by Clemens B. Caspar, Shoshana Levy, and Ronald Levy Idiotype Vaccines for Non-Hodgkin's Lymphoma Induce Polyclonal Immune Responses That Cover Mutated Tumor Idiotypes: Comparison of Different Vaccine Formulations by Clemens B. Caspar, Shoshana Levy, and Ronald Levy Blood Volume 90(9):3699-3706 November 1, 1997 ©1997 by American Society of Hematology

Id constructs used for the vaccination: (a) Id protein coupled to KLH 50 μg injected subcutaneously twice 2 weeks apart, (b) scFv-muGM-CSF protein 50 μg injected intraperitoneally 3 times 2 weeks apart, (c) Id plasmid DNA (with human IgGκ constant regions) ... Id constructs used for the vaccination: (a) Id protein coupled to KLH 50 μg injected subcutaneously twice 2 weeks apart, (b) scFv-muGM-CSF protein 50 μg injected intraperitoneally 3 times 2 weeks apart, (c) Id plasmid DNA (with human IgGκ constant regions) 100 μg injected IM 3 times 1 week apart, and (d) AdV containing the sequence encoding the scFv injected once IM at a dose of 3 × 107 pfu. Clemens B. Caspar et al. Blood 1997;90:3699-3706 ©1997 by American Society of Hematology

Reactivity of tumor-derived Id proteins with the treatment MoAb 7D11 and with sera from immunized mice. Reactivity of tumor-derived Id proteins with the treatment MoAb 7D11 and with sera from immunized mice. Sera from individual mice were absorbed against polyclonal human Ig and on normal Ig from patient C.J. The absorbed sera were then reacted at a final dilution of 1:50 in 2% BSA/PBS with the 7D11-idiotope–positive proteins (2A12 and 1H1), with mutated 7D11-idiotope–negative Id variants (1B11 and a to h), and with class-matched irrelevant human Ig or with C.J. normal Ig. Bound murine Ig was detected with HRPO-conjugated goat anti-murine IgG Abs (human-absorbed). The mean OD at 405 nm per vaccine group of 5 mice is given for each Id protein. Reactivity with the treatment MoAb 7D11 controlled for the presence of the specific idiotope in Id protein 2A12 and 1H1 and its absence in the Id variants a to h, 1B11, and the human control Igs. Sera from mice immunized with irrelevant Igs were negative in this assay. Clemens B. Caspar et al. Blood 1997;90:3699-3706 ©1997 by American Society of Hematology

Anti-Id titers in vaccinated and control mice measured by ELISA Anti-Id titers in vaccinated and control mice measured by ELISA. Sera were diluted 1:20 in BSA 2% in PBS, preabsorbed on polyclonal human Ig, and then serially diluted on plates with 1 of 4 Id proteins (7D11-idiotope–positive: 2A12, 1H1, and 2C12; 7D11-idio... Anti-Id titers in vaccinated and control mice measured by ELISA. Sera were diluted 1:20 in BSA 2% in PBS, preabsorbed on polyclonal human Ig, and then serially diluted on plates with 1 of 4 Id proteins (7D11-idiotope–positive: 2A12, 1H1, and 2C12; 7D11-idiotope–negative: 1B11) or with 1 of 3 class-matched irrelevant human Igs (same VH family as the Id). The irrelevant Igs were tested separately but are shown as a single bar since they were all equally negative. Reactivity of the target proteins with treatment MoAb 7D11 is shown. Clemens B. Caspar et al. Blood 1997;90:3699-3706 ©1997 by American Society of Hematology

Relative contribution of IgG isotypes to the anti-Id immune response measured by ELISA comparing the different vaccine formulations. Relative contribution of IgG isotypes to the anti-Id immune response measured by ELISA comparing the different vaccine formulations. Id protein was indirectly captured on the plates, and the sera of 5 mice per group were pooled and serially diluted. Bound murine IgG was detected with subclass-specific HRPO-conjugated Abs. Results are presented as relative units. Clemens B. Caspar et al. Blood 1997;90:3699-3706 ©1997 by American Society of Hematology

Protein sequence of the VH of Id proteins derived from the original and the relapsed tumor. Protein sequence of the VH of Id proteins derived from the original and the relapsed tumor. Amino acids were deduced from DNA sequences of the respective clones and compared against the previously published consensus sequence.10 Dashes in the sequence indicate identity with the consensus sequence, and differences are indicated by single letters. X indicates undetermined amino acids. Positions of the CDR are marked. Changes in CDR2 thought to be critical for the loss of binding to treatment antibody 7D11 are underlined twice. Clemens B. Caspar et al. Blood 1997;90:3699-3706 ©1997 by American Society of Hematology