michael Dechant et al. Blood 2002;100:4574-4580 ©2002 by American Society of Hematology