image_1_Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming.PDF

<p>Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C⁺FcεRIγ^- long-lived “memory” NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor’ HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.</p

The gp41CHRTM antigen used for immunization.

<p><b>A</b>) Schematic representation of gp41 and of the regions present in gp41CHRTM. FP, fusion peptide; HR1, N-terminal heptad repeat; C-C loop, cysteine loop, HR2, C-terminal heptad repeat; MPER, membrane proximal external region; TM, transmembrane region. The residue numbers at the domain/region boundaries are given. <b>B</b>) Gel filtration chromatogram of recombinant gp41CHRTM, which elutes at 13.3 ml from the column, similar to the elution profile of a marker protein of 158 kDa. This indicates that gp41CHRTM is most likely trimeric and may have an elongated structure. The inset shows a Coomassie stained SDS-PAGE gel with the gp41CHRTM protein band at the left and a protein marker at the right with the marker protein sizes in kDa indicated at the right.</p

Molecular modeling of the 2H10-gp41 peptide interaction suggests that 2H10 W100 is oriented towards the membrane.

<p><b>A</b>) Molecular model of the gp41 peptide interaction with 2H10 produced by HADDOCK. Salt bridges and hydrogen bonds present in the top model and found in most of the top10 models are shown as dashed lines. <b>B</b>) Superpositioning of the Cα atoms of the gp41 peptide derived from the HADDOCK 2H10-peptide model onto the structure of a late fusion intermediate of gp41 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003202#ppat.1003202-Buzon1" target="_blank">[29]</a> shows that the CDR3 W100 is oriented towards the membrane and found in the same plane as gp41 residues W678, W680 and Y681. Note that there are no clashes between the 2H10 VHH and gp41.</p

Epitope mapping of 2H10 and binding to helical peptides.

<p><b>A</b>) The peptide Sym-903 was used for the replacement analysis with 2F5 and 2H10. Underlined cyan colored residues are recognized by the antibodies. <b>B</b>) Full substitution analysis of 2H10 epitope on gp41 MPER. The binding activity of VHH 2H10 at 2 ng/ml with a peptide is shown as a vertical line proportional to the Pepscan ELISA signal. Each group of 19 lines corresponds to the replacement set of all residues except cysteine for each amino acid position in the original 15-mer cyclic peptide: KNEQELLELDKWASL. Within each group of 19 lines the substitutions are in alphabetical order: based on the one-letter amino acid code (ADEFGHIKLMNPQRSTVWY) and the reactivity of the original nonapeptide is shown as a red line. The peptides contained an N- and C-terminal cysteine and were cyclised by addition of a T2-CLIPS <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003202#ppat.1003202-Timmerman1" target="_blank">[115]</a>. These results show that EQ at position 3 and 4 and LE at position 7 and 8 and K at position 11, cannot be replaced by another amino acid without loss of binding and are thus essential for binding 2H10. <b>C</b>) Binding of VHH 2H10 (100 ng/ml) and <b>D</b>) Mab 2F5 (1 ng/ml) to gp41-MPER peptides grafted in GCN4-like helical templates that contain a stabilizing Ile/Leu heptad repeat (top panel) and binding to peptides in which the zipper motif is mutated to destabilizing glycine residues (lower panel). Peptide sequences are all based on hybrids of HIV-1 gp41 and GCN4. Alignment is shown of peptide sequence with heptad repeat positions (top line). Residues essential for 2H10 binding according to the substitution analysis are underlined and residues essential for 2F5 binding are in green. Mutations are shown with one-letter codes and dots indicated that the residue is not changed.</p

2H10, bivalent 2H10 and mutant bi-2H10 IC50 titers against HIV-1 in TZM-bl cells.

<p>Neutralization activities of 2H10, bivalent 2H10, W100A mutant bi-2H10 and as a control 2F5 were determined against the indicated viruses. The type, clade, tier and the accession code of the envelope protein sequence are given for each virus strain. The wild-type bi-2H10s are named bi-15GS and bi-17GS and have a 15 and 17 residue linker respectively. The W100A mutant bi-2H10 is named W100A bi-15GS and has a 15 residue linker. The column labeled Tier indicates whether the virus is classified for 1,2, or 3 assessment of neutralizing antibodies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003202#ppat.1003202-Mascola3" target="_blank">[116]</a>. The column labeled Epitope sequence, shows the sequence that aligns with the part of the HBX2 sequence to which 2H10 is directed. The underlined, boldfaced residues are essential for the binding of 2H10 and are equal to the residues at the equivalent positions in HXB2, which was used for immunization. Residues in grey are at the position of an essential residue, but deviate from the HBX2 equivalent. • indicates IC50>50 µg/ml; the empty cells represent non-performed measurements.</p

Data collection and refinement statistics.

<p>Numbers in parentheses refer to the highest resolution shell. R_merge = Σ|I _ 〈I〉|/〈ΣI〉, where I = observed intensity. R_cryst = Σ|Fo _ Fc|/Σ|Fo|, where |Fo| = observed structure factor. Amplitude and |Fc| = calculated structure factor amplitude. R_free is R_cryst for 3% of reflections excluded from the refinement.</p

Surface plasmon resonance affinity measurements reveal essential and non-essential 2H10 residues for gp41 interaction.

<p><b>A</b>) 2H10 single domain mutants. The curves are labeled with the mutant codes in colors corresponding with the colors of the curves. The mutants R56A, R96A, E98A and R100A do not bind at all to gp140-92UG037. Fitting the curves with the two-state reaction algorithm yielded K_Ds of 8.0 nM for 2H10 WT and 10.1 nM for 2H10-W100A. The other fits, except for those of the non-binding mutants, yielded K_Ds between 5.5 nM for 2H10-S29F and 18.7 nM for 2H10-R71S <b>B</b>) bi-2H10-W100A mutant and wild type bi-2H10 on immobilized gp140-92UG037. Because the maximum responses units are slightly different for the two bi-2H10 molecules, the curves were normalized to the maximum response, to be able to compare the dissociation rates well.</p

Amino acid and DNA sequence of 2H10.

<p><b>A</b>) Amino acid sequence. Secondary structure assignment is based on the crystal structure. β-strands are depicted as blue arrows. CDR regions are indicated with CDR1 to CDR3 and are underlined. Residues depicted in bold were mutated. Mutants that still showed binding are shown in green and mutants that do not bind to the antigen anymore are shown in red. <b>B</b>) DNA sequence alignment of 2H10 with the germline V-gene from which it originated. The asterisks indicate identical nucleotides. The CDR1 and CDR2 coding regions are underlined. Codons with at least two point mutations are in boldface.</p