The Impact of FcγRI Binding on Immuno-PET

Abstract

Antibodies are promising vectors for PET imaging. However, the high uptake of radioimmunoconjugates in nontarget tissues such as the liver and spleen hampers their performance as radiotracers. This off-target uptake can lead to suboptimal tumor-to-background activity concentration ratios, decreasing the contrast of images and reducing their diagnostic utility. A possible cause of this uptake is the sequestration of radioimmunoconjugates by immune cells bearing Fc-γ-receptors (FcγR) that bind to the Fc regions of antibodies. Methods: Since the heavy chain glycans influence the affinity of FcγR for the Fc domain, we set out to investigate whether radioimmunoconjugates with truncated glycans would exhibit altered binding to FcγRI and, in turn, improved in vivo performance. Using the HER2-targeting antibody trastuzumab, we synthesized a series of desferrioxamine-bearing immunoconjugates with differing glycosylation states and interrogated their FcγRI binding via surface plasmon resonance, enzyme-linked immunosorbent assay, and flow cytometry. Furthermore, we labeled these immunoconjugates with ⁸⁹Zr and explored their biodistribution in athymic nude, NSG, and humanized NSG mice bearing human epidermal growth factor receptor 2-expressing human breast cancer xenografts. Results: We observed a strong correlation between the impaired in vitro FcγRI binding of deglycosylated immunoconjugates and significant decreases in the in vivo off-target uptake of the corresponding ⁸⁹Zr-labeled radioimmunoconjugates (i.e., liver activity concentrations are reduced by ∼3.5-fold in humanized NSG mice). These reductions in off-target uptake were accompanied by concomitant increases in the tumoral activity concentrations of the glycoengineered radioimmunoconjugates, ultimately yielding improved tumor-to-healthy organ contrast and higher quality PET images. Conclusion: Our findings suggest that the deglycosylation of antibodies represents a facile strategy for improving the quality of immuno-PET in animal models as well as in certain patient populations.

Keywords: Fc receptor; Fc region; FcγRI; PET; glycans; radioimmunoconjugate.

FIGURE 1.

Antibody structure and FcγRI binding. (A) Detailed structure of an antibody with a magnified view of the glycans. (B) Cartoon depicting the influence of deglycosylation on the structure of the Fc region of an antibody and its binding to FcγRI.

FIGURE 2.

Preparation of the trastuzumab immunoconjugates. (A) Magnified view of cleavage sites for PNGaseF and EndoS. (B) Scheme of synthesis of the non–site-specifically and site-specifically modified immunoconjugates.

FIGURE 3.

Flow cytometry and ELISA analyses of DFO-bearing immunoconjugates. (A) Flow cytometry with BT474 cells to verify HER2-binding. (B) ELISA of the immunoconjugates (0.5 or 50 μg/mL) with huFcγRI (10 μg/mL) and mFcγRI (10 μg/mL). (C) Flow cytometry of the immunoconjugates with human U937 macrophages and mouse RAW 264.7 macrophages. Data represent mean ± SD; each immunoconjugate was tested in triplicate (distinct samples). MFI = mean fluorescence intensity. Data were analyzed by unpaired, 2-tailed Student t test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 4.

SPR analysis of interaction between the immunoconjugates and recombinant huFcγRI. (A) Binding affinity and kinetic rate constants for each immunoconjugate are shown in sensorgram plots. (B) Bar graphs illustrating correlation between deglycosylation and binding affinity (K_D), half-life (t_1/2), on-rate (k_a), and off-rate (k_d). Statistically significant relationships are indicated with asterisks. * = P < 0.05, ** = P < 0.005, *** = P < 0.0005, **** = P < 0.00005, ***** = P < 0.000005.

FIGURE 5.

PET imaging in nude and NSG mice. Planar (left) and maximum-intensity-projection (MIP, right) PET images of nude (A) and NSG (B) mice bearing subcutaneous BT474 xenografts at 24, 48, and 120 h after injection. Values in white represent tumoral activity concentrations in %ID/g ± SD as determined via region-of-interest analysis (n = 4).

FIGURE 6.

In vivo biodistribution profile for ⁸⁹Zr-DFO-^nsstrastuzumab, ⁸⁹Zr-DFO-^nsstrastuzumab-PNGaseF, and ⁸⁹Zr-DFO-^sstrastuzumab-EndoS at 24, 48, and 120 h after injection in NSG mice bearing subcutaneous BT474 xenografts. Data represent mean ± SD, n = 4. Data were analyzed by 1-way ANOVA: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 7.

In vivo results in humanized NSG mice. (A) Planar (left) and maximum-intensity-projection (MIP, right) PET images of humanized NSG mice bearing subcutaneous BT474 xenografts at 24, 48, and 120 h after injection. Values in white represent tumoral activity concentrations in %ID/g ± SD as determined via region-of-interest analysis (n = 4). (B) In vivo biodistribution profile for ⁸⁹Zr-DFO-^nsstrastuzumab and ⁸⁹Zr-DFO-^nsstrastuzumab-PNGaseF at 120 h after injection in humanized NSG mice bearing BT474 tumors. Data represent mean ± SD, n = 4. Data were analyzed by unpaired, 2-tailed Student t test with a Welch’s correction: *P < 0.05, **P < 0.01.