Dimerization of an Immunoactivating Peptide Derived from

Article pubs.acs.org/bc

Dimerization of an Immunoactivating Peptide Derived from Mycobacterial hsp65 Using N‑Hydroxysuccinimide Based Bifunctional Reagents Is Critical for Its Antitumor Properties Karel Bezouška,*,† Zuzana Kubínková,† Jiří Stříbný,† Barbora Volfová,† Petr Pompach,†,‡ Marek Kuzma,‡ Milada Šírová,‡ and Blanka Ř íhová‡ †

Department of Biochemistry, Faculty of Science, Charles University Prague, Hlavova 8, CZ-12840 Praha 2, Czech Republic Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, CZ-14220 Praha 4, Czech Republic

‡

S Supporting Information *

ABSTRACT: We have shown previously that a short pentapeptide derived from the mycobacterial heat shock protein hsp65 can be highly activating for the immune system based on its strong reactivity with the early activation antigen of lymphocytes CD69. Here, we investigated an optimal form of presentation of this antigen to the cells of the immune system. Four different forms of the dimerized heptapeptide LELTEGY, and of the control inactive dimerized heptapeptide LELLEGY that both contained an extra UV active glycine-tyrosine sequence, were prepared using dihydroxysuccinimidyl oxalate (DSO), dihydroxysuccinimidyl tartarate (DST), dihydroxysuccinimidyl glutarate (DSG), and dihydroxysuccinimidyl suberate (DSS), respectively. Heptapeptides dimerized through DST and DSG linkers had optimal activity in CD69 precipitation assay. Moreover, dimerization of active heptapeptide resulted in a remarkable increase in its proliferation activity and production of cytokines in vitro. Furthermore, while DST and DSG dimerized heptapeptides both significantly enhanced the cytotoxicity of natural killer cells in vitro, only the DSG dimerized compound was active in suppressing growth of melanoma tumors in mice and in enhancing the cytotoxic activity of tumor infiltrating lymphocytes ex vivo. Thus, while the dimerization of the immunoactive peptide caused a dramatic increase in its immunoactivating properties, its in vivo anticancer properties were influenced by the chemical nature of linker used for its dimerization.

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INTRODUCTION

activation-induced cell death has been described only in monocytes or eosinophils.7−9 CD69 is expressed at cell surface as homodimeric receptor belonging to C-type lectin family.3 The physiological ligand for this receptor is not known, although our results indicate that CD69 is a functional lectin able to bind calcium and Nacetylhexosamines.10 Introduction of charged groups into Nacetylhexosamine sequences increases significantly the affinity for CD69.11 Sialylated oligosaccharides bearing negatively charged groups in Siaα2→6Gal or Siaα2→6GalNAc (SiaTn) sequences are among such high-affinity ligands for the CD69 receptor. Since these sequences form an integral part of tumor surface sialomucins,12 they may be among the ligands initiating activation-induced apoptosis in killer lymphocytes. This process could be, in principle, inhibited by monomeric SiaTn. However,

Natural killer (NK) cells and cytotoxic T cells (CTL) are both essential components of the immune response against tumors. They mediate cytotoxicity and produce chemokines and inflammatory cytokines. Cytotoxic cells express a large number of surface receptors that activate or inhibit their effector function.1 CD69 occupies a specific place among these receptors since, unlike the prototype NK cell receptor NKRP12 or the specific T cell receptor for antigen, it is also expressed by most other leukocytes for which it may serve as the universal triggering receptor.3 The role of this widespread leukocyte receptor in natural killing remains controversial.4 Originally, it was reported as an important activation receptor of NK cells.5 However, it was shown by Sanchez-Madrid and colleagues that CD69−/− mice have increased resistance to lymphomas.6 One reason for this controversy may be the hyperactivation of CD69+ cytotoxic cells upon their contact with tumors, followed by their increased sensitivity for apoptotic death. However, until now such CD69 dependent © 2012 American Chemical Society

Received: February 5, 2012 Revised: August 27, 2012 Published: September 18, 2012 2032

dx.doi.org/10.1021/bc300056x | Bioconjugate Chem. 2012, 23, 2032−2041

Bioconjugate Chemistry

Article

Scheme 1. Synthesis of Dimeric Heptapeptides Using Disuccinimidyl Reagentsa

a

Reagents and conditions: (a) 80% DMSO, 20% 50 mM ethylmorpholine acetate pH 7.5, 45 °C, 16 h.

NMR spectra of peptides 3, 4, 5, and 6 were measured on a Bruker AVANCE III 600 MHz spectrometer (600.23 MHz for 1 H, and 150.93 MHz for 13C) in DMSO-d6 (99.9 atom % D, Sigma-Aldrich, Steinheim, Germany) at 303 K. Residual signal of solvent was used as an internal standard (δH 2.500, δC 39.60). 1H NMR, 13C NMR, gCOSY, gTOCSY, gHSQC, gHMBC, and 1D-TOCSY spectra were measured using standard manufacturers’ software. The proton spin systems of each amino acid unit were assigned by gCOSY, gTOCSY, and 1D-TOCSY. The assignment was transferred to carbons by gHSQC. The quaternary carbons were identified by gHMBC correlations, which also allowed determination of peptidic bonds. The amount of compound 6 did not allow us to measure gHMBC. Chemical shifts are given in δ-scale [ppm] and coupling constants in Hz. Digital resolution enabled us to report chemical shifts of protons to three, coupling constants to one, and carbon chemical shifts to two decimal places. Some proton and carbon chemical shifts were read out from TOCSY and gHSQC and are reported to two decimal places.14 Mass spectra were measured on a matrix-assisted laser desorption/ionization reflectron time-of-flight (MALDI-TOF) mass spectrometer BIFLEX (Bruker-Franzen, Bremen, DE) equipped with a nitrogen laser (337 nm) and gridless delayed extraction ion source. Ion acceleration voltage was 19 kV and the reflectron voltage was set to 20 kV. Spectra were calibrated externally using the monoisotopic [M+H]+ ions of matrix peak 379.1 m/z. A saturated solution of α-cyano-4-hydroxy-cinnamic acid or 2,5-dihydroxybenzoic acid in MeCN/0.3% aq acetic acid (1:1) was used as a MALDI matrix. A 1 μL of matrix solution was mixed with a 1 μL of sample diluted in acetonitrile or THF and a 1 μL of premix was loaded on the target, the droplet was allowed to dry at ambient temperature. The MALDI-TOF spectra were collected in reflectron mode. General Scheme for the Dimerization of the Immunoactive and Control Peptides via N-Hydroxysuccinimide Chemistry (Scheme 1). Analytical reactions were set at 5 μmol scale related to the starting peptide. Five micromoles of the active or control heptapeptide (Leu-GluLeu-Thr-Glu-Gly-Tyr or Leu-Glu-Leu-Leu-Glu-Gly-Tyr, respectively) were dissolved in 280 μL of DMSO, and diluted with an additional 200 μL of solvent mixture containing DMSO

this disaccharide is expensive, not easily available in pure form, and unstable both in vitro and in vivo. Therefore, we have been searching for additional high-affinity ligands for CD69. We have recently identified the immunoactivating pentapeptide derived from the mycobacterial heat shock protein hsp65, and found that CD69 is the target receptor for this peptide due to the interaction mediated through negatively charged carboxylic functions that are essential for the binding activity.13 The immunoactive pentapeptide sequence can be active on cells of the immune system per se, or as a part of a larger polypeptide or even in the environment of the entire protein. However, while the pentapeptide completely retains the binding activity of larger peptide or hsp65 subunit, it has very little immunostimulatory activity in the monomeric form.13 These results led us to evaluate the degree of clustering or conjugation that is necessary for attainment of optimal immunological activity. Here, we describe our evaluation of the immunological activity of the active peptide and control peptide that have been conveniently dimerized using the unique amino group within their peptide sequence. We have evaluated the effect of the active peptide component as well as of the length and chemical nature of the linker on several biochemical and immunological activities, and on the anticancer activity evaluated in vivo and ex vivo.

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EXPERIMENTAL PROCEDURES Materials and Instrumentation. Chromatographical solvents were from Merck, Darmstadt, Germany. Synthesis of the immunoactive LELTEGY and control LELLEGY peptides was performed on 100 mg scale by Xaia, Lund, Sweden, and the synthesized peptides were purified by HPLC and supplied with MALDI-MS spectra by the manufacturer. Bifunctional reagents used for protein cross-linking DSO, DST, DSG, and DSS were from Pierce. Monoclonal antibodies against mouse CD69 (clones H1.2F3 and G235−2356) were from BD Pharmingen. HPLC system used for separation of peptide dimers was from Beckman-Coulter, and consisted of 126 Solvent Delivery Module, 166 Detector, manual Rheodyne injector, and SC100 Fraction Collector. The system was operated using Gold chromatographic software. 2033



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for these compounds are provided in Supporting Information Tables 1 to 4. Synthesis of Dimeric Peptides Coupled via DSS. Compounds 7 and 8 were prepared at analytical scale using the general reaction scheme described above. 18.5 μg and 19.7 μg of conjugates 7 and 8, respectively, were obtained using reaction scheme (b) providing the highest yield amounting 4.5% and 4.7% of the amount of the input peptide, respectively. These yields were based on spectrophotometry (ε = 1490 M−1 cm−1) and quantitative amino acid analysis using the AccuTaq system (Millipore). MALDI-MS, [M+H+], m/z: 1783.8 and 1807.8 for 7 and 8, respectively. Binding and Inhibition Experiments. For all binding and inhibition experiments, the lyophilized peptides and dimerized peptides dried in vacuo were dissolved in ddH2O, and the concentration was verified and adjusted to 2 mM using spectrophotometry at 280 nm. Soluble dimeric human CD69 was expressed in Escherichia coli, refolded, and purified essentially as described previously.5,10 This protein was radioiodinated with a carrier-free Na125I (Amersham) to a specific activity of 107 cpm per μg protein. Binding and inhibition assays were performed as described previously13 with minor modifications. Briefly, 96-well poly(vinyl chloride) microplates (Titertek Immuno Assay-Plate, ICN Flow, Irvine, UK) were coated overnight at 4 °C with 50 μL of GlcpNAc17BSA (10 μg/mL, Sigma) in TBS+C buffer (10 mM Tris-HCl pH 8.0 with 150 mM NaCl, 1 mM CaCl2, and 1 mM NaN3). Plates were blocked with 1% BSA (Sigma) in TBS +C for 2 h at 4 °C, and incubated with an amount of the radiolabeled protein corresponding to half of the saturation amount and the indicated dilutions of the tested compounds in a total reaction volume of 100 μL. Plates were washed three times with TBS+C, drained, dried, and 100 μL of a scintillation solution was added to each well. Radioactivity in wells was determined in a β-counter (Microbeta, Wallac). All experiments were performed in duplicate. The degree of inhibition was calculated relative to wells without any inhibitor (addition of water). The results are average values from duplicate experiments within the range indicated by error bars. Equilibrium Dialysis. Peptides and dimerized peptides were radiolabeled using carrier-free Na125I (Amersham) to a specific activity of 500 GBq/mmol) and, when appropriate, diluted with the unlabeled compound according to the required specific activity. To set up equilibrium dialysis experiments, a rotating apparatus with glass blocks containing separate sealable chambers with external access was used as described previously.15 200 μL aliquots of 0.1 μM solutions of CD69 proteins in 10 mM MES pH 5.8 with 49 mM NaCl and 1 mM NaN3 were incubated with varying amounts of ligand at 278 or 300 K for 48 h. After equilibration, 100 μL aliquots were withdrawn from the control and from the protein-containing chambers. The total ligand concentration was determined by liquid scintillation, and the bound ligand was calculated as the difference between the amount of peptide in the chamber containing the protein and the control chamber. The results were calculated and plotted according to Scatchard as described previously.15 Precipitation Assays with the Soluble CD69 Receptor. Each ligand was dissolved in water at concentrations of 200, 60, 20, 6, and 2 nM. The 125I-labeled protein (20 nM, 50 μL) was added to each sample (50 μL) in 96-well microtiter plates. Mixtures were incubated at 4 °C for 30 min, and then 100 μL of the solution of PEG 8000 (20% (w/v)) was added. The

and 50 mM ethylmorpholine acetate buffer pH 7.5 using 4 different ratios: (a), 33 μL:167 μL; (b), 100 μL:100 μL; (c), 150 μL:50 μL; and (d), 175 μL:25 μL. Thereafter, 2.3 μmol of the respective bifunctional reagent (DSO, DST, DSG, or DSS) was added in 20 μL of DMSO, and the mixture was closed in 1.5 mL plastic tubes under argon. Under these conditions, the final content of DMSO in the reaction mixture was 67%, 80%, 90%, and 95% in the above reaction conditions (a) to (d), respectively. Tubes were placed onto a heating block, and incubated for 16 h at 45 °C. Thereafter, the reaction was stopped by the addition of 500 μL of 0.1% TFA, and the tubes were centrifuged at 18 000 g for 30 min at room temperature. The entire reaction mixture was then injected onto a semipreparative reverse-phase column Vydac C-18 (10 × 250 mm, 300 Å, 10 μm, Dionex) connected to the HPLC system that had been equilibrated in 0.1% TFA in water, and run at 2 mL/min. The column was washed for 10 min with the starting solvent, and then eluted for 40 min with acetonitrile gradient increasing its content by 2% per min. The elution was monitored continuously at 280 nm, and the chromatograms were integrated using the Gold software in order to evaluate the amount of compounds in individual peaks. The molar extinction coefficients at 280 nm were determined experimentally using the separated compounds, the concentrations of which were independently verified by quantitative amino acid analysis.5,10 Synthesis of Dimeric Peptides Coupled via DSO. Compounds 1 and 2 were prepared at analytical scale using the general reaction scheme described above. 19.7 μg and 21.3 μg of conjugates 1 and 2, respectively, were obtained using reaction scheme (b) providing the highest yield amounting to 4.8% and 5.2% of the amount of the input peptide, respectively. These yields were based on spectrophotometry (ε = 1490 M−1 cm−1) and quantitative amino acid analysis using the AccuTaq system (Millipore). MALDI-MS, [M+H+], m/z: 1702.8 and 1726.8 for 1 and 2, respectively. Synthesis of Dimeric Peptides Coupled via DST. Compounds 3 and 4 were prepared at analytical scale using the general reaction scheme described above. 147 μg samples of conjugates 3 and 4, respectively, were obtained using reaction scheme (b) providing the highest yield amounting 36% and 35% of the input peptide, respectively. These yields were based on spectrophotometry (ε = 1490 M−1 cm−1) and quantitative amino acid analysis using the AccuTaq system (Millipore). MALDI-MS, [M+H+], m/z: 1762.8 and 1786.8 for 3 and 4, respectively. At preparative (50 μmol) scale, 1.4 mg samples of compounds 3 and 4 were obtained as white powders after lyophilization corresponding to 34% and 33% of the input peptide. Complete 13C and 1H NMR data for these compounds are provided in Supporting Information Tables 1 to 4. Synthesis of Dimeric Peptides Coupled via DSG. Compounds 5 and 6 were prepared at analytical scale using the general reaction scheme described above. 139 μg and 151 μg of conjugates 5 and 6, respectively, were obtained using reaction scheme (b) providing the highest yield amounting to 34% and 36% of the input peptide, respectively. These yields were based on spectrophotometry (ε = 1490 M−1 cm−1) and quantitative amino acid analysis using the AccuTaq system (Millipore). MALDI-MS, [M+H+], m/z: 1742.8 and 1766.8 for 3 and 4, respectively. At preparative (50 μmol) scale, 1.2 and 1.4 mg of compounds 5 and 6 were obtained as white powders after lyophilization corresponding to 30% and 34%, respectively, of the input peptide. Complete 13C and 1H NMR data 2034



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mixture was left to precipitate for one hour at 4 °C. After centrifugation (10 min, 4 °C, 3800 gav), the supernatant was carefully removed, and the pellet was resuspended in 100 μL of 10% (w/v) solution of PEG 8000 using sonication. This procedure was repeated three times to wash the precipitate. After additional centrifugation and removal of the supernatant, the precipitates were dried overnight at 37 °C, and measured by liquid scintillation counting. The amount of precipitated protein was expressed as the % of total, and the complete precipitation curves were constructed as described previously.11 Preparation of Cells. The cell lines used in this study were mouse lymphoma YAC-1, mouse mastocytoma P815, human erythroleukemia K562, human lymphoma RAJI, and low metastasis mouse melanoma B16F1, all of which were obtained from American Tissue Culture Collection, and routinely maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (GIBCO), antibiotics, and 2 mM Lglutamine (complete RPMI 1640) at 37 °C in a humidified atmosphere containing 5% CO2. Human peripheral blood mononuclear cells (PBMC) were isolated from fresh heparinized peripheral blood of healthy donors by centrifugation on Histopaque 1077 (Sigma) gradient, washed, and cultivated in complete RPMI 1640 medium. For proliferation assays and cytokine production, PBMC were used without further purification.16 Inositolphosphate Production. [3H] inositol phosphates were separated and quantified by the methods described previously.13 Briefly, incorporation of [3H] inositol into the phospholipid was achieved by incubating human peripheral blood mononuclear cells (PBMC) (107 cells/mL) with 100 μL of [3H] inositol (1.48 TBq/mol, 37 MBq/ml; GE Healthcare) for 3 h at 37 °C, followed by extensive washing, and resuspension at 108 cells/mL. 50 μL of this suspension containing 5 × 106 cells in complete RPMI 1640 with 10 mM Hepes pH 7.4 was mixed with 50 μL of the tested compounds, and incubated at 37 °C for the indicated times. The reaction was stopped by rapid transfer of the reaction mixture to 100 μL of 10% trichloroacetic acid. The reaction was neutralized by the addition of 50 μL of triethylamine; 20 μL of 50% aqueous slurry of Dowex l-X8, 100−200 mesh (Sigma) in formate form was then added. The supernatant was collected, and inositolbisphosphates and inositoltrisphosphates were eluted by the addition of 50 μL of 0.3 and 0.6 M ammonium formate pH 7.0, respectively. The eluent was dried in a thinwalled 96-well plate, and the radioactivity was counted in 100 μL of Biodegradable Counting Scintillant (GE Healthcare) using the Microbeta counter (Wallac). Intracellular Calcium Monitoring. Human PBMC were loaded with the calcium-sensitive fluor Indo-1 by incubating 107 cells/mL in 5 pM Indo-1, AM (Molecular Probes, Eugene, OR) in complete RPMI 1640 with 25 pM 2-ME at 37 °C.13 Cells were washed twice and resuspended at 5 × 106 cells/mL in fresh medium. The fluorescence of the cell suspension was monitored with a Safire2 spectrofluorometer using an excitation wavelength of 349 nm and emission wavelength 410 nm. The signal was calibrated for each experiment by lysing the Indo-1loaded cells with Triton X-100 (0.07%) for maximum fluorescence. The minimum fluorescence was determined after the addition of 10 mM EGTA and sufficient Tris base to raise the pH to >8.3. Intracellular calcium concentration was calculated using the formula: [Ca2+] (nmol) = 250 × [(F − Fmin)/(Fmax − F)] where F is the measured fluorescence and 250 (nM) is the dissociation constant of Indo-1. Signaling was

measured in the absence of extracellular calcium in medium containing 1 mM EGTA. Where indicated, ionomycin (Sigma) was added as a positive control to a final concentration of 1 μM. Measurement of Lymphocyte Proliferation and Cytokine Production. PBMC were cultivated in 96-well flat-bottomed plates (Nunc) at 2 × 105/well in complete RPMI 1640 medium, together with different concentrations of peptides (in a range from 250 μg/mL to 250 ng/mL), either without additional stimulation or with phorbol myristate acetate (PMA, Sigma; 50 ng/mL) and calcium ionophore A23187 (Sigma; 500 ng/mL). Proliferation of cells was determined using standard [3H]-thymidine incorporation assay. The plates were harvested by automated harvester (Tomtec) and radioactivity measured by Microbeta scintilation counter (Wallac). Stimulation index was calculated as a ratio SI = cpm of stimulated cells/cpm of unstimulated cells. For cytokine production, PBMC were cultivated under the same conditions, and supernatant samples were collected after 24 h, pooled from 3 parallels, and kept frozen in aliquots at −20 °C until the assays. Cytokine levels (IL-2, IL-4, IL-10, TNFα, IFNγ, IL-12) were measured by ELISA (Cytokine Module Set, Bender MedSystems) according to the manufacturer instructions. Apoptosis Assays. Cells were resuspended at 2 × 106/mL in complete RPMI 1640 medium, aliquoted into roundbottomed 96-well plates, and the tested concentrations of compounds were added into duplicate test wells. Individual tested compounds were added 12 and 6 h before the estimation of the percentage of apoptotic cells using Annexin V-FITC/ Hoechst 33258 staining and flow cytometry. The percentage of apoptotic cells (Annexin V+/Hoechst 33258−) observed in the presence of PBS alone or in the presence of 5 × 10−6 M arsenite were used as the negative and positive controls, respectively.13 Natural Killing. The standard 51Cr release test was performed as described previously.17 Briefly, 104 chromiumlabeled target cells in 100 μL complete RPMI 1640 was mixed in triplicate with the tested compounds in 50 μL of RPMI 1640 in round-bottomed 96-well plates. Thereafter, the appropriate amount of effector cells (mouse spleen cells or isolated tumorinfiltrating lymphocytes) was added in 100 μL of complete RPMI 1640, and the plate was incubated at 37 °C, 5% CO2 for 3 h. 50 μL of 1% Triton X-100 was added into the maxima release wells, and the incubation continued for another 1 h. Plates were cooled on ice bath, and 100 μL of the supernatant was used for radioactivity measurements. % of specific lysis was calculated using a formula % = [ (exp − spont)/(max − spont)] × 100, where exp are the counts in experimental wells, spont are counts in wells containing medium instead of the effector cells, and max are counts in wells containing 1% Triton X-100. Complete killing curves were constructed, from which the lytic units counts for individual experiments were calculated. Lytic efficiency was defined as the inverse of the lytic units count.17 Tumor Animal Models and Therapies. Young (6−8week-old) female C57BL/6 mice were purchased from Charles River (Montreal, Quebec, Canada) and handled under the guidelines of the Institute of Microbiology CAS Animal Care protocol. Mice were shaved in the right flank area, and were given subcutaneous injections of 2.5 × 104 viable low metastasis variant B16F1 cells18 in the final volume of 100 μL of PBS. The tested peptides (0.1 mg in 100 μL PBS), or monoclonal antibody against mouse CD69 H1.2F3 (0.5 mg in 100 μL of 2035



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PBS), or monoclonal antibody against mouse CD69 G235− 2356 (0.3 mg in 100 μL PBS), or PBS only were injected in a single intravenous dose on day 20. Tumor growth was followed by Vernier caliper measurement on day 30 after transplantation. All of the experiments included ten mice/group. Tumor area was calculated according to the formula A = (a × b)/2, where a = the largest superficial diameter and b = the smallest superficial diameter. The tumor infiltrating lymphocytes were isolated from animals at day 25 and day 30 (5 and 10 days after the injection of dimerized peptides), and their cytolytic activity was measured as described above. Tumor infiltrating lymphocytes were isolated as described previously.19

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RESULTS The starting peptides for dimerization were based on the immunoactivating peptide sequence Leu-Glu-Leu-Thr-Glu, and Table 1. Structure and Yield of the Synthesized Compounds yield using reaction conditionsa compd.

structure

(a)

(c)

(d)

1

(TyrGlyGluThrLeuGluLeu)2-NHCO-CO-NH(TyrGlyGluLeuLeuGluLeu)2-NHCO-CO-NH(TyrGlyGluThrLeuGluLeu)2-NHCO-CHOH-CHOH-CO-NH(TyrGlyGluLeuLeuGluLeu)2-NHCO-CHOH-CHOH-CO-NH(TyrGlyGluThrLeuGluLeu)2-NHCO-(CH2)3-CO-NH(TyrGlyGluLeuLeuGluLeu)2-NHCO-(CH2)3-CO-NH(TyrGlyGluThrLeuGluLeu)2-NHCO-(CH2)6-CO-NH(TyrGlyGluLeuLeuGluLeu)2-NHCO-(CH2)6-CO-NHCOOHTyrGlyGluThrLeuGluLeu-NH2 COOHTyrGlyGluLeuLeuGluLeu-NH2

2.6

4.8

3.9

3.5

2.0

5.2

3.6

3.4

30

20

7.8

25

8.2

23

6.5

25

6.4

2.3

36 (34)b 35 (33)b 34 (30)b 36 (34)b 4.5

3.8

2.5

2.4

4.7

3.8

2.5

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

2 3 4 5 6 7 8 9 10

34 26 30

(b)

Figure 1. Evaluation of the binding of the synthesized compounds to the target receptor CD69. (A) The synthesized compounds were evaluated as inhibitors of binding of recombinant human CD69 to microtiter wells coated with the high affinity ligand, GlcNAc23BSA. (B) Compounds 3, 5, 7, and 9 were tested for their affinity to CD69 receptor using a direct binding experiment. (C) Selected compounds were tested for their ability to precipitate soluble CD69 receptor when added in equimolar amounts (0.1 nmol of both per precipitation reaction). The data in (A) and (C) are the means ± SD of three independent experiments all performed in duplicates.

a

hydroxysuccinimide groups of the bifunctional reagent, and reaction at elevated temperature (45−50 °C) for prolonged reaction time (16−20 h) (Scheme 1). The reaction was terminated by acidification, and the mixture was applied onto C18 reverse-phase column, on which the individual peptide forms were separated using a standard acetonitrile gradient in 0.1% TFA. Under these conditions, a very good separation of the unreacted peptide from the reagent containing only a single peptide residue, and the desired peptide dimer could be achieved (Supporting Information Figure 1). Fractions that were active at 280 nm were collected, evaporated, and analyzed for molecular size using MALDI-MS (Supporting Information Figure 2). This analysis confirmed that the peptide dimer eluted in the last peak at approximately 22−23 min under the given experimental conditions. Using the initial conditions containing 50:50 (by volume) DMSO:buffer mixtures, only very low yields of the target dimerized peptides (10−20% of the input peptide) could be observed. Therefore, we have changed the concentrations of DMSO in the reaction mixture to reach 67%, 80%, 90%, and 95% (reaction conditions (a) to (d), respectively). Increasing the content of DMSO in the reaction mixture resulted in significant increase in the yield reaching optimum for 80% DMSO after which there was a drop in the yield using reaction

Yield is expressed as the % of the input peptide recovered in the purified conjugate. bPreparative (50 μmol) yields under optimal reaction conditions; n.a., does not apply.

control inactive sequence Leu-Glu-Leu-Leu-Glu identified previously.13 Since we have shown that this sequence remains active as a part of large peptide or protein sequence, we decided to extend both the active and the control sequence by two additional amino acids, Gly and Tyr. This additional sequence included the tyrosine residue that is convenient for the purpose of UV detection (strong signal at 280 nm), as well as radiolabeling of the synthesized peptide. The glycine residue, on the other hand, was used as an inactive amino acid connecting the tyrosine to the rest of the peptide. Such modified peptides were dimerized using commercially available bifunctional reagents based on the reaction of a unique Nterminal amino group in the dimerized peptides with the Nhydroxysuccinimide reactive group of the reagent. The starting conditions for individual syntheses were selected on the basis of protocols optimized for protein cross-linking using the bifunctional reagents. Thus, we have employed the previously tested20 N-ethylmorpholine acetate buffer pH 7.5 in combination with DMSO that facilitated the solubility of the peptides, 2.2 molar excess of the peptide over the N2036



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significantly lower yields compared to middle length (DST and DSG providing 4 and 5 carbon length linkers, respectively) reagent (Table 1). Altogether, microgram amounts of the dimerized peptides 1 to 8 could be obtained, and analyzed by spectrophotometry, quantitative amino acid analysis, and MALDI-MS (Table 1). Synthesized peptide dimers were initially used in biochemical assays in order to verify the interaction of the synthesized ligands with its target receptor, recombinant human CD69 expressed as a stable, soluble protein refolded in the form of noncovalent dimers.5,10 We first tested the synthesized compounds as the inhibitors of binding of the above recombinant receptor to its high-affinity ligand, GlcNAc23BSA. To validate these experimental assays, we tested the monomeric starting peptides 9 and 10, and found them to have affinities corresponding to those reported previously13 (Figure 1A). Also, the inhibitory activities of control compounds 2, 4, 6, and 8 bearing the nonreactive peptide were very low. Of the newly synthesized dimerized peptides, those linked by DSO and DSS (compounds 1 and 7, respectively) had very little inhibitory activity. On the other hand, active heptapeptide 3 dimerized using DST had inhibitory activity comparable with the starting immunoactivating peptide, and the one dimerized using DSG (compound 5) had even somewhat higher inhibitory activity. These results were further corroborated using the direct binding assays in which the interaction of the 125I-radiolabeled immunoactive peptide with the receptor is followed using equilibrium dialysis. This assays revealed the same hierarchy of reactivities already observed using the plate inhibition assays: while the immunoreactive peptide 9 interacted with the receptor with nanomolar value of dissociation constant, the interaction of DSG dimerized peptide 5 was more then 1 order of magnitude stronger, and the DST dimerized peptide 3 interacted about three times more strongly with the receptor (Figure 1B). DSS dimerized peptide 7 had affinity comparable with the monomeric peptide 9 (Figure 1B). We have further tested the selected peptide dimers using the precipitation assays with the soluble CD69 receptor. In order to mediate any immune activity, the immunoactive compound must cross-link the target receptor at the surface of immune cells. Since CD69 has two ligand binding sites per receptor dimer, the reaction of its soluble form with equimolar concentrations of the bivalent soluble ligand may result in extensive receptor cross-linking under conditions that mimic the reaction occurring with the cellular (membrane bound) form of the receptor. In the precipitation assays, both the monomeric peptides 9 and 10 (immunoactive as well as control) and the dimerized control peptides 4, 6, and 8 provided very little precipitation (compounds 1 and 2 were not examined because of their low inhibition activities). On the other hand, the active heptapeptide 3 dimerized using DST precipitated nearly 40% of the soluble receptor present in the reaction mixture, while the active heptapeptide 5 dimerized through DSG could precipitate nearly 100% of the receptor (Figure 1C). Therefore, we further synthesized compounds 3 and 5, and their respective controls 4 and 6, at preparative scale using optimized reactions providing most favorable yields. The yields of preparative reactions were similar to those performed at analytical scale allowing us to obtain milligram quantities of these peptide dimers suitable for detailed NMR analyses (Supporting Information Tables 1−4), and for further biological testing.

Figure 2. Evaluation of the ability of the synthesized compounds to stimulate human PBMC and initiate their apoptosis. (A,B) Production of inositol bisphosphates and inositoltrisphosphates, respectively. (C) Elevation of the levels of intracellular calcium. (D) Apoptosis of human PBMC 6 and 12 h after the addition of compounds. % apoptotic cells was defined as the percentage of the entire population bearing annexin V+/propidium iodide − phenotype.13 All compounds were tested at 10−8 M concentrations. The results are the means ± SD of three independent experiments all performed in duplicates.

Figure 3. Evaluation of the selected compounds for their ability to modulate the proliferation of normal (A) and activated (B) human PBMC. The stimulation index was calculated relative to the basal levels that were 1678 ± 85 cpm for nonstimulated control cells (PBMC) in panel (A), and 4394 ± 127 cpm for PBMC activated by PMA and calcium ionophore A23187. The results are the means ± SD of three independent experiments all performed in triplicate.

mixtures with less than 10% of buffer. Moreover, the yield of the dimerized peptide depended much on the linker length of the employed cross-linking reagent: both short (two carbon, DSO) and long (six carbon, DSS) cross-linkers provided 2037



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Figure 4. Analysis of the effect of selected peptides and dimerized peptides on the production of cytokines by normal human PBMC.

In order to test the ability of the dimeric peptide to activate human PBMC, we monitored the generation of inositolbisphosphates, inositoltrisphosphates, and the increase in the intracellular calcium (Figure 2A, B, and C, respectively). Both dimeric peptides that were active in the receptor precipitation assays caused lymphocyte activation (compounds 3 and 5). Since extensive cross-linking of CD69 may lead to apoptosis of lymphocytes bearing this receptor, we also tested the ability of these compounds to induce apoptosis of human PBMC using surface measurements of annexin V by flow cytometry. Surprisingly, while compound 3 caused significant apoptosis, compound 5 was completely inactive in this respect (Figure 2D). We have further investigated the ability of the dimerized peptide to increase the proliferation of both normal and activated human lymphocytes. Using normal human lymphocytes, we could observe a significant enhancement in the proliferation of these cells that was maximal at approximately 3 μg per mL for both compounds 3 and 5. Active heptapeptides dimerized using both DST and DSG had comparable activity, and both enhanced the proliferation nearly a hundred times compared to PBS control (Figure 3A). Dimerized control peptides 4 and 6 (Figure 3A) or monomeric peptides (not shown) remained completely inactive. On the other hand, the remarkable activity of compounds 3 and 5 observed in normal lymphocytes was not paralleled in experiments using activated lymphocytes (PMA plus calcium ionophore), where we could

not observe any increase in proliferation (Figure 3B). Moreover, compounds 3 and 5 were also active in cytokine production, again using normal nonstimulated human peripheral lymphocytes. Both compounds significantly enhanced the production of IL-2, IL-4, IL-12, TNF-α, and IFN-γ at concentrations as low as 3 to 30 μg/mL (Figure 4). IL-10 was highly stimulated by dimeric peptide 5, while dimeric peptide 3 stimulated the production of this cytokine much less. Similarly, conjugate 5 stimulated the production of IL-12 (and, to lesser extent, IL-2) at lower concentrations compared to conjugate 3. The dimerized immunoactive peptide 3 and, in particular, 5 caused a significant increase in the production of IL-2 and IL12. As both these cytokines are known to activate NK cells and cytotoxic CD8+ T cells, we investigated the direct effect of these compounds using the 4 h tumor cell killing assays. Interestingly, both 3 and 5 significantly enhanced the killing of tumor targets, both sensitive and resistant for natural killing in mouse (Figure 5 A,B), and human cells (Figure 5C,D), although compound 5 appeared superior to compound 3. For this reason, it appeared interesting to test the anticancer effect of these compounds in experimental animal models. We have selected the model of mouse B16F1 melanoma that turned out useful in our previous studies. Compound 5 that appeared most active in receptor binding assays and in vitro cytotoxicity test provided the highest antitumor effects also in vivo in mice bearing melanomas (Table 2). In order to further substantiate these results, we tested the 2038



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Figure 6. Survival of C57BL/6 mice bearing syngeneic B16F10 melanoma, and treated with a single injection of 0.1 mg of the tested compounds 10 days after the injection of melanoma cells. Displayed data represent a typical result of independent experiments (P < 0.05).

protection of mice against melanomas, only caused a prolonged survival (Figure 6). Compounds 4 and 5 had only moderate effects, and compound 3 had essentially no activity since its curve was similar to that of the PBS control (Figure 6).

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DISCUSSION We have recently identified the immunoactivating pentapeptide derived from the mycobacterial heat shock protein hsp65, and found that CD69 is the target receptor for this peptide due to the interaction mediated through negatively charged carboxylic functions that are essential for the binding activity.13 While the pentapeptide completely retained the binding activity of a larger peptide containing this sequence internally, or even the entire hsp65 subunit, it had to be presented in multimeric form to achieve optimal immunostimulating activity. Previously, we presented this sequence as a part of a complex cyclic dekapeptide raft.13 We now show that the dimerization of the target peptide through a simple synthetic linker, either hydrophobic or hydrophilic, is sufficient to sustain its high biological activity. However, as noted by other laboratories,21 in order to achieve an optimal biological activity of such conjugates, a careful optimization with respect to both linker length and linker chemistry was needed. When we dimerized the immunoactive heptapeptide with two short linkers composed only of two carbonyl groups, or two long linkers composed of six methylene groups, the inhibitory activity was not optimal. In the former case, the linker may have been too short to secure good access of the ligand into the binding site of the receptor, while in the latter case, a linker that is too long most probably provided excessive flexibility to the ligands and supported the mutual interaction between the ligands at the expense of efficient binding to the receptor. As a result of these initial biochemical investigations, the immunoactive heptapeptide dimerized through DST (compound 3) or DSG (compound 5), and thus containing either hydrophilic or hydrophobic links composed of 2−3 methylene or hydroxymethylene groups, appeared to be optimal. Subsequent immunological evaluations have confirmed such an assumption, since both compounds 3 and 5 turned out to be highly active in enhancing the proliferation of normal unstimulated human lymphocytes, and increasing the production of several cytokines critical for the production of activated killer cells and enhanced cytotoxicity. These results point to similar effects that we have observed recently (both in vitro and in vivo) with other dimeric high-affinity ligands for CD69.21−24 The cytokine production in the heterogeneous PBMC

Figure 5. Effect of selected compounds on the cytotoxicity (natural killing) by lymphocytes in vitro and ex vivo. (A,B) Effect on % specific cytotoxicity by fresh mouse lymphocytes was tested using the sensitive and resistant cell lines YAC-1 and P815; (C,D) effects on the % specific cytotoxicity by human PBMC to sensitive and resistant tumor cell lines K562 and RAJI was tested; (E,F) cytotoxic activity of tumor infiltrating lymphocytes isolated from tumor bearing animals treated with the indicated compounds was tested against the resistant P815 cell line 5 days and 10 days after the injection of the compounds, respectively. Effector:target cell ratio was 300:1 in (A) and (B), 100:1 in (C) and (D), and 30:1 in (E) and (F).

Table 2. Evaluation of the Antitumor Activity of the Synthesized Compounds Using the Mouse Melanoma B16F1 Modela compound PBS MAb1 MAb2 (3) (4) (5) (6)

tumor size [cm2] day 30 1.3 1.1 0.8 0.7 1.2 0.2 1.3

± ± ± ± ± ± ±

0.3 0.2 0.2 0.1 0.1 0.2 0.2

a

MAb1 = monoclonal anti-mouse CD69 antibodies clone H1.2F3. MAb2 = monoclonal anti-mouse CD69 antibodies clone G235-2356.

effect of both compounds (3 and 5) on the cytotoxic activity of tumor infiltrating lymphocytes ex vivo. Interestingly, while compound 5 appeared to enhance the activities of these lymphocytes in the tumor microenvironment significantly, compound 3 had no effect (Figure 5E). The ex vivo effect of compound 5 that was observed at day 5 after injection was sustained, since at day 10, this compound had an even higher effect on the enhancement of killer activity of tumor infiltrating lymphocytes than on day 5 (Figure 5E,F, respectively). Similar effects were also observed when we tested the influence of these compounds on the survival of tumor bearing mice. Compound 5 was most effective, although it did not produce a permanent 2039



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for further development of even more active immunostimulating compounds providing a permanent protection for tumor bearing animals.

population could not be related to one particular subpopulation, but conforms well to the wide expression of CD69 on lymphocytes. The enhanced release of cytokines parallels the significantly stimulated proliferation, and encompasses both Th1 (IL-2, IL-12, IFNγ, TNFα), and Th2 cytokines (IL-4, IL10). It is generally accepted that activation of the Th1 subset of cells and relevant cytokine production is related to antitumor immune mechanisms.25 The activation of tumor killing activity by IL-2 was thorougly documented, including activation of tumor-infiltrating lymphocytes.26−28 IL-12 dictates the orchestration of immune response toward proimmunogenic nature,29 and generation of protective antitumor response by stimulating T cells and NK cells to produce IFNγ, which is involved in tumor cell killing.30 IL-12 can induce a substantial increase of cytotoxicity in NK cells cultured in the absence of additional stimuli,31 and increases expression of activation markers CD69 and CD25 on highly purified human NK cells.32 The balance between Th1 and Th2-cytokine activities is predominantly important in the tumor microenvironment, and could affect the outcome of the antitumor immune responses. Release of Th2related cytokines IL-4 and, namely, IL-10 by PBMC was more pronounced in peptide 5 than in peptide 3-stimulated cells. IL10 is a pivotal immunoregulatory cytokine suppressing macrophage and dendritic cell functions, thereby switching off immune system activation,33 and dampening antitumor immune response.34 However, the IL-10 impact is determined by the timing and localization of its release,35 which could not be determined in detail. Recently, it was shown that IL-10 could even be important in tumor immune surveillance36 pointing to a complex role of this cytokine in the antitumor response. In addition to producing the enhanced levels of cytokines potentiating the cytotoxic activities of natural killer cells and lymphocytes, compounds 3 and 5 had a significant direct effect on killer lymphocytes as shown by results of in vitro cytotoxicity assays. Thus, the effect of both compounds most probably is complex, including the direct effect via binding to the target receptor CD69 on killer cells, and indirect effect via inducing an array of cytokines. Reciprocal activating interactions between human NK and dendritic cells, which are the principal cell population activating specific antitumor immune responses, were recently described.37 These interactions may also play an important role in the outcome of the melanoma therapy with the peptides.

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ASSOCIATED CONTENT

* Supporting Information S 13

C and 1H NMR data of dimeric peptides 3, 4, 5, and 6 (Supporting Tables 1 to 4). HPLC separation of the synthesized peptide dimers (Supporting Figure 1). MALDIMS spectra of the individual synthesized compounds (Supporting Figure 2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel. +420-2-2195-1272; Fax. +420-2-2195-1283. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dedicated to Professor Gustav Entlicher on the occasion of his 70th birthday. The authors are indebted to Pavlı ́na Jungrová, Helena Mišurcová, David Adámek, and Alan Kádek for their invaluable technical support, and to Petr Novák and Jakub Tomala for their help at initial stages. This work was supported by grants from Ministry of Education of Czech Republic (MSM_21620808, 1M0505 and AVOZ50200510 to K.B.) from Czech Science Foundation (303/09/0477 and 305/09/H008 to K.B.), by Charles University Prague (projects 263209/2009 and UNCE 204025/2012), and by EU Project Spine 2 (contract LSHG-CT-2006-02/220 to K.B.).

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REFERENCES

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CONCLUSIONS Detailed examination of the direct binding and precipitation data, as well as the in vitro cytotoxicity data revealed that compound 5 had actually a much stronger interaction with the CD69 target receptor than compound 3. In good concordance with these results is the in vivo antimelanoma activity that was significant in compound 5 and still detectable, but much weaker in compound 3. Moreover, the tumor infiltrating lymphocytes from mice treated with compound 3 had extremely low cytotoxicity ex vivo. We also documented that compound 3 but not compound 5 induced a significant level of apoptosis in human PBMC cells. Taking into consideration all these results, we can speculate that the interaction of compound 3 with CD69 can caused a hyperactivation of CD69 positive cells leading to their activation induced cell death, a process that has been described previously for monocytes and eosinophils,7−9 and recently by us for lymphocytes.22,38 The most effective compound 5 did not possess these undesired side effects, and would thus represent a suitable lead and promising candidate 2040



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Dimerization of an Immunoactivating Peptide Derived from

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