Figure 1.
DARPins interact specifically with CD4 and compete with gp120 for binding to CD4.
(A) Binding of DARPins, in the form of crude bacterial lysates, to CD4-IgG2 (black) is determined by ELISA and compared with binding to the capture antibody alone (gray). DARPins A–F show specific binding to CD4-IgG2, a property that was confirmed by further tests using the purified proteins. DARPins G and H reveal nonspecific binding whereas I and J are examples of library members that do not recognize the target protein. (B) Competition ELISA using soluble gp120 as competing ligand. Shown is binding of 200 nM of the CD4-specific DARPins 1.1 to 6.1 in competition with 0 nM, 50 nM and 800 nM gp120. Binding of DARPins alone was defined as 100% and background binding as 0%. (C) Binding of the DARPins to cellular CD4 was tested using A2.01 cells (CD4−), the CD4 expressing lines A3.01 cells, TZM-bl cells, CEM 5.25 cells and CD8-depleted PBMC as source of primary CD4 T cells. CD4-specific DARPins D3.1 (blue), D23.2 (red), and of a control DARPin, E3_5 (black), an unselected library member binding to the various cell types are shown. PE-labeled CD4-antibody (clone Q4120, Sigma) (green) was used as positive control and a PE-labeled goat-anti-mouse antibody (shaded in gray) as negative control. The shifts in fluorescence intensity correspond to the differences in affinities of the DARPins for CD4 (see Supporting Table S1). Representative data from 2–4 independent experiments are shown.
Table 1.
Dissociation constants of CD4 specific DAPRins as determined by surface plasmon resonance (SPR).
Figure 2.
The selected CD4-specific DARPins potently inhibit HIV entry.
(A) Effect of the six selected CD4-specific DARPins of the 1st series (D1.1–D6.1) on the entry of replication-competent viruses (7 R5 and 3 X4 users) on PBMC. 70% inhibitory concentrations (IC70) derived in representative individual experiments are reported. (B) Inhibition of HIV entry by 1st and 2nd series DARPins: CD4-specific DARPins of the 2nd series (red to yellow) are more potent inhibitors of HIV entry as DARPins of the 1st series (dark blue to light blue). Inhibition of JR-FL, SF-162 and NL4-3 infection in a pseudotyped virus entry assay on TZM-bl cells (upper panels) and the respective replication competent viruses in a PBMC based assay (lower panels) was probed in parallel. The unselected DARPin E3_5 (gray) was used as control in the TZM-bl based assay. Data points are means of virus replication measured in two replicate wells. See Supporting Table S1 for a summary of the derived IC50 and IC70 values in these assays.
Figure 3.
CD4-specific DARPins efficiently inhibit entry of both subtype B and C viruses.
(A) Graphical representation of the IC50 values of a 1st series DARPin, D3.1, and a 2nd series DARPin, D55.2, tested using env-pseudotyped viruses of subtype B (n = 9) and subtype C (n = 4) on TZM-bl cells. The following median IC50 values were determined: 45.4 nM for DARPin 3.1 (67.1 nM and 28.2 nM for clade B and C viruses, respectively) and 1.3 nM for DARPin 55.2 (1.3 nM and 0.7 nM for clade B and C viruses, respectively). (B) Inhibition curves of JR-FL pseudovirus used to assess synergy by analyzing combination indices (CI) in Figure 3C. Equipotent stocks of inhibitors were used to obtain comparable inhibition curves. Inhibitory effects of DARPin 25.2 (gray square) and the 2F5 mAb (gray triangle) alone are shown aside by side with the calculated (light gray, open triangle) and the actual observed inhibitory effect (black circles) of a 1∶1 mixture of the two inhibitor stocks. (C) DARPin 25.2 shows potent synergy in JR-FL pseudovirus inhibition in combination with neutralizing mAbs or entry inhibitors. CI for the inhibitory concentrations 70% and 90% (CI70, CI90) are represented for DARPin 25.2 in combination with mAbs IgG-b12, 2F5, 2G12, 4E10, the fusion inhibitor T-20, the anti-CCR5 mAb Pro140 and CD4-IgG2. Means from three independent experiments are shown. Error bars indicate the standard error of the mean.
Table 2.
Competition between DARPins and CD4-specific antibodies for binding to CD4.
Figure 4.
Characterization of the binding domain of CD4 specific DARPins.
(A) Competition between the fluorescently labeled DARPins D29.2HLX and D57.2HLX with unlabeled DARPins was analyzed by flow cytometry using CD4+ A3.01 cells. Compared to the control with no competitor (shaded in gray) competitive binding was observed for all CD4-specific DARPins (E3_5: blue; D23.2: light blue; D25.2 red; D27.2: orange; D29.2: brown; D55.2: green; D57.2: purple). The autofluorescence control is shown as dotted line (B) Binding of the same DARPins to human CD4 (hCD), murine CD4 (mCD4) and chimeric human CD4 containing murine D1 domain in the human CD4 context (hCD4mD1), indicating that all tested DARPins bind to the D1 domain of human CD4. The same coloring scheme for DARPins as in Figure 4A was used. MAb OKT4, specific for human CD4-D3, is shown as black dotted line.
Figure 5.
Interaction of DARPins with CD4 has no detectable effect on cell viability and stimulation.
(A) PBMC stimulation with IL-2 and OKT3 to induce proliferation was not altered in presence of the CD4-specific DARPin 55.2 (red), the non-binding DARPin E3_5 (blue) or absence of DARPin (gray) over a 4 day period. Proliferation was monitored by flow cytometry by determining CFSE dilution as a result of cell division. One representative experiment out of two is shown. (B) Activation of dendritic cells (DC) as determined by CD80 expression. Neither addition of DARPin 55.2 (red), nor of control DARPin E3_5 (blue), resulted in detectable upregulation of CD80 on DC over a 24 h period. One out of two independent experiments is depicted. (C) Prolonged treatment of T lymphocytes (4 days) and immature monocyte derived DC (24 h) with DARPin 55.2 (CD4 specific) and E3_5 (unselected control DARPin) has no effect on cell viability. Viability was determined by propidium iodide staining. DARPin concentration in the lymphocytes and DC cultures were 500 nM and 375 nM, respectively. (D) Interaction of DARPins with CD4 does not result in downregulation of surface CD4. Untouched peripheral blood CD4+ T cells were cultured in presence or absence of the indicated DARPins for 1 h, 3 h or 18 h at either 37°C (red line) or 4°C (blue line). Cells were stained for CD4 and the expression of surface CD4 was analyzed by flow cytometry. Shown is one representative experiment out of four.
Figure 6.
Effect of DARPin on T cell function and MHC class II interaction.
(A) The effect of the DARPin∶CD4 interaction was assessed in a binding assay based on rosette formation between CD4 and MHC class II expressing cells. Rosette formation was blocked by all tested CD4 specific DARPins (200 nM, shown are D25.2 and D55.2) or by the CD4-specific mAb Q4120 but not by the control DARPin E3_5. One out of two representative experiments is shown. (B) ELISpot assay to detect IFN-γ production by activated T cells showed no interference of DARPin 55.2 with CD4+ T cell activation. The response of two donors against CMV or streptokinase/streptodornase (SKSD) antigen was tested without DARPin (gray) and with nonspecific (blue) or CD4-specific DARPin (red) at 200 or 250 nM. One out of two independent experiments is depicted.
Figure 7.
Human CD4 specific DARPins can crossreact with macaque CD4 and inhibit entry of SIV.
(A) PBMC from rhesus macaques were incubated with the indicated DARPins at 4°C, labeled with an anti-His-tag antibody and detected by flow cytometry. MFI (mean fluorescence intensity) of DARPin staining on CD3+ cells, corrected for the negative control is shown. Standard deviations are indicated and represent n = 4 animals. (B) DARPin 25.2 potently inhibits entry of SIVmac239 in macaque PBMC. Shown are the IC90 values which were derived from neutralization assays performed using DARPin 25.2 and the control E3_5 on PBMC from three different animals.