Bespoke Pretargeted Nanoradioimmunotherapy for the Treatment of Non-Hodgkin Lymphoma

Non-Hodgkin lymphoma (NHL) is one of the most common types of hematologic malignancies. Pretargeted radioimmunotherapy (PRIT), the sequential administration of a bispecific antibody-based primary tumor-targeting component followed by a radionucleotide-labeled treatment effector, has been developed to improve the treatment efficacy and to reduce the side effects of conventional RIT. Despite the preclinical success of PRIT, clinical trials revealed that the immunogenicity of the bispecific antibody as well as the presence of competing endogenous effector molecules often compromised the treatment. One strategy to improve PRIT is to utilize bio-orthogonal ligation reactions to minimize immunogenicity and improve targeting. Herein, we report a translatable pretargeted nanoradioimmunotherapy strategy for the treatment of NHL. This pretargeting system is composed of a dibenzylcyclooctyne (DBCO)-functionalized anti-CD20 antibody (α-CD20) tumor-targeting component and an azide- and yttrium-90-(⁹⁰Y) dual-functionalized dendrimer. The physicochemical properties of both pretargeting components have been extensively studied. We demonstrated that an optimized dual-functionalized dendrimer can undergo rapid strain-promoted azide–alkyne cycloaddition with the DBCO-functionalized α-CD20 at the physiological conditions. The treatment effector in our pretargeting system can not only selectively deliver radionucleotides to the target tumor cells but also increase the complement-dependent cytotoxicity of α-CD20 and thus enhance the antitumor effects, as justified by comprehensive <i>in vitro</i> and <i>in vivo</i> studies in mouse NHL xenograft and disseminated models

Analysis of “Headless” AD-Cterm (3xThr) and GFP-AD-Cterm (3xThr) Assembly by EM

<div><p>(A and B) The scale bars are 100 nm, and all panels are on the same scale. (A) The “headless” AD-Cterm (3xThr) tail fragment assembled for 2 h. (B) Three images of GFP-AD-Cterm (3xThr) assembled for 2–5 min.</p> <p>(C) Analysis of GFP AD-Cterm (3xThr) (open circles) and GFP-AD-Cterm (3xAsp) (open squares) assembly by sedimentation.</p></div

Localization of GFP-RLC-Tail Fragment Constructs in Live Dividing <i>Dictyostelium</i> Cells

<p>The localization of several GFP-RLC-myosin tail fragments during and just after cytokinesis in live <i>Dictyostelium</i> cells are shown. GFP-myosin (row 1) is clearly localized to the early and late cleavage furrow of the dividing cell and to the back end of the resulting daughter cells. By contrast, GFP-RLC-AD-Cterm (3xThr) (row 2) is localized correctly only at the late stages of cytokinesis and in the back end of one daughter cell. GFP-RLC-AD-Cterm (3xAla) (row 3) is localized to the furrow as well as to the back end of a daughter cell, while GFP-RLC-AD-Cterm (3xAsp) (row 4) shows diffuse localization throughout cytokinesis. The scale bar is 10 μm and the time is indicated in min:sec.</p

Design and Assembly Characteristics of AD-Cterm-GFP

<div><p>(A) The AD-Cterm charge distribution (top) is aligned with the reverse charge distribution (bottom), showing the overall symmetry of the 196-a.a. charge repeat in this region of the tail.</p> <p>(B) Analysis of assembly by EM. The AD-Cterm (3xThr) tail fragment has GFP on the C-terminus (AD-Cterm-GFP [3xThr]). The scale bar indicates a distance of 100 nm. Shown are three images of AD-Cterm GFP (3xThr), assembled 2–5 min.</p> <p>(C) Analysis of assembly by sedimentation. The solubility of AD-Cterm-GFP (3xThr) and AD-Cterm-GFP (3xAsp) tail fragments constructs are compared to GFP-AD-Cterm (3xThr) and “headless” AD-Cterm (3xThr) tail fragments.</p></div

Domains and Charge Distribution within the Myosin Tail

<div><p>(A) The myosin head (1–818) and light chains are shown at the N terminus. In the coiled-coil tail, Ala 1 is red (1,348–1,530), the AD is blue (1,531–1,824), Ala 2 is purple (1,825–1,966), the C-terminal domain is green (1,967–2,116), and the remainder is black (819–1,347). Phospho-threonines (at positions 1,823, 1,833, and 2,029) are indicated by the letter T.</p> <p>(B and C) Plots of the average charge of each tail domain color coded as in (A). The y-axis is average charge; the x-axis is tail position. Aspartic acid and glutamic acid are assigned –1, lysine and arginine are assigned +1, and all other a.a. are assigned 0. The average charge in (B) was determined with a window size of 14 a.a., and the average charge in (C) was determined with a window size of 28 a.a.. Arrows highlight the 28 a.a. charge repeat in (B) and the 196 a.a. charge repeat in (C).</p> <p>(D) “Headless” AD-Cterm (3xThr) (1,531–2,116).</p></div

Multiple protein sequence alignment of FLASH and Lsm11 N-terminal domains.

<p>(A) Lsm11 N-terminal domain. (B) FLASH N-terminal domain. Alignment was carried out with Clustal Omega [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186034#pone.0186034.ref060" target="_blank">60</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186034#pone.0186034.ref061" target="_blank">61</a>] and the results displayed with ESPript [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186034#pone.0186034.ref062" target="_blank">62</a>]. Secondary structure for FLASH is based on the structure of human FLASH NTD, while that for Lsm11 is based on Psipred [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186034#pone.0186034.ref063" target="_blank">63</a>] secondary structure prediction of human Lsm11. Conserved residues are highlighted in red with white fonts, semi-conserved residues in red fonts, and other residues in black fonts. Blue dots indicate residues at the FLASH dimer interface. Gaps are indicated by dotted lines. Species abbreviations: Hs, <i>Homo sapiens</i> (human); Dr, <i>Danio rerio</i> (zebrafish); Dm, <i>Drosophila melanogaster</i> (fruit fly).</p

Characterization of “Headless” AD-Cterm Tail Fragments

<div><p>(A) Analysis of assembly by sedimentation. Fraction of soluble protein as a function of NaCl concentration is plotted for the constructs depicted adjacent to the graph. The solubility of “headless” AD-Cterm (3xThr) and AD-Cterm (3xAsp) are compared to the solubility of unphosphorylated and phosphorylated full-length myosin having the globular motor domain (full-length myosin data from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020356#pbio-0020356-Cote1" target="_blank">Cote and McCrea [1987]</a>).</p> <p>(B) Sedimentation equilibrium analysis of 52 μM “headless” AD-Cterm (3xThr) and AD-Cterm (3xAsp) in buffer containing 500 mM NaCl. The top graphs show the concentration distribution, fit, and residuals for AD-Cterm (3xThr), while the bottom graphs show the same data for AD-Cterm (3xAsp). The molecular weight obtained from the fit was 130 kDa for AD-Cterm (3xThr) and 120 kDa for AD-Cterm (3xAsp).</p> <p>(C) Thermal melts of “headless” AD-Cterm (3xThr) and AD-Cterm (3xAsp) in buffer containing 500 mM NaCl are shown as fraction of protein denatured as a function of temperature. The open circles are data for 50 μM “headless” AD-Cterm (3xThr), and the open squares are data for 50 μM “headless” AD-Cterm (3xAsp).</p></div

Tail Fragments Used for Truncation Analysis

<p>For tail fragments that include more than one domain, the name is determined by the first and last domain in the tail fragment. Phosphorylation sites are indicated in parentheses. 1xThr indicates that the fragment contains the threonine at a.a. 1,823; 2xThr indicates that the fragment contains the threonines at a.a. 1,823 and 1,833; and 3xThr indicates that the fragment contains the threonines at a.a. 1,823, 1,833, and 2,029. The same scheme is used to describe aspartic acid-containing constructs in the paper, except threonine is substituted with aspartic acid.</p

Molecular weights for FLASH NTD, Lsm11 NTD and their complex from SEC-MALS experiments.

<p>Molecular weights for FLASH NTD, Lsm11 NTD and their complex from SEC-MALS experiments.</p

Structures of FLASH NTD C54S/C83A dimer.

<p>(A) Structure of FLASH NTD C54S/C83A crystal form 1 (resolution 2.1 Å) showing FLASH dimer without the presence of a disulfide bond. (B) Structure of FLASH NTD C54S/C83A crystal form 2 (resolution: 2.6 Å) showing observable residues 52–70 that adopt a helical structure on protomer 1, and are less ordered in protomer 2. The LDLY motif essential for binding the HCC is shown as sticks. (C) Superimposition of the structures of FLASH NTD C54S/C83A crystal forms 1 (cyan) and 2 (gray) with wild-type FLASH NTD dimers (green).</p