Volume 3, Issue 5, Pages 768-778 (May 2001) Specific Depletion of Human Anti-adenovirus Antibodies Facilitates Transduction in an in Vivo Model for Systemic Gene Therapy  Amena Rahman, Van Tsai, Ann Goudreau, Jeremy Y. Shinoda, Shu Fen Wen, Muralidhara Ramachandra, Rob Ralston, Dan Maneval, Drake LaFace, Paul Shabram  Molecular Therapy  Volume 3, Issue 5, Pages 768-778 (May 2001) DOI: 10.1006/mthe.2001.0316 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Adenovirus capsid column can remove anti-adenoviral antibodies. Depletion of anti-Ad-Ab following affinity chromatography with Ad-capsid column. Ad-primed serum and anti-Ad-capsid depleted serum were used as primary antibodies in Western analysis of pooled DEAE/hydroxyapatite chromatography purified capsid components (hexon, fiber, penton, and IIIA) (lane 1) or whole adenovirus particle (lane 2). MW marker is SeeBlue prestained standard from Invitrogen (lane 3). Molecular Therapy 2001 3, 768-778DOI: (10.1006/mthe.2001.0316) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Adenovirus capsid column can reduce titer of neutralizing antibodies. (A) Neutralizing Ab titers of immune sera primed either once or twice with adenovirus (5 × 1010 particles total) by subcutaneous administration. Sera were collected and pooled from animals 7–10 days postinjection (for single injection) (black bars) or alternatively 14 days after injection (both one and two adenovirus administrations, white and gray bars, respectively). Titers from undepleted immune sera, adenovirus capsid column depleted sera, and sera from vehicle control-injected mice are shown. This is representative of three similar experiments. (B) Comparison of adenovirus capsid column with protein A or KappaLock column. Neutralizing activity was seen to be most effectively reduced by passing adenovirus-primed serum (single injection, serum harvested 14 days after injection) through an adenovirus capsid column. KappaLock, which removes both IgG and IgM, was seen to be more effective than protein A, which primarily removes IgG. Molecular Therapy 2001 3, 768-778DOI: (10.1006/mthe.2001.0316) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Heterogeneity of rAd antibodies in murine immune sera. (A) Effect of individual capsid components in reducing neutralizing Ab titer. Columns were made with a mixture of major capsid proteins, individual indicated capsid components, or a nonspecific protein, BSA. The ability of each column to reduce nAb titer was tested and neutralizing activity was measured in duplicate samples with equivalent amounts of adenovirus-specific antibody) of both the depleted sera and the antibodies eluted off the columns. Variations were negligible in each group, hence error bars are not shown. (B) Isotyping of anti-adenovirus antibodies bound to individual capsid components. A capsid-affinity column or individual capsid column was used to purify anti-adenoviral antibodies from adenovirus-immunized mouse sera. Equal amounts of the affinity-purified antibodies obtained from this procedure were subsequently used in an ELISA-based isotyping kit. Since equal volumes of antibody were used for each assay condition, the spectrophotometric OD readings can be used to gauge the relative distribution of the various immunoglobulin types tested. Molecular Therapy 2001 3, 768-778DOI: (10.1006/mthe.2001.0316) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Removal of adenovirus nAb using an adenovirus capsid column shows transduction in vivo after BGCG systemic delivery. (A) Neutralizing activity assay of passively immunized naïve mice. Naïve mice were passively immunized with adenovirus capsid-depleted serum, adenovirus-primed serum, or affinity-purified adenovirus antibodies (40 and 80 μg) and a vehicle control. Neutralizing activity is depicted as the titer at which 50% neutralization of adenovirus occurs. Serum was collected and assayed for neutralizing activity following passive immunization (black bars), challenge with 5 × 1010 of BGCG (gray bars), and finally 3 days postchallenge with BGCG (diagonally hatched bars). (B) Transgene expression. Livers were harvested from mice in all four experimental cohorts to assay for transgene expression. X-gal-stained sections of livers from mice passively immunized with vehicle, adenovirus capsid-depleted serum, primed serum (undepleted serum), and antibody eluted from Ad-capsid column (Ad-Ab) are shown. Insets show 10× original magnification, whereas the panels depict 100× original magnification. (C) Quantitative PCR and RT-PCR analysis of β-gal transgene to assess transduction efficiency. PCR (DNA, gray bar) and RT-PCR (RNA, black bar) results from livers collected 3 days post BGCG challenge from animals passively immunized with vehicle control, adenovirus-primed immune serum (rAd-primed sera), protein A-depleted primed serum, and capsid-depleted primed serum. β-gal DNA and RNA are expressed as MEQ/mg of liver tissue (see Materials and Methods). Molecular Therapy 2001 3, 768-778DOI: (10.1006/mthe.2001.0316) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Sera from normal donors display various patterns of anti-adenovirus neutralizing antibody titers. Serum samples were collected from 52 normal donors at three different times. Serum from each donor was assayed for anti-adenovirus neutralizing activity. Three patterns of neutralizing titers emerged: titers greater than 320, titers less than 320, and undetectable titers. Molecular Therapy 2001 3, 768-778DOI: (10.1006/mthe.2001.0316) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 6 Adenovirus capsid-affinity column significantly lowers anti-adenovirus neutralizing antibody titers in human sera. Sera from normal donors were pooled according to their titer range. Those with titers less than 320 are designated low titer and those with titers greater than 320 are designated high titer. Adenovirus capsid-affinity column can deplete anti-adenovirus neutralizing antibodies in both low- and high-titer groups. Antibodies eluted from the capsid columns constitute most of the neutralizing activities seen in these different pools of human serum. Molecular Therapy 2001 3, 768-778DOI: (10.1006/mthe.2001.0316) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 7 Correlation of in vitro nAb titers and in vivo transduction. NAb titers were assessed at different times in sera of SCID/Beige mice that were passively immunized with human sera pre- and post-capsid column following systemic administration of BGCG. Neutralizing antibody titers in sera of SCID/Beige mice passively immunized with pooled low-titer human sera (A) and pooled high-titer sera (B) are shown. 3 h post-passive immunization (gray bars), 2 h post-iv challenge with 5 × 1010 BGCG (white bars), and 3 days post-iv challenge with adenovirus (black bars). Vehicle controls, undepleted serum, protein A-depleted serum, and Ad-capsid-depleted serum are shown. Livers were harvested from mice in all four experimental cohorts. PCR and RT-PCR data are expressed as MEQ (see Materials and Methods) for the quantification of β-galactosidase. DNA copies (gray bar) and RNA copies (black bar) are from mice passively immunized with low-titer serum (C) and high-titer serum (D). Vehicle controls, undepleted serum, protein A-depleted serum, and Ad-capsid-depleted serum are shown. This is representative of three experiments. Molecular Therapy 2001 3, 768-778DOI: (10.1006/mthe.2001.0316) Copyright © 2001 American Society for Gene Therapy Terms and Conditions