There was also no apparent decrease in cell-surface levels of CD4, although we could not accurately quantify levels of cell surface NONMEM software was used to perform the analysis of the data with the IV 2-compartment model

By the six-hour time level, the plasma levels of DARPin 57.two ended up already beneath the level of detection of the assay (<50 pM) for the low dose group of macaques.870281-34-8 Free DARPin was still detectable in the plasma of animals that received the high dose of DARPin 57.2 at 6 hours post-injection, but fell below the detection limit by 24 hours post-injection. The log scale DARPin 57.2 concentration vs. time plot did not yield a straight the rate of clearance of DARPin 57.2 from plasma is rapid and dose-dependent. Free DARPin 57.2 concentration in macaque plasma sampled at various times post injection was measured using a sandwich ELISA. Concentrations (mean6SEM) for both DARPin 57.2 doses are shown at each indicated time point. 6 animals received 300mg/kg and 3 animals received 30mg/kg of DARPin 57.2 line, suggesting a 2-compartment pharmacokinetic model (Figure 2). The clearance rate (CL), volume of distribution (Vd) and a-half-life (T1/2) of DARPin 57.2 all increased with the increase in input DARPin dose (Table 3). Since we used macaques that were previously infected with SHIV-RT, we looked at the plasma viral loads at various time points after the injection with the high dose of DARPin 57.2 to check if the one-time DARPin treatment had an effect on the infection. 3 of 6 animals had high pre-treatment virus loads (.105 range 130,00040,000 copies/ml), 2 of 6 animals had intermediate virus loads (,104 range 9,000,500 copies/ml), and 1 animal had only 200 copies/ml (Table 1). As predicted, a single dose of DARPin 57.2 did not alter the plasma viral load compared to the DARPin E3_5 controls (Figure 3). Decreases of up to 1 log in plasma SIV RNA were observed in some animals but this was not sustained and seen in both groups of animals. To be sure that the lack of effect on SHIV-RT in vivo was not due to an inability of DARPin 57.2 to block SHIV-RT infection, the inhibition of SHIV-RT infection of activated PBMCs by DARPin 57.2 was measured. DARPin 57.2 also blocked the infection of macaque PBMCs with SHIV-RT (IC90,10nM, Figure S2). There were also no sustained changes in numbers of circulating CD4+ T cells (data not shown).To investigate whether the circulating CD4-specific DARPin would bind CD4+ cells in vivo we sampled blood and lymph nodes at various time after injection. Initially, PBMCs were co-stained for CD4 and penta-His. Virtually all cells in the CD4+ gate were DARPin-positive and expressed similar levels of DARPin to that Table 3. Pharmacokinetic parameter estimates for DARPin 57.2 bound in vitro 30 minutes after DARPin 57.2 injection (Figure 4A). The level of staining was dependent on the input dose of DARPin 57.2, as seen in our in vitro binding studies (Figure 1E). The staining was weaker by 6 hours post-injection (but all CD4+ cells were DARPin positive) and was down to background levels by 24 hours for the low dose group. Weak staining was still detected at the 24 hour mark after the injection with 300mg/kg of DARPin 57.2 (Figures 4A and S3). To ensure that the DARPin binding detected in the PBMCs was not simply due to the cell-free DARPins in the plasma binding to cells during the overnight shipment of the blood to the laboratory, we carried out the His staining on whole blood samples immediately upon collection from the animals. Similar to the PBMC results, His-positive cells were detected in whole blood 30 minutes after injection with DARPin 57.2, with the level of staining decreasing by 6 hours and being undetectable after 24 hours (Figure 4B and S3). Therefore, there was little contribution from cell-free DARPins in the plasma DARPin binding to cells during the shipment. In order to determine if the DARPins circulated from the blood to the lymph nodes, superficial lymph nodes were first sampled 24, 48 and 168 hours after the injection of DARPin 57.2 vs the control DARPin E3_5. Lymph node cells were stained for CD4 and penta-His. After 24 hours, binding by DARPin 57.2, but not by DARPin E3_5, was detected on CD4+ cells (Figure 4C). As seen in the peripheral blood, no DARPin binding was seen at later time points. The level of His staining (DARPin binding) was comparable to the levels seen at the 24 hour time point in the blood (compare the MFIs in panel 4A and 4C for the 300mg/kg dose). Therefore, we examined earlier time points to ascertain whether higher levels were also present in the lymph nodes earlier (as in the blood). To do this, a separate set of animals were injected with 300mg/kg DARPin 57.2 and lymph nodes and blood collected 0.5, 6, and 24 hours later. Analysis of these acute time points revealed that CD4+ cells in the lymph nodes were already bound by DARPin 57.2 30 minutes after the injection (Figure 4D). The levels of DARPin binding to the cells was lower in the lymph nodes than in the peripheral blood after 30 minutes, but was at similar levels after 6 and 24 hours (Figure 4D). As in blood, the entire CD4+ T cell population in the lymph nodes bound the DARPins. To better understand the mechanism by which the levels of cell bound DARPin 57.2 were decreasing with time, we isolated macaque PBMCs 30 minutes after injection with DARPin 57.2 and cultured them in vitro. Levels of cell-bound DARPin 57.2 decreased with time during the in vitro culture (Figure 5A). The cells retained DARPin 57.2 longer in culture than they did in the peripheral blood: some DARPin-bound cells were still present after 5 days in culture, while none were detected in the peripheral blood by 48 hours (Figures 4B and 5A). The decrease in DARPin binding was not due to a change in CD4+ cell numbers (Figure 5B). There was also no apparent decrease in cell-surface levels of CD4, although we could not accurately quantify levels of cell surface NONMEM software was used to perform the analysis of the data with the IV 2-compartment model. Means (SD) for the following parameters are shown: t1/2, half-life (given for the two phases, as indicated) Vd, volume of distribution, CL, clearance rate AUC, area under the concentration-time curve. AUC was calculated using the GraphPad Prism software.A single injection with a CD4-specific DARPin 57.2 does not lead to a change in plasma RNA viral load in rhesus macaques infected with SHIV-RT. Plasma samples were taken from the macaques at indicated timepoints after an injection with 300mg/kg of DARPin 57.2 and E3_5. SIV RNA copy numbers were determined by qRT-PCR. Each line represents an individual animal.CD4 expression, because DARPin 57.2 partially blocks binding by CD4 MAbs (Figures 5C and S1). More extensive analyses were performed on the blood cells to establish which CD4+ cells bound the DARPins in vivo. Multicolor staining was performed on blood cells collected at different times post DARPin injection. In vivo, the pattern of DARPin 57.2 binding to cells resembled that seen in vitro (Figures 1B and 6). All CD4+ (but not CD42) cells bound DARPin 57.2 proportional to the level of surface CD4 expressed. On all cell types, the greatest binding was seen at the earliest time point (30 minutes) after injection, the amounts decreasing similarly thereafter irrespective of cell type. DARPin 57.2 binding only persisted on CD4+ T cells after 24 hours, albeit at low levels. Therefore, DARPin 57.2 functions normally in vivo, binding to target CD4+ cells in a specific, dose-dependent manner.Small proteins such as DARPins may prove to be a useful alternative to antibodies, which have disadvantages such as high cost, low stability and poor tissue penetration. Experiments done in vitro demonstrated that DARPins can bind their targets with high affinity and high selectivity [8,146]. A proof of concept study was needed to investigate whether these small proteins demon-injection of macaques with DARPin 57.2 results in transient binding to CD4+ cells. A. Blood samples were taken from macaques at indicated timepoints after an injection with DARPin. PBMCs were isolated, labeled with antibodies to CD4 and to penta-His and analyzed by flow cytometry. The MFI (mean6SEM) of His staining on CD4+ cells within a total leukocyte gate is shown for animals injected with 300mg/kg DARPin 57.2 (n = 6), 30mg/kg DARPin 57.2 (n = 3) or DARPin E3_5 (n = 5). Data from both doses of DARPin E3_5 was combined for simplicity. B. Whole blood samples taken after DARPin injection were immediately labeled with the anti-His antibody and analyzed by flow cytometry. The MFIs (mean6SEM) of His+ cells are shown for animals injected with 300mg/kg DARPin 57.2 (n = 6), 30mg/kg DARPin 57.2 (n = 3) or DARPin E3_5 (n = 5). Lymph nodes (C and D) and blood (D) were taken from macaques at indicated timepoints after an injection with DARPin. Isolated cells were labeled with antibodies to penta-His and CD4 and analyzed by flow cytometry. MFIs (mean6SEM) of gated CD4+ cells are shown for animals injected with 300mg/kg DARPin 57.2 (C, n = 7 D n = 4) or DARPin E3_5 (C, n = 6).Bound DARPin 57.2 is not retained long term on macaque cells. PBMCs were collected 30 minutes after DARPin injection and cultured in vitro for 7 days. Cells were harvested at the indicated timepoints, labeled for His and CD4 and analyzed by flow cytometry. (A) The MFIs (mean6SEM) of His staining on CD4+ cells are shown for animals injected with 300mg/kg DARPin 57.2 (n = 4) or DARPin E3_5 (n = 3). Baseline samples were taking from the same animals immediately before the injections (pre). The percentage of CD4+ cells (B) and the MFIs of CD4 expression (C) for the samples described in panel A were also measured over time strate comparable qualities in vivo. We previously described the identification of human CD4-specific DARPins that specifically bound to human and macaque CD4 and inhibited HIV and SIV infection in vitro [8]. In this study, we characterized a more potent second series CD4-specific DARPin 57.2 for macaque reactivity and investigated its activity after a single intravenous injection into rhesus macaques. DARPin 57.2 was chosen due to its higher levels of binding and more effective inhibition of SIV infection in vitro. Here we demonstrate that it rapidly binds CD4+ cells in vivo. DARPinbound cells were detected 30 minutes after the intravenous injection not only in the peripheral blood, but also in the lymphoid tissue. DARPin could be reaching the tissue in its free form and binding resident cells there and/or we could be detecting cells that have bound DARPin in the periphery and migrated to the lymph node. If only the latter scenario were true, one would expect to find at least some tissue-resident CD4+ cells that are DARPin-negative, while we find that all of the CD4+ cells in the lymph node are bound by the DARPin 57.2. Therefore, it is likely that free DARPins can rapidly reach the lymphoid tissue and bind resident cells there. We found that the clearance of DARPin 57.2 from the plasma was rapid, but the rate increased with the increase in dose, suggesting that DARPin elimination is a saturable process. Similarly rapid plasma clearance rate was also observed when a HER2-specific DARPin of a comparable size was used in mice [17]. The dynamics of the loss of CD4+ cell-bound DARPins were slower, but generally similar to that of the free DARPin in the plasma. The binding of DARPin 57.2 to primary cells was also short-lived when cells were cultured in vitro. Additional studies are needed to determine what happens to the CD4-specific and other DARPins after they bind their targets. The binding levels of the DARPin 57.2 were determined by the levels of cell surface CD4 expression. Hence, binding of the DARPin 57.2 to CD4 on various cell types was likely not affected by possible cell type-dependent differences in CD4 conformations or associations of CD4 with other cell surface molecules. The vast majority of DARPin 57.2-binding cells in macaques were CD4+ T cells, which also had the highest levels of CD4. Unlike human monocytes, macaque monocytes have relatively low levels of CD4 expression, which resulted in low levels of binding by DARPin 57.2. In contrast, macaque CD123+ pDCs had higher levels of CD4 expression than monocytes or myeloid DCs [18] and consequently high levels of DARPin 57.2 binding. The functional importance of CD4 expression by these cell types and the precise role these cell types play during retroviral infection is not yet fully understood. Monoclonal antibodies to CD4 can potently inhibit infection of T cells by primary HIV-1 isolates in vitro, including strains that are resistant to other types of HIV inhibitors [19]. One of the CD4specific MAbs, Ibalizumab, substantially reduces HIV-1 RNA levels in infected people and is currently in advanced clinical trials [20]. However, single infusions with Ibalizumab did not lead to decreases in plasma viral loads at doses below 3mg/kg [21]. In our study the highest dose of DARPin 57.2 was 300mg/kg, which is slightly lower than 3mg/kg of antibody in terms of molarity. Hence, given the faster plasma clearance rate of the DARPins, a reduction in the plasma viral load was not a likely outcome. Since DARPin 57.2 binding to macaque leukocyte cell types in vivo. PBMCs were prepared from blood samples taken from macaques at 30 minutes, 6 hours or 24 hours after an injection with 300mg/kg of DARPin 57.2 (n = 6) or DARPin E3_5 (n = 4). Cells were stained the various antibody combinations (same as in Figure 1C) to define the indicated subsets. An average of MFIs (mean6SEM) of the anti-His staining for each indicated cell type is shown the animals used in the study were already infected with SHIVRT, we checked their plasma RNA levels and confirmed our predictions that a single injection of DARPin 57.2 had no effect on the SHIV-RT plasma RNA levels. CD4-specific DARPin 57.2 did not cause a rapid flux of CD4+ T cells from the lymphoid tissue into peripheral blood, which was seen in Ibalizumab-treated people and macaques [21,22]. 10676638Possibly this is caused by the cellular reaction to rapid internalization of CD4, which is induced by antibody binding but not by DARPin binding [8,23]. Antiviral therapies targeted at CD4 can potentially have an undesired immunomodulatory effect, by either signaling through CD4 or blocking the interactions with MHC class II. CD4-specific DARPins were shown to have no effect on T cell proliferation, DC activation or overall cell viability in vitro [8]. Although unexpected, we were not yet able to assess DARPin 57.2 effect on immunological functions in vivo due to the limitations of the animal samples, but it will be looked at closer in the future studies. Overall, our data demonstrates that DARPins can rapidly, potently and selectively bind their targets in vivo, even in the tissues, opening the doors for future development for novel treatment of HIV and other conditions. The high stability and low production costs could also make antiviral DARPins a promising candidate for the HIV prevention treatment, sorely needed due to the lack of effective vaccines. Topical vaginal microbicides can prevent mucosal transmission of the virus [11,246]. Blocking the gp120 interaction with CD4 by a small molecule BMS-378806 protected most macaques from vaginal SHIV challenge, verifying that the CD4-envelope interaction is a possible target for a microbicide [25]. There are few CD4+ cells in the vaginal mucosal tissue, which makes it difficult to reliably quantify the kinetics and the effectiveness of CD4-specific DARPin binding after it is applied topically.