CD4+ levels control the odds of induction of humoral immune responses to tracer doses of therapeutic antibodies.

Rapidly increasing number of therapeutic antibodies are being repurposed to imaging probes for noninvasive diagnosis, as well as monitoring during treatment or disease recurrence. Though antibody-based imaging involves tracer doses (~3 log lower than therapeutic doses), and immune responses are severely reduced in patients with impaired immunity, formation of anti-tracer antibodies (ATA) has been observed hampering further diagnostic monitoring. Here, we explored the potential to develop humoral responses to intravenously administered tracer dose of a monoclonal antibody F(ab΄)2 fragment, and associated with host related immune measures in 49 rhesus macaques categorized into healthy (uninfected controls), SIV-progressors, SIV non-progressors, or total body irradiated (TBI). Antibody fragment administered in tracer amount (~100μg) induced immune responses with significantly lower odds in SIV-progressors or TBI macaques (P<0.005) as compared to healthy animals. Peripheral blood (PB) CD4+ cell counts, but not CD20+ cell levels, were associated with significantly higher risk of developing a humoral response (P<0.001). Doubling the PB CD4+ counts is associated with an odds ratio of developing an immune response of 1.73. Among SIV-infected animals, CD4+ cell count was a stronger predictor of immune response than plasma SIV-RNA levels. Both SIV-progressors and TBI macaques showed higher odds of responses with increasing CD4+ counts, however when compared to healthy or SIV non-progressors with similar CD4+ count, they were still functionally incompetent in generating a response (P<0.01). Moreover, presence of ATA in systemic circulation altered the in vivo biodistribution by increasing hepatic uptake and decreasing plasma radiotracer clearance, with minimal to no binding detected in targeted tissues

Correlation between radio-HPLC and plasma binding assay.

<p>Strong correlation was observed between plasma binding assay and HPLC assay. Samples were assayed with either F(ab΄)₂-CD4R1 antibody (brown dots) or F(ab΄)₂-huOKT4A antibody (red dots). For samples assayed with F(ab΄)₂-CD4R1, the bootstrap estimate and 95% confidence interval of the Spearman rank correlation between PBA-ratio and HPLC-HM-ratio was -0.88 (-0.91, -0.84), and between PBA-ratio and HPLC-Fab’2-ratio was 0.90 (0.85, 0.93). Similar results were observed for samples assayed with F(ab΄)₂-huOKT4A.</p

Immune response outcome of total 118 exposures for all 49 macaques receiving F(ab΄)₂ radiotracers.

<p>Immune response outcome of total 118 exposures for all 49 macaques receiving F(ab΄)₂ radiotracers.</p

Maximum intensity projection SPECT images.

<p>Rhesus macaques that developed antibody response to the radiotracer after baseline exposure were subsequently imaged while anti-tracer antibodies were present in the plasma in (A) healthy macaque imaged at 48 hours post-radiotracer injection with intact-huOKT4A labelled with ¹¹¹In and scanned using Triad88 (Trionix) camera, (B) SIV-TK infected non-progressor imaged at 4 hours post-radiotracer injection with F(ab΄)₂-huOKT4A labelled with ^99mTc and scanned using Triad88 (Trionix) camera, and (C) healthy macaque that underwent total body irradiation prior to 2^nd exposure and imaged at 4 hours post-radiotracer injection with F(ab΄)₂-CD4R1 labelled with ^99mTc and scanned using Symbia T2 (Siemens) camera. Images show increased hepatic uptake and altered biodistribution with minimal to no binding observed in secondary lymphoid organs when imaged in the presence of anti-tracer antibodies. Within each panel, images were adjusted (standardized) for injected dose and body weight. Tissue uptakes were converted to RAINBOW color scale as shown in color bar.</p