WO1991009613A1

WO1991009613A1 - Synthetic peptides as modulators of functional responses of intact cells

Info

Publication number: WO1991009613A1
Application number: PCT/US1990/007312
Authority: WO
Inventors: Michail V. Sitkovsky
Original assignee: The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
Priority date: 1989-12-22
Filing date: 1990-12-19
Publication date: 1991-07-11
Also published as: AU7142391A

Abstract

The purpose of this invention is to provide methods for selectively inhibiting the INTRACELLULAR biochemical processes that lead to cellular responses through use of specifically designed synthetic peptide substrates (pseudosubstrates) or inhibitors of protein kinases or peptides that interfere with the action of DNA-binding proteins. These synthetic peptides will block activities of enzymes by competing with natural substrates or by preventing functioning of DNA binding proteins. This invention provides a novel means of modulation of intact cells by using specifically designed synthetic peptides which serve as substrates (pseudosubstrates) or inhibitors of protein kinases which are able to strongly inhibit activation of unpermeabilized cells. Functional responses of lymphocytes in vitro as appropriate model systems are particularly illustrated.

Description

Title : SYNTHETIC PEPTIDES AS MODULATORS OF FUNCTIONAL RESPONSES OF INTACT CELLS

The purpose of this invention is to provide methods for selectively inhibiting the INTRACELLULAR biochemical processes that lead to cellular responses through use of specifically designed synthetic peptide substrates (pseudosubstrates) or inhibitors of protein kinases or peptides that interfere with the action of DNA-binding proteins . These synthetic peptides will block activities of enzymes by competing with natural substrates or by preventing functioning of DNA binding proteins . Previously the use of synthetic peptides has been restricted to experimental studies of artificially permeabilized cells because no method was known to facilitate entry of sufficient amounts of inhibiting peptide into the intact cell interior . This invention provides a novel means of modulation of intact cells by using specif ically designed synthetic peptides which serve as substrates (pseudosubstrates) or inhibitors of protein kinases which are able to strongly inhibit activation of unpermiablized cells . Functional responses of lymphocytes in vitro as appropriate model systems are particularly illustrated .

BACKGROUND OF THE INVENTION It has been widely accepted that phosphorylation/dephos- phorylation reactions mediated by protein kinases and phospha tases regulate cellular responses (Edelman, et al., Ann. Rev. Biochem, 56, 567-613 (1987)) . Activation of T- lymphocytes and cytolytic T-lymphocytes (CTL) also was known to involve protein kinases. (Baniyas, et al., J. Biol. Chem. 263, 18225- 18231 (1988) ; Kincaid, et al., Nature 330. 176-178 (1987) and Takayama, et al., J. Biol. Chem. 263, 2330-2336 (1988)) . Previously synthetic peptides which interfere with intercellular protein kinases were used only in systems using permeabilized muscle cells (e.g. SMI inhibitor of myosin light chain kinase (MLCK) (Itoh, et al, Nature 338, 164-167)) or with permeabilized T-lymphocytes (Pseudosubstrate of protein kinase C (PK-C) (Alexander, et al., Biology of Cellular transducing signals '89 Abstract #70 (Ninth International Washington spring Symposium, May, 1989)). However, these investigators worked only with permeabilized cells. Experiments with permeabilized cells can not serve as an adequate model for the testing of the intact modalities needed for pharmaceutical biological structures for obvious reasons. No effect of such peptides on unpermeabilized cells was described.

Earlier studies suggest the use of regulatory anti-sense RNA as a method of altering intracellular acitivty cells. The use of peptides dsigned to compete for the recognition of the antigen (for immunomodulation) has also been described. However, no use of synthetic peptides designed to interfer with intracellular activation of lympyocytes was suggested. The closest known technology using immunomodulation as therapy is found in the use of Cyclosporin A (CsA) for immunosuppression. The major disadvantage of CsA technology is the severity of side effects, including nephrotoxicity. The use of synthetic peptides could avoid such effects, since peptides are not toxic. However, the inability to

unpermeabilized cells has proven to be a barrier to advance of this technology for treatment modalities. In order to move forward to more definitive studies of cellular function and to pharmacological manipulations it is essential that a means be found to o

t unpermeabilized cells using synthetic peptides.

FIGURE LEGENDS

Fig . 1. Synthetic peptides substates and inhimiors of protein kinases inhibit proliferation and cytotoxicity of CTL clone. Ficoll-purified CTLs (clone OE4, 10⁶ cells/ml, day 5 after expansion with Ag-bearing, irra diated DBA-2 spleen celis) were incubated alone (basal level of proliferation), with 4 units/ml of recombinant IL-2 (rIL-2 induced proliferation) in the presence or absence of different concentrations of peptides. Amino acid sequences and abbrevitions of names of used peptides are shown in the legends. Three- and one-lecer abbreviations of amino acids are as follows: Gly, G; Ala, A; Val, V; Leu, L; lle, I; Ser. S, Thr, T; Asp, D; Asn, N; Lys, K; Gla, E; Gln, Q; Arg, R; His, H; Phe. F; Cys, C; Trp, W; Tyr, Y; Met, M; and Pro, P. ³H-thymidine incorporation into CTL was tested after incubation in DMEM-5% FCS containing media. In pretiminary experiments, we determined that pr ollferation response in CTL clone OE 4 can be achiev ed eith er by incubation with immobilized anti-TCR mAb alone (low level of stimulation) with rIL-2 alone or with anti-TCR mAb and rIL-2 together. No changes in -viability of CTL during the time of the assay were noticed, as measured by visual observations, cell counring and trypan blue exclusion test. The intensity of proliferation dramatically depeaded on the time after Ag-stimulation (''feeding") and the length of the "expansion" phase (cutrure in the presence of rIL-2). In this experiment, rIL-2 dependent proliferation was studied Ordinate. ³H-thymidine incorporation, cpm x10^-3; Abscissa, concentration of pepddes.

Panel A: (-O-), - Effect of syntberic pcpdde substrate (CaMPK-S) of a multifunctional CaM-dependent protein ki nase (Ca M PK)^8,9 on CTL's proliferation. CaMPK-S (PLSRTLSVSS-NH₂) was used as amide form. The basal level of ³HTdR incorporarion (no rIL-2, no pepdde) in control triplicates was 25.7±1.6 x 10³ cpm. In the absence of pepride, rIL-2 induced 135.5±22 x 10³ cpm of ³HTdR incorporatio n Even 2.0 and 4.0 mM CaMPK-S were not significantly inhibitory and allowed for

134±9 x 10³ and 95±13 x 10³ cpm of ³H-TdR incorporation, respectively. ( ), - Effect of synthetic peptide E11G1 (EDAEYAARRRG], substrate of tyrosine protein kinase^{10,1 1}. Basal level of ³HTdR incorporation was 16±1 x 10³ cpm. ³HTdR incorporation below the basal level was observed only at very high concentration, 4.0 mM E11G1 (9.8±0.5 x 10³ cpm). In response to rIL-2 (no peptide present) ³HTdR incorporation was 93±2 x 10³ cpm. (-O-), - Effect of peptide PKA-S (RGYSLG), substrate for PKA (cAMP dependent protein kinase)^{1,12- 15}. Basal level of ³HTdR incorporation was 21.5±0.7 x ³ cpm. The proliferarion below the basal level was not observed even at 2.0 mM PKA-S (80±3 x 10³ cpm) in the presence of rIL-2. In response to rIL-2 (no pepd de present) - 116±5 x 10³ cpm; (-Δ-), - Effect of synthetic peptide RR-src (RRL IFDAEYAARG), a substrate for src-tyrosine-specific protein kinase^{5 - 7}. Basal level of ³ΗTdR incorporarion was 16±0.7 x 10³ cpm. The proliferation below the basal level in the presence of rIL-2 was observed at 1.0 and 2.0 mM RR-src (6±2 x 10³ and 0-5±0.1 x 10³ cpm, respectively. In response to rIL-2 (no peptide) ³Η-TdR incorporation was 108±8 x 10³ cpm.

Panel B: The same as in panel A, except different peptides were tested. (Δ), - Effect of PKG-I (RKRARKE) peptide inhibitor⁴ of PKG (cGMP-dependent protein kina se). Basal ³HTdR incorporation was 19±0.9 x 10³ cpm.³HTdR incorporation below the basal level (129±0.3 x 10³ cpm) was observed al 0.5 mM and higher concentration of PKG-I. rIL-2 induced ³H TdR incorporation (no peptide) was 115±3 x 10³ cpm. (-O-), - Effect of PKG-S (RKRSRAE), pepdde substrate of PKG¹. Basal ³HTdR incorporation 22±2 x 10³ cpm, in response to rIL-2 was 106±4 x 10³ cpm. Even at 0.25 mM PKG-S inhibited proliferation to levels (12.7±0.1 x 10³ cpm) substantially below the basal level. ( ) - Effect of peptide MLCK-S (KKRPQRATSNVFS-NH₂ amide form)^2,3, substrate for the smooth muscle myosln light chain kinase. Basal ³HTdR incorporation was 18±2 x 10³ cpm. MLCK-S at 0.5 mM inhibited the proliferation (14.7±6 x 10³ cpm) below the basal level. In response to rIL-2 (no pepride) ³H-TdR incorporation was 127±24 x 10³ cpm. ( ) - Effect of peptide kemptide (LRRASLG)^1,12-15. Basal ³H TdR incorporation was 20±0.7 x 10³ cpm. Kemptide did not substantially inhibit proliferation even at 4.0 mM (90±4.7 x 10³ cpm).

In response to rIL-2 (no peptide) ³HTdR incorporation was 120±11 x 10³ cpm.

Fig. 2. The synthetic peptide PKG-S can preferentially inhibit one of two different CT L's responses triggered by the same stim uli, while the peptide RR-src can differentially inhibit the same response of CTL that was triggered by partially different stimuli. CTL OE4 (10⁶ cells per well) were incubated in several different parallel assays, in the same incubation condlrions in 96-well plate with the same concentration of immobilized anti-T CR mAb. Some of the wells also received 4 units/ml of rIL-2 and different concentrations of peptides. This allowed simultaneous comparison of the effect of pepd des on such different responses as TCR- triggered exocytosls of grannles, TCR-triggered γ-interferon secretion (data will be reported elsewhere) and TCR- or TCR- and IL-2 triggered proliferation.

Panel A: Peptid e RR-src differentially inhibits proliferation of CTL-triggered by anti-TCR mAb plus rIL -2 and by anti-TCR mAb only. Peptide RR-src was added at different co ncentrations to the wells of 96 well plate with immobilized anti-TCR raAb (5 μg/ml of purified F23.1 mAb per well were used during immobilization). In such conditions, F23.1 mAb induced "low" intensity response, 2500±120 cpm of ³H-TdR incorporation per 5x10⁴ cells (background in the absence of F23.1 was 290 cpm). Effect of peptide (-O-) is presented is percent of control response (where 100% is the response in the absence of pepdde). In parallel wells the same number of CTL was incubated with immobilized anti-TCR mAb (20 ng/ml of purified F23.1 mAb per well during immobilization) and rIL-2. Peptide RR-src was added at different concentrarions in these weils after addition of 4 un irs/m l of rIL-2 and effect of peptide on anti-TCR and rIL-2 indrred protiferation (•) was evaluted. Four units/ml of rIL-2 alone induc ed 3700+300 cpm, while anti-TCR mAb plus rIL -2 triggered 18,500±1,800 cpm ³H-TdR incorporation.

Panel B: Peptide PKG-S differendally inhibits exocytosis and proliferation of cells triggered by the same signal. Peptide PKG-S was added at different concentrations to the wells with imp obilized anti-TCR mAb as indicated on the figure and exocytosis of granules detected by the release of granule enzyme was estimated.

Parallel samples were set up in wells incubared with identical concentrations of anti-TCR mAb and were tested for TCR-triggered CTL proliferation. In the absence of pepdde, 45.8% of granule enzyme BLT-esterase was released due to triggering by anti-TCR mAb and this level of secretion was taken as control 100%. Inhibition of exocytosis of granules by peptide PXG-S (-O-) is presented as percent of control. Proliferation (³HTdR incorporation background was 293±10 cpm) of CIL. in response to anti-TCR mAb (in the absence of added rIL-2) was sdmulared 8.27 times in the absence of PKG-S. Inhibition of ³H-TdR incorporztion by PKG-S (-O-) is presented as percent of ³H-TdR incorporation (2424±119 cpm) that was triggered by immobilized anti-TCR mAb in the absence of peptide.

Fig. 3. Pretreatment of cells with peptide protein Kiruse substrate is sufficient to cause the inhibition of CTL incubated with ⁵¹C r-labeled target cells in the absence of peptide d uri n g the assay.

CTL clone OE4 was preincubatel for indicated length of time with PKG-S. Supernatant with "used" peptide was carefully removed and CTL were washed to remove the traces of unbound peptide. Treated CTL were tested in comparison with control CTL (identically treated excepc for omission of peptide) for the ability to kill Ag-bearing targets. The "used" peptide solu tion was ested for inhibition of freshly set ⁵¹ Cr-release assay with untreated CTL and compared with control peptide solution of the same concentration.

Papel A: At least 60-90 min preincubation of CTL with peptide PKG-S are needed to observe inhibition of CTL-mediated cytotociciry. CTL OE4 were Ficoll purified, washed and incubated different times (5, 15.30, 60 and 90 min) with 1mM PKG-S at 37 C in CO ₂ incubator. After preincubation was finished, tubes were gently centrifuged, supematants carefully re moved, cells were washed, counted and added to the wells of 96-well plate, followed by the adfftion α^{- 5 1}Cr- labelled P815 target ceils (P815 spontaneous release in this ⁵¹Cr-assay was 7 2%) at different effector to target cell ratios. Results obtained after 5 and 15 min preinrabation of CTL were practically indistinguishable from control (-■-) - experime nt (target cell killing by untreated CTL) and are not presented in the figure. Effect of pepdde preteatment is compared with the inhibition by the same peptide PKG-S. 1mM ( ) that is present throughout an ⁵¹ Cr-rekase assay of cytotoxicity of untreated CTL. (O), -30 min pretrentrnent; (-Δ-), - 60 min pretreatmeng (-O-), - 90 min pretreatment.

Panel B: Peptide solution. used during the pretreadpent of CTL. remins almost the same inhibitory potential when tested in a cytotoxicity assay with untreated CTL. Results are shown; -O-, Control, cytotoxictry of untreated CTL OE4; -O-, inhibition of cytotoxicity by the "fresh" pepdde, continuously present during assay, ♡, inhibition of cytotoxicity with peptide solution ("used" pepdde) obtained as a CTL's supern arant after 90 min incubation with 1mM PKG-S.

Fig. 4. Pretreatmen t of CTL with PKG-S for 150 m in results in more effct ent inhibition of their activity corppare d to the observed when pcpdde is present throughout the ^{5 1}Cr-release assay.

Cells (CTL OE4) were treated as described in the legend to Fig. 3 and effect of 150 min. pretreatment with lmM PKG-S (♤) was compared with control (-O-, untreated) cells or with the effect of the continuous presence ( ■ ) of the peptide during assay.

DESCRIPTION OF THE INVENTION

It is the purpose of this invention to provide a mean for designing synthetic peptides which will serve as substrates (pseudosubstrates) or inhibitors of protein kinases and wherin are able to inhibit activation of unpermeabilized cells . synthetic peptides can also be designed on the basis of the knowledge of the amino acid sequences of nuclear factors i nuclear factor inhibiting proteins ( e. g . NF-KB) so that t regulation of the activity of DNA transcription factors will be af fected . The peptides have use in controlling cellula activity in cell culture and in vivo and as specificall targete inhibitors of protein kinases both in vitro and in

It is a further obj ect of this invention to proxy de selective inhibitors of protein kinases that will effect unpermeabilized cells to preferentially inhibit parti lar responses of the cells such as proliferation of norme or tumor cells , secretion, cytotoxicity, and/or mechanical bi chemical activities (e . g. conjugate formation between target lls and T- killer cells . )

Because the invention provides means of inf ncing unpermeabilized cells , influencing cellular activi by interfering with already expressed functional proteins is ssible . However, this approach may also be used to block the transcription/translation and protein synthesis-dependent rocesses , since protein kinases (targeted by described synthetic eptides) do regulate these processes . It is also deemed portable to use synthetic peptides which will interfere with the process of activation (de-inhibition of nuclear factors , e . g . NF-K beta) which are able to regulate gene expression . The method described is qualitatively different from the " antisense message" procedure because it involves targeting proteins instead of nucleic acids . The method will avoid the known shortcomings of antisense message , which are summarized in the conclusions of the prior art . (See M. Ratner, "Can Antisense Message be Delivered? " Biotechnology , Vol 7 p . 207 Nature Publishing Company ( 1989 ) )

It is now possible to affect intracellular responses by incubating intact cells with peptides . It had been widely believed that peptides could not enter the intact cells in amounts sufficient to influence cellular responses ; that only permeabilization of cells would allow effects of peptides to be realized . It is now disclosed that unpermeabilized cells can be influenced by exposure to synthetic peptides which serve as substrates or inhibitors of protein kinases . The synthetic peptides can, therefore, be used to selectively inhibit cell responses to peptides in, for example, cloned and polyclonal lymphocyte populations .

To achieve the selective inhibition of responses of intact cells lymphocytes were incubated with 50-500UM of purified peptides in RPMI culture medium supplemented with 5% fetal calf serum. Peptides may be washed away after the required incubation or left present during the assay as described herein . Inhibition by peptides is seen in all functional assays tested, including Ag-driven proliferation, Ag-specific cytotoxicity and Agtriggered secretion. The examples indicate known functional responses of lymphocytes could be selectively inhibited by synthetic peptides using amino acid sequences constructed by the method taught herein . Peptides for selective inhibition of targeted response could be

found , for example , to inhibit cytotoxicity of lymphocytes without affecting their proliferation . By contrast , other peptides could be used when only proliferation , but not cytotoxicity or secretion , should be inhibited . Thus , the described invention for manipulation of the effector functions is exemplified in lymphocytes and could be summarized as follows : i) The panel of synthetic peptide substrates , pseudosubstrates and inhibitors of protein kinases is chosen ( See Table I and Table II) . ii) This panel of peptides is screened with cloned lymphocyte lines , peripheral blood lymphocytes or lymphocytes from other tissues of patients with transplants of organs or autoimmune diseases in vitro for the identification of the most efficient inhibitory peptide . iii) The half-life of the amino acid is increased by use of amino acid analogues . (Amino acid sequences that had been modified through use of protective groups and cyclic peptides are such analogues . )

Second messenger dependent protein kinases are shown to have inhibitory domains with pseudosubstrate sequences . Examples include cAMP , cGMP-dependent protein kinases , Ca^{2 +}/CaM- dependent protein kinases and protein kinase C . It should be stressed that these are examples only used to illustrate the method of selecting, designing, and modifying the amino acid sequences to obtain the benefits taught herein. Sequences of amino acids near the phosphorylation sites of protein substrates o f these prote in nases or in the inhibitory oma ins o f enzym es are selected for the design of synthetic peptides . Synthetic peptides are constructed to combine two properties . 1) Amino acid sequences of substrates , pseudosubstrates , or inhibitors of a given protein kinase are mimicked ( "approximation sequences approach" ) using peptides which are expected to interfere with the phosphorylation of the natural substrates , 2 ) The peptides are specifically designed to include features (e . g . inclusion of positively charged basic residues at the aminoterminal of the peptide) that facilitate interaction of such peptides with the cell surface and , consequently , internalization , so that peptides act as efficient inhibitors of protein kinases in local cellular environment . The data presented demonstrates that such peptides do inhibit various cellular functions .

Refinements of the "approximation sequences" approach are possible . By adjusting for species to differences between amino acid sequences even in highly selected and evolutionary conserved proteins . Thus , if the goal is to manipulate a human ' s cell responses , it will be possible to accomplish such manipulation even when peptides with amino acid sequences derived from amino acid sequences of chicken analogue of a given protein substrate .

However, the selectivity of the synthetic peptides can be improved by the "taylored sequences approach" if the natural human protein substrate of this enzyme is sequenced. Examples of enzymes to be targeted for inhibition of response of intact peptides and their amino acid sequences are presented in Table 1 and Table II In order to practice the "approximation" approach to " f ine tune" the manipulation of intracellular enzymes and to practice the "taylored sequences approach" it is necessary to ( 1 ) identify proteins whose phosphorylation is changed in response to changes in the cellular level of the second messengers in the cell type that is implicated in the particular disease ; 2 ) sequence the amino acid sequences near the phosphorylation sites of those proteins which are changed both in response to the second messenger level and during the physiological response in normal and pathological conditions ; and 3 ) construct synthetic peptides on the basis of the knowledge of the exact a mino acid sequences .

The inventive process was followed to provide synthetic peptide shown in Table I and Table II . The peptides were strongly inhibitory in T cell receptor (TCR) and interleukin 2 ( IL-2 ) triggered proliferation of cytolytic T-lymphocyte (CTL) clones . Other tested peptides , including substrate for multifunctional CaM dependent protein kinase , (peptide CaMPK-S ) substrate for tyrosine-Specif ic protein kinase (peptide E11G1) substrate for cAMP dependent protein kinase (peptide PKA-S ) and kemptide had no effect on proliferation . Some , but not all , of the peptides that inhibited CTL clone proliferation , were also efficient in inhibition of such TCR-mediated response of CTL as cytotoxicity and exocytosis of granules (Figures 1 and 2 ) . Opposite effects of other peptides tested in parallel in different CTL activation assays provided an internal control for the specificity of inhibition . Peptide PKG-S , for example , was much more efficient in inhibition of proliferation of CTL than in inhibition of cytotoxicity of the same cells. The results clearly indicate that selective modulation of the particular cellular response by synthetic peptide substrates, pseudosubstrates, and inhibitors of protein kinases is appropriate as a means of providing targeted cell modulation activity.

METHODS:

VβS-speclfle anti-TCR antibody F23.1³⁰ (mouse IgG2a), which reacts with OE4 CTL clone, was purified by affinity chromatography using a protein-A column. The peptides used in these studies were synthesized according to the solid phase synthesis method of Merrifield and were further purified by HPLC. The purity of peptides was vigorously verified by ansdytical HPLC, paper electrophoresis, amino acid analysis and thin layer chromatography. According to the results of such quality control studies performed by the peptide synthesis facilities, the peptides are more than 95% pure, with correct amino acid content and amino acid sequence and their purity was confirmed by the appearance of only one spoc on thin layer chromatography and paper electrophoresis. The composition of peptides, abbreviations of their name, references, and en zymes, for which they are designed as substrates or inhibitors, are described in legends to Figs. 1 and 2. CTL clone OE4³⁰ was maintained in modified Dulbecco's minimum essential medium containcg 5% FCS and supplemented with irradiated sti mulator cells and an innterknkin-2 (IL -7)-con taining culture supernatant of EL4 cells as described in refs.22 and 25. CTL clones were used on day 4 or 5 after stim ulation with irradiated spleen cells and IL-2. CTL- clones were purified from dead cells by Ficoll-Hypaque centrifugat ion shordy before using them in the assays. R-Scombinant DL-2 (rEL-2) was 1 gift from Biogen Research Corporation and was used at a final concentration of 4U per ml in triplicate 100 μl CTL cultures. Peptides were dissolved in DMEM without FCS, 2-fold dilutions were made and dilutions of filter-sterilized peptide solutions were added to achieve the indicated concentrations. Control wells received medium (no IL-2) or medium without FCS (no peptide) to ensure uniform culture conditions. Detailed preliminary experiments revealed that intensity of proliferative response to anti-TCR mAb and rIL-2 strongly depends on the density of immobilized anti-TCR mAb F23.1, and allowed to establish condition for the "low- and "high" intensity ³H -TdR incorporation by CTL done GE4. Proliferatica of CTL was estimated by the routine procedure. Briefly, wells were pulsed with 1 μCi

³H-thymidi ne overnight and cells were harvested onto glass fiber filters for scintillation counting 20 hours after addition, of rIL-2 and peptides to the wells. To evaluate the effect of peptides on CTL-medfated cytotoxiciry, after 3 days in culture with irradiated spleen cells. Ficoll-Hypaque centrifugation and washed twice in medium before use. Ficoll-parified CTL clones were mixed with ⁵¹Cr-labelled target cells in the standard assay with minor modifficarions as was described earlier with the same CTL clone ^22,25. The percent of specific ⁵¹Cr release was calculated as 100 X (a-b)/(t-b), where a is ⁵¹Cr release in the presence of CTL, b is spontaneous release from labeled target ceils in the absence of CTL, and t is the total ^{5 1}Cr content in the target cells.

CTL clone OE4 cells were prepared as descrlbed in the legend to Fig. 2. ³H-TdR incorporarion was estimated as described in the legend to the Fig. 1, except 48hr assay was performed. The wells of a 96-well microtiter plate were coated with monoclonal anti-TcR antibodies by incubating 50 μl of antibody solurion per well in sterile pbosphate-buffered saline at 37 C for 16hr (Costar plates) or 1hr (Dynatech plates). After incubation, each well was washed with medium. The amount of the secreted BLT esterase was determined²⁵, either after mixing 1 x 10⁵ CTL with target cells in 0.1 ml of RPMI 1640 (supplemented with 10 mM HEPES, 5% FCS) in a 96-well microtiter plate, or after incubation of CTL in wells coated with immobilized mAb F23.1. After a 4h incubation at 37 C under 5% CO ₂, cells were resuspended by gentle pipetting and centrifuged at 200 x g for 0.5 min,, Fifty-μl aliqnots of supernatant were used to assay BLT esterase activity. Total cellular content of the enzyme was determined using 0.1% Triton X-100-solubilized cells. D etermi nations were carried out in triplicate. Data are presented as specific percent of enzymatic activity released calculated from the equation: % release = 100 X (E-S)/(T-S); where E represents the number of enzyme units in the supernatant of the experimental well, S represents the number of enzyme units in the su pernarants of the well containing no stimuli, T represents total number of units of BLT esterase in CTL per well. Activity of BLT esterase was measured as described earlier ^22,25. To assess the contribution of cell death during incubation on BLT esterase secretion in the cell supernatants, we measured activity of cytosolic enzyme lactatc dehydrogenase in the supernatants according to the procedure described in ref.25. It was shown by simuhaneous EXAMPLES:

Example 1.

Several peptides were simultaneously tested for the ability to inhibit the proliferation of CTL clone OE4 in response to recombinant IL-2 (Fig. 1A,B) . Peptides CaMPK-S, E11G1, PKA-S and kemptide had no effect en proliferation even at ImM concentrations while peptides RR-src, MLCK-S, PKG-S, and PKG-1 were strongly inhibitory. Inhibitory peptides blocked not only IL-2 induced activity, but even blocked basal level of ³HTdP. incorporation in CTL (See legend to Figure 1.) . Interestingly, peptide E11G1, which share with inhibitory peptide RR-src the sequence around the phosphorylatable tyrosine (EDAEYAAR) was not inhibitory at all (Fig. 1) , suggesting the important role of aminoterminal arginines in the inhibition by RR-src. Even miner differences in peptide amino acid sequence result in dramatic changes in their effects on intact cells. This observation is supported by the comparison of the effects of PKG-s/PKG-I peptides (at 125 uM and 250 uM, Fig. 1B). Noticeable inhibition of CTL proliferation with RR-src peptide was observed at 250 uM and strong inhibition at 500 uM. The Km of this peptide was higher (3-4 mM) when tested with solubilized particulate fraction of lymphoma cell line. (See Casnellie, et al,, Proc. Natl. Acad. Sci. USA 79, 282-286 (1982)).

The peptide PKG-I (RKRARKE) is a non-phosphorylatable analogue of a substrate peptide PKG-S (RKRSRAE) . Peptide PKG-I was less efficient as an inhibitor of proliferation of CTL (Fig. 13) when compared to peptide PKG-S. PKG-I was also less inhibitory than PKG-S when compared for ef fects on CTL-meciated cytotoxicity at up to ImM concentration (Fig . 1C) . Hence , it appears that the total positive charge of the peptide ( four basic residues in PKG-S and five in PKG-I) is not likely to be the sole determinant of the inhibitory potential of peptides in intact cells . Example 2 :

The effects of two different peptides on two different responses was demonstrated and illustrates the possibility of obtaining the selectivity of inhibition of a particular response (e . g. proliferation or cytotoxicity) by constructing an appropriate peptide . Figure 2 gives data obtained when the same peptide ( in this case , RR-src) was tested simultaneously in parallel assays . Data shows different inhibitory potential in the same response (proliferation) triggered by partially different activating signals . The ability of the same peptide to have very different efficiency of inhibition of different responses (proliferation or exocytosis ) of the same cells that are , in this case, activated by the same signal , is also demonstrated in experiments described in Figure 2B . Up to 250 uM of PKG-S had little affect TCR-triggered exocytosis . The same concentration of PKG-S inhibited 85% of prolif erative response to the same activating ligand, indicating selectivity of response in a concentration-dependent manner.

Antimalignant effects may be obtained by using peptides which selectively block activity of protein kinases or transcriptional factors of malignat cells . It is demonstrated that peptides developed according to the inventive process can dramatically effect cellular responses of in vitro cultured tumor T-lymphoma cells (helper T-cell hybridoma ) .

A more intensive response (proliferation) triggered by combined addition of anti-TCR mAb and IL-2 was inhibited in a similar manner by the peptide RR-src and by a combination of anti-TCR mAb and IL-2 . While the peptide was needed in the assay if the cells had been preincubated less that 60 minutes , a period of 150 minutes of incubation resulted in high inhibitory activity in cells that had been incubated with the peptide even though the peptide was not present in the assay (Fig . 3 and 4 ) .

It has been accepted that phosphorylation/dephosphcrylation reactions mediated by protein kinases and phosphatases regulate cellular responses in general and activities of T- lymphocytes and CTL in particular . It is now found that amino terminal positive charge on peptides due to the presence of basic residues such as lysine or arginine influences their interactions with the negatively charged cell surface and facilitates their internalization . Hence , " arginization" cr " lys inization" of peptides is deemed a preferred method of rendering a peptide capable of entry into the intact cell .

Peptides of the invention are , therefore , prepared by the following method :

1) Potential phosphorylation sites on the targeted protein are identified by presence of serine , theonine or tyrosine, 2 ) Sequences which meet the requirements of ( 1) having are chosen .

3 ) The peptides chosen in ( 2 ) may be rendered more positively charged . In an preferred embodiment of the invention , sequences are modified so that at least the first two bases at the amino terminus of the peptide are arginine or lysine .

The half-life of the amino acid sequence can be increased by rendering the sequences less susceptible to the action of proteases by modification . A standard means of making such modifications is by substitution with protective groups used in the peptide art. Furthermore , the inhibitory effect may be increased by cyclization of peptides . Cyclic peptides w ould , however , retain structures of amino acid sequences that are able to interfere with protein kinase or activity of DNA binding proteins .

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Claims

1 A method of producing peptides capable of entering intact cells comprising modifying the peptide so that at least the first two amino acids at the amino terminus are positively charged amino acids.

2, A method of claim 1 wherein the positively charged amino acids are chosen from lysine and arginine.

3. A method of selectively inhibiting responses in intact cells comprising the steps of:

1) Identifying the protein whose activity is to be inhibited;

2) Identifying phosphorylation sites which could be important for regulation of protein functional activities;

3) Identifying amino acid sequences near the phosphorylation site (surrounding serine, theonine, or tyrosine);

4) Construction ofg synthetic peptides consisting of 4 to 40 amino acids which mimicl phosphorylation sites of the targeted natural protein;

5) Rendering the peptide positively charged at its amino terminal by including positively charged amino acids at that terminous during peptide synthesis or by chemical modification of the peptide of the natural source.