Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases

Abstract

The voltage-gated Kv1.3 K(+) channel is a novel target for immunomodulation of autoreactive effector memory T (T(EM)) cells that play a major role in the pathogenesis of autoimmune diseases. We describe the characterization of the novel peptide ShK(L5) that contains l-phosphotyrosine linked via a nine-atom hydrophilic linker to the N terminus of the ShK peptide from the sea anemone Stichodactyla helianthus. ShK(L5) is a highly specific Kv1.3 blocker that exhibits 100-fold selectivity for Kv1.3 (K(d) = 69 pM) over Kv1.1 and greater than 250-fold selectivity over all other channels tested. ShK(L5) suppresses the proliferation of human and rat T(EM) cells and inhibits interleukin-2 production at picomolar concentrations. Naive and central memory human T cells are initially 60-fold less sensitive than T(EM) cells to ShK(L5) and then become resistant to the peptide during activation by up-regulating the calcium-activated K(Ca)3.1 channel. ShK(L5) does not exhibit in vitro cytotoxicity on mammalian cell lines and is negative in the Ames test. It is stable in plasma and when administered once daily by subcutaneous injection (10 mug/kg) attains "steady state" blood levels of approximately 300 pM. This regimen does not cause cardiac toxicity assessed by continuous EKG monitoring and does not alter clinical chemistry and hematological parameters after 2-week therapy. ShK(L5) prevents and treats experimental autoimmune encephalomyelitis and suppresses delayed type hypersensitivity in rats. ShK(L5) might prove useful for therapy of autoimmune disorders.

Fig. 1

Generation of a selective Kv1.3 blocker. A, molecular model of ShK based on the published NMR structure. The Lys²², critical for channel blockade, is highlighted in orange. l-pTyr was attached to the α-amino group of Arg¹ of ShK (highlighted in cyan) through an Aeea linker (right). The structures of the linker and l-pTyr were modeled with AM1 in Hyperchem. B, effect of ShK (top) and ShK(L5) (bottom) on Kv1.3 and Kv1.1 currents in stably transfected cells. C, dose-dependent inhibition of Kv1.3 (open symbols) and Kv1.1 (closed symbols) by ShK (blue) and ShK(L5) (red). K_d values on Kv1.3 = 10 ± 1 pM (ShK) and 69 ± 5 pM (ShK(L5)); K_d values on Kv1.1 = 28 ± 6 pM (ShK) and 7.4 ± 0.8 nM (ShK(L5)). D, time course of wash-in and wash-out of ShK(L5) on Kv1.3. Cells were held at a holding potential of -80 mV and depolarized for 200 ms to 40 mV every 30 s. E, K_d values shown for inhibition of Kv1.3 and Kv1.1 by ShK analogs. K_d values for ShK-F6CA and ShK-Dap²² are from published sources (Kalman et al., 1998; Beeton et al., 2003; Chandy et al., 2004).

Fig. 2

ShK(L5) preferentially suppresses the proliferation of human T_EM cells. Human PBMCs (A) and a human T_EM line (B) were stained with antibodies against CD3, CD45RA, and CCR7. Staining intensities of CD45RA and CCR7 were determined by flow cytometry in the CD3⁺-gated population. C, dose-dependent inhibition by ShK (blue) and ShK(L5) (red) of [³H] thymidine incorporation by PBMCs (open symbols, a mixture of naive/T_CM cells) and T_EM cells (closed symbols) stimulated for 48 h with anti-CD3 antibody. D, preactivated human PBMCs (naive/T_CM cells) that up-regulate KCa3.1 expression (Ghanshani et al., 2000) become resistant to ShK(L5) inhibition when reactivated with anti-CD3 antibody. These cells have previously been shown to become sensitive to the K_Ca3.1-specific inhibitor TRAM-34 (Ghanshani et al., 2000).

Fig. 3

ShK(L5) preferentially suppresses the proliferation of rat T_EM cells. A, CD45RC staining of rat splenic T cells (left) and PAS T cells (right) detected by flow cytometry. B, Kv1.3 currents exhibited by quiescent (top) and myelin antigen-activated (bottom) PAS T cells. C, flow cytometry profiles of ShK-F6CA-staining in quiescent (top) and myelin antigen-activated (bottom) PAS T cells. Unstained cells (black lines) and cells stained with ShK-F6CA (green filled). Competition of ShK-F6CA staining by unlabeled ShK(L5) is red-filled. D, confocal images of Kv1.3 immunostaining in quiescent (top) and myelin antigen-activated (bottom) PAS T cells. Statistical analysis was carried out using the Mann-Whitney U test. E, dose-dependent inhibition by ShK (blue) and ShK(L5) (red) of [³H]thymidine incorporation by rat naive/T_CM (open symbols) and T_EM (closed symbols) cells activated with Con A (1 μg/ml). F, dose-dependent inhibition by ShK (blue) and ShK(L5) (red) of IL2 secretion by PAS T cells 7 h after stimulation with MBP. G, ShK(L5)-induced inhibition of myelin-antigen triggered [³H]thymidine incorporation by PAS T cells (open symbols) is reversed by the addition of 20 units/ml IL2 (closed symbols).

Fig. 4

Circulating half-life and stability of ShK(L5). A, known amounts of ShK(L5) were added to rat serum (Δ) or to PBS (■) and blocking activity was determined on Kv1.3 channels stably expressed in L929 cells. B, a single dose of 200 μg/kg of ShK(L5) was injected subcutaneously into four rats. Blood was drawn at the indicated times and serum was tested by patchclamp to determine the amount of ShK(L5). C, data fit to a single exponential decay. Half-life ≈ 50 min. D, five Lewis rats received single daily subcutaneous injections of 10 μg/kg/day ShK(L5) for 5 days. Blood was drawn each morning (24 h after the previous injection) and tested for blocking activity on Kv1.3 channels by patch-clamp. E, rats received a single dose of 10 μg/kg ShK(L5) either subcutaneously (open bars; n = 4) or intravenously (closed bars; n = 4). Blood was drawn at the indicated times. Serum was tested by patch-clamp to determine the amount of ShK(L5) in blood. F, a half-blocking dose of ShK(L5) was added to rat plasma (□) or to PBS containing 2% rat plasma (■) and incubated at 37°C for varying duration. Aliquots were taken at the indicated times and blocking activity determined on Kv1.3 channels.

Fig. 5

ShK-L5 prevents DTH and acute adoptive EAE in Lewis rats. A, prevention of EAE. PAS T cells were activated in vitro, washed, and injected intraperitoneally on day 0. Clinical scoring of EAE: 0 = no clinical signs, 0.5 = distal limps tail, 1 = limp tail, 2 = mild paraparesis or ataxia, 3 = moderate paraparesis, 4 = complete hind limb paralysis, 5 = 4 + incontinence, 6 = death. Rats (n = 6/group) were injected subcutaneously with vehicle (□; n = 6) or ShK(L5) (■; n = 6; 10 μg/kg/day) from day 0 to day 5. B, treatment of EAE. PAS T cells were activated in vitro, washed, and injected intraperitoneally on day 0. Treatment with ShK(L5) at 10 μg/kg/day was started when rats developed clinical signs of EAE and was continued for 3 days. C, DTH reaction was elicited against ovalbumin and rats (n = 6/group) were treated with ShK(L5) 10 μg/kg/day for 2 days, after which ear swelling was measured. Statistical analysis was carried out using the Mann-Whitney U test.