OPTIMIZATION OF PEPTIDE-MELANIN BINDING

Abstract

The invention relates to the use of melanin, complexed with a peptide, in particular containing epitopes for use as an immunostimulatory composition, wherein the peptide that has been modified as to increase nucleophilicity.

Claims

1. A method for obtaining a composition comprising melanin bound to a modified peptide, the method comprising: a) providing a synthetic melanin obtained by an oxidative polymerization of a melanin precursor; and b) mixing the synthetic melanin with a peptide that has been modified by addition of one or more amino acids containing a nucleophilic residue to obtain the composition comprising melanin bound to the modified peptide.

2. The method of claim 1, wherein the synthetic melanin is a soluble melanin.

3. The method of claim 1, wherein the peptide is an immunologically active peptide.

4. The method of claim 1, wherein the one or more amino acids added to the peptide are selected from cysteine, acetylcysteine, methionine, proline, hydroxyproline, histidine, lysine, and a combination thereof.

5. The method of claim 1, wherein the one or more amino acids are added to an N-terminus of the peptide.

6. The method of claim 1, wherein a cysteine is added to an N-terminus of the peptide.

7. The method of claim 1, wherein the peptide modified by addition of the one or more amino acids containing a nucleophilic residue is selected from SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 33.

8. The method of claim 1, wherein the melanin precursor is selected from L-dopa, DHICA, DHI, L-tyrosine, D-dopa, 6-hydroxy-Dopa, dopaquinone, cyclodopa, dopachrome, dopamine-o-quinone, dopamine, leukodopaminochrome, and dopaminochrome.

9. The method of claim 8, wherein the melanin precursor is L-Dopa.

10. The method of claim 1, wherein the oxidative polymerization is performed in presence of oxygen, H.sub.2O.sub.2, or persulfate.

11. The method of claim 1, wherein the oxidative polymerization is performed in presence of tyrosinase.

12. The method of claim 1, wherein the synthetic melanin is first purified by filtration on a 10 kDa filter before being mixed with the peptide.

13. The method of claim 1, wherein an immune adjuvant is added to the composition comprising melanin bound to a modified peptide before administration to a host.

14. The method of claim 1, further comprising conditioning the composition for administration to a host.

15. An immunostimulatory composition obtainable by the method of claim 1.

16. The immunostimulatory composition of claim 15, wherein the immunostimulatory composition is a vaccine that protects or treats a human or an animal against a disease implicating cells expressing an antigen expressing an epitope.

17. The immunostimulatory composition of claim 16, wherein the disease is a cancer, a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

18. The immunostimulatory composition of claim 17, wherein the disease is a low or high grade glial tumor.

19. The immunostimulatory composition of claim 18, wherein the peptide is selected from SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 34, and SEQ ID NO: 35.

20. A peptide comprising SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 34, or SEQ ID NO: 35.

21. The peptide of claim 20, comprising SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 34, or SEQ ID NO: 35 at its N-terminus.

22. The peptide of claim 20, consisting of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 34, or SEQ ID NO: 35.

Description

DESCRIPTION OF THE FIGURES

[0177] FIG. 1: CTL response after subcutaneous immunizations in 5-weeks old, C57BL/6, mice. Peptides (10 μg/mouse) were mixed with L-Dopa (weight ratio peptide/L-Dopa=1/4 and 1/6 for gp100 and EphA2 respectively) and incubated under the above described conditions (see table 1). Phosphorothioate oligonucleotide CpG-28 (5′-TAAACGTTATAACGTTATGACGTCAT, SEQ ID NO: 21), were added to vaccine formulations (10 μg/mouse) just before the immunizations. Mice were then immunized sub-cutaneously with gp100-Mel+CpG (“gp100-Mel”), or with previously synthesized melanin mixed with gp100 and CpG (“Mel+gp100”); EphA2-Mel+CpG (“EphA2-Mel”), melanin+EphA2+CpG (“Mel+EphA2”). Mice were sacrificed on day 8 and the CTL response was performed as described in Carpentier; 2017. Briefly, splenocytes were re-stimulated in vitro with the corresponding MHC class 1-epitope (non-conjugated to melanin) and the numbers of IFNg-SFCs (Spot forming cells) were measured and expressed as Mean+/−S.E.M. (n=8 mice/group with pooled data from 2 different experiments of 4 mice each. Student-T test: gp100-Mel vs Mel+gp100: p<0.001; EphA2-Mel vs Mel+EphA2: p<0.001)

[0178] FIG. 2: CTL response after subcutaneous immunizations in C57BL/6 mice. The gp100 peptide (10 μg/mouse) was mixed with L-Dopa (weight ratio peptide/L-Dopa=1/4 and incubated at pH 8.5 in aerobic conditions for 2 hours at 60° C. to generate gp100-Mel. Alternatively, L-Dopa (0.8 mg/ml) underwent an oxidative polymerization at pH 8.5 in aerobic conditions for 2 hours at 60° C. The reaction mixture was then filtered on a 10 kDa filter, and the retentate containing the synthetic melanin was resuspended at pH 7.5 in phosphate buffer. Peptides (gp100 (SEQ ID NO: 1) or C-gp100 (SEQ ID NO: 17); 10 μg/mouse) were then added (at the weight ratio peptide/L-Dopa=1/4) and the mixtures (Mel+gp100 or Mel-C-gp100) were rapidly (<1 hour) used for subcutaneous immunizations in mice. Phosphorothioate oligonucleotide CpG-28 (5′-TAAACGTTATAACGTTATGACGTCAT, SEQ ID NO: 21) was added to vaccine formulations (10 μg/mouse) just before the immunizations. Mice were sacrificed on day 8 and the CTL response was performed as described in FIG. 1. (n=12 mice/group with pooled data from 3 different experiments of 4 mice each. Student-T test: gp100-Mel vs Mel+gp100: p<0.01; Mel+gp100 vs Mel+C-gp100: p<0.05)

EXAMPLES

Example 1. Preparing Immunogenic Compositions According to WO2017089529

[0179] Vaccine formulations combining antigens and synthetic melanin were prepared and tested for their ability to trigger cytotoxic T-lymphocyte (CTL) immune response (Carpentier et al, PLoS One. 2017 Jul. 17; 12(7):e0181403, WO2017089529). In these studies, short synthetic peptides (8-35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides that can be efficiently used as a vaccine to trigger immune responses in mice. The binding of the antigens to synthetic melanin appeared critical to trigger immunity. Indeed, if the antigens (for ex the peptides gp100, EphA2) are added not before, but just after L-Dopa is polymerized in melanin, minimal binding of the peptides to melanin is seen in SDS-page analysis (Table 1), and the ability of the vaccine formulation to trigger a CTL (CD8) response in mice is lost (FIG. 1).

TABLE-US-00001 TABLE 1 Percentage of gp100 (KVPRNQDWL SEQ ID NO: 1) or EphA2 (FSHHNIIRL, SEQ ID NO: 2) binding to melanin (Tricine-SDS-PAGE analysis). Peptide-Mel Mel + peptide KVPRNQDWL (SEQ ID NO: 1) 100 +/− 0% 23 +/− 8% FSHHNIIRL (SEQ ID NO: 2)   93 +/− 12%  9 +/− 6%

[0180] Peptides were mixed with L-Dopa (weight ratio peptide/L-Dopa=1/4 and 1/6 for gp100 and EphA2, respectively) then incubated for 2 hours at pH 8.5 and 60° C. under agitation to ensure oxygenation of the solution to generate nanoparticles of melanin-bound peptides (Peptide-Mel). Alternatively, L-Dopa alone (0.8 mg/ml) was polymerized into melanin for 2 h at pH 8.5 and 60° C. under agitation, before the peptides (weight ratio peptide/L-Dopa=1/4 and 1/6 for gp100 and EphA2, respectively) were added to the solution (Mel+peptide). Tricine-SDS-PAGE analysis was performed as described in Carpentier; 2017 (op. cit.). Briefly, samples (peptide-Mel or peptide alone) were loaded on acrylamide gels. Following electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 and imaged with the ChemiDoc XRS+ system (Bio-Rad Laboratory), allowing the quantification of the free peptide in the gel. The binding of peptides to melanin was expressed as the ratio: [amount of unbound peptide in samples Peptide-Mel/amount of peptides in control samples containing peptides alone]

Example 2. Optimization of Peptides Binding to Melanin

[0181] As shown above, efficient binding of the antigenic peptide to melanin plays a critical role to obtain biological properties. This binding that can be conveniently achieved by mixing the peptide during polymerization of L-Dopa as disclosed in WO2017089529.

[0182] Yet, concerns exist that peptides can be degraded during this oxidative process, which generates reactive oxygen species. A method of grafting/binding peptides on synthetic melanin (after synthetic melanin was obtained by oxidative polymerization of L-Dopa) was developed.

[0183] The radical moieties involved in melanin binding were first studied. Different peptides, all containing the basic sequence SIYRYYGL (SEQ ID NO: 3), were mixed with L-Dopa then incubated for 2 hours at pH 8.5 under agitation to generate nanoparticles of melanin-bound peptides (Peptide-Mel). As seen in the first column (Peptide-Mel) of table 3, melanin binding seems to depend upon the presence within peptides of nucleophilic moieties:

1) No binding was seen when the terminal NH2 was blocked by an acetyl residue (Acetyl-R-SIYRYYGL, SEQ ID NO: 4), pointing out the important and possibly critical role of the —NH2-terminal end of peptides (and a less important role of the terminal —COOH moiety) in melanin binding.
2) Nucleophilic Proline or Hydroxyproline can be added at the NH2-terminal amino acid.
3) Lateral chain of some nucleophilic amino acids such as Lysine and Cysteine, also allowed a significant binding even when the NH2-terminal end of the peptides were blocked

[0184] Most surprisingly, even when L-Dopa alone was first polymerized into melanin, before the peptides were added to the solution, then incubated for 2 hours at room temperature (Mel+Peptide), some binding can be seen if some specific amino-acids (Cysteine, and to a minor degree Hydroxy Proline) are included in the peptide (Table 2, second column).

TABLE-US-00002 TABLE 2 Bindinq of peptides on synthetic melanin (Tricine-SDS-PAGE analysis): Peptide- Mel + Mel peptide Reference SIYRYYGL (SEQ ID 90% 0% NO: 3) Acetyl-R SIYRYYGL  0% 17%  (SEQ ID NO: 4) Nucleophile P SIYRYYGL (SEQ ID 87% 7% NO: 5) HydroxyP SIYRYYGL 95% 23% (SEQ ID NO: 6) Nucleophile Acetyl-R C SIYRYYGL 74% 53%  (SEQ ID NO: 8) Acetyl-R H SIYRYYGL 60% ND (SEQ ID NO: 9) Non Acetyl-R T SIYRYYGL 10% 0% nucleophile (SEQ ID NO: 12) Acetyl-R F SIYRYYGL  5% 0% (SEQ ID NO: 13) Acetyl-R W SIYRYYGL 56% 0% (SEQ ID NO: 14)

[0185] First column (Peptide-Mel): L-Dopa (0.8 mg/ml) was mixed with peptides (weight ratio peptide/L-Dopa=1/4) then incubated for 2 hours at pH 8.5 and 60° C. to generate nanoparticles of melanin-bound peptides. Alternatively (second column; Mel+peptide), L-Dopa alone (0.8 mg/ml) was first polymerized into melanin for 2 h at pH 8.5 and 60° C. under vigorous agitation, before the peptides (weight ratio peptide/L-Dopa=1/4) were added to the solution and incubated for 2 hours at room temperature. In both cases, the percentage of peptides that bound to melanin was then quantified with SPS-page analysis, as described in table 1. (ND=not done)

Incubation Conditions

[0186] We further investigated the impact on melanin binding of various incubations procedures. L-Dopa underwent oxidative polymerization; the reaction mixture was then filtered, and the retentate containing the synthetic melanin was then mixed with different peptides for various periods of time and temperature. Table 3 shows that limited binding of peptides is seen when the incubation time is short (approx. 10 minutes) as disclosed in the literature (Carpentier 2017). Yet, binding increased with incubation time, pH (table 3) or temperature (not shown). This binding is particularly seen when the peptide contained either a free NH2-terminal moiety, or one of the following amino acids: Lysine, Cysteine, Proline, HydroxyProline. Interestingly, for Cysteine, the impact of higher pH on melanin binding appeared limited.

TABLE-US-00003 TABLE 3 Bindinq of peptides on synthetic melanin; impact of various physico-chemical conditions. Mel + Mel + Mel + peptide 2 peptide peptide hours 18 hours 18 hours at pH 7.4 at pH 7.4 at pH 8.5 refer- SIYRYYGL (SEQ 0% 28% 31% ence ID NO: 3) nucleo- P SIYRYYGL 7% 22% 41% phile (SEQ ID NO: 5) hydroxyP 23%  39% 74% SIYRYYGL (SEQ ID NO: 6) nucleo- Acetyl-K 0% 31% 62% phile SIYRYYGL (SEQ ID NO: 7) Acetyl-R C 53%  46% 42% SIYRYYGL (SEQ ID NO: 8) Acetyl-R S 0% 12% ND SIYRYYGL (SEQ ID NO: 10) Acetyl-R M 0% 13% 32% SIYRYYGL (SEQ ID NO: 11) non Acetyl-R 17%   2%  0% nucleo- SIYRYYGL (SEQ phile ID NO: 4) Acetyl-R T 0% ND ND SIYRYYGL (SEQ ID NO: 12) Acetyl-R F 0% ND ND SIYRYYGL (SEQ ID NO: 13) Acetyl-R W 0% ND ND SIYRYYGL (SEQ ID NO: 14)

[0187] L-Dopa (0.8 mg/ml) underwent an oxidative polymerization at pH 8.5 in aerobic conditions for 2 hours at 60° C. The reaction mixture was then filtered on a 10 kDa filter, and the retentate containing the synthetic melanin was resuspended at pH 7.5 in phosphate buffer. Peptides were then added (at the weight ratio peptide/L-Dopa=1/4) and the mixture was incubated for various periods of time and or pH. The percentage of peptides that bound to melanin was then quantified with SPS-page analysis, as described in table 1. (ND=not done).

[0188] A similar experiment was carried out on a family of gp100 peptides (basic sequence: KVPRNQDWL (SEQ ID NO: 1), see also Example 1): (Table 4). Again, when melanin is synthetized before the peptides are added, the melanin binding of peptides is enhanced 1) by increasing the incubation time, and b) when peptides contained specific amino acids such as Lysine, Cysteine, hydroxyproline or methionine.

TABLE-US-00004 TABLE 4 Bindinq of peptides on synthetic melanin; impact of incubation time and amino-acids. Mel + Mel + Mel + peptide peptide peptide 10 2 18 minutes hours hours refer- KVPRNQDWL 6% 22%  89% ence (SEQ ID NO: 1) nucleo- P KVPRNQDWL 13%  11% 100% phile (SEQ ID NO: 15) HydroxP 12%  35% 100% KVPRNQDWL (SEQ ID NO: 16) C KVPRNQDWL 9% 54%  99% (SEQ ID NO: 17) M KVPRNQDWL 7% 43% 100% (SEQ ID NO: 18)

[0189] L-Dopa (0.8 mg/ml) underwent an oxidative polymerization at pH 8.5 and 60° C. for 2 hours. The reaction mixture was then filtered on a 10 kDa filter, and the retentate containing the synthetic melanin was resuspended at pH 7.5 in phosphate buffer. Peptides were then added (at the weight ratio peptide/L-Dopa=1/4) and the mixture was incubated for various periods of time. Binding of the peptides was then quantified with SPS-page analysis, as described in table 1. (ND=not done)

[0190] Similar results were also obtained with various peptides of different length: adding a Cysteine at the NH2-terminal end of the peptide significantly increased the binding on synthetic melanin (data not shown).

[0191] When the amino-acid promoting the melanin binding was placed at the COOH terminal end, instead of the NH2-terminal end of a peptide containing an epitope (for example X-VYDFFVWL (SEQ ID NO: 19) vs VYDFFVWL-X (SEQ ID NO: 20)), the peptide binding to melanin was similar (51% vs 55%).

[0192] Cysteine can be either in oxidized or reduced states, and binding to melanin was studied in both cases. L-Dopa underwent oxidative polymerization; the reaction mixture was then filtered, and the retentate containing the synthetic melanin was then mixed with the Cgp100 (SEQ ID NO: 17) (either in a oxidized or reduced state) peptide for 2 hours at room temperature. Binding was observed in both cases, although more favorable when cysteine is in the reduced state.

[0193] Finally, it was checked that one of the above-described formulations in which peptides had a good melanin binding (>50%) has a good biological activity. As shown in FIG. 2, immunization of mice with one of such formulations induced an immune response of the same magnitude as historical controls in which peptides were mixed to L-Dopa before oxidative polymerization.

CONCLUSION

[0194] Altogether, these experiments show that addition of nucleophilic moieties contained in amino-acids such as Cysteine, Proline, hydroxyProline, Lysine, Methionine to peptides increases binding thereof to melanin. The immunological efficacy of these formulations are similar to the one disclosed in the prior art (WO2017089529), where peptides are added in the reaction mixture before L-Dopa got oxidized.

[0195] The co-incubation of peptides with a previously synthetized melanin, when a nucleophilic amino acid has been added to the peptide is thus an efficient way to increase binding to melanin, and the immune response resulting therefrom. It also presents the advantage of the better control of both the quality of the synthetic melanin (which can be reliably characterized, which is an advantage for regulatory matters) and the quantity of the peptide actually bound to the melanin. This process also provides a better protection of the integrity of the peptide than the one of the prior art, where such peptide is submitted to the oxidizing agent used for polymerizing the melanin precursor.

Example 3. Use of Other Peptides

[0196] CTL response was evaluated after subcutaneous immunizations in humanized female HLADRB1* 0101/HLA-A*0201 (HHD DR1) mice.

[0197] L-Dopa (0.8 mg/ml) underwent an oxidative polymerization at pH 8.5 in aerobic conditions for 2 hours at 60° C. The reaction mixture was then filtered on a 10 kDa filter, and the retentate containing the synthetic melanin was resuspended at pH 7.5 in phosphate buffer.

[0198] Peptides (A10, SEQ ID NO: 29; A8, SEQ ID NO: 34; A30, SEQ ID NO: 31, 10 μg/mouse) were then added (at the weight ratio peptide/L-Dopa=1/4).

[0199] Mixtures (either Mel+peptides or Peptides alone) were rapidly (<1 hour) used for subcutaneous immunizations in mice. Phosphorothioate oligonucleotide CpG-28 (5′-TAAACGTTATAACGTTATGACGTCAT, SEQ ID NO: 21) was added to vaccine formulations (10 μg/mouse) just before the immunizations. Mice were sacrificed on day 8 and the CTL response was performed as described in Carpentier; 2017. Briefly, splenocytes were re-stimulated in vitro with the corresponding peptide (non-conjugated to melanin) and the numbers of IFNg-SFCs (Spot forming cells) were measured and expressed as Mean+/−S.E.M. (n=4 to 8 mice/group). More than 10 spots is considered as a positive immune response

TABLE-US-00005 Protein Peptide Formulation Mean SEM PTPRZ1 A8 (SEQ ID NO: 34) Pept + CpG-28 5 1 A8 (SEQ ID NO: 34) Mel + pept + 29 9 CpG-28 A30 (SEQ ID NO: 31) Mel + pept + 182 35 CpG-28 hTERT A10 (SEQ ID NO: 29) Pept + CpG-28 18 10 A10 (SEQ ID NO: 29) Mel + pept + 150 57 CpG-28

TABLE-US-00006   peptide A10 SEQ ID NO: 29: CKSVWSKLQSIGIRQH, peptide A30 SEQ ID NO: 31: CKVFAGIPTV, peptide A08 SEQ ID NO: 34: CKVFAGIPTVASDTV, peptide combining SEQ ID NO: 30 and SEQ ID NO: 24, modified with C at N-terminus. SEQ ID NO: 35: CKVFAGIPTVSKSVWSKLQSIGIRQH, peptide combining SEQ ID NO: 30 and SEQ ID NO: 24, separated by a Serine (S). SEQ ID NO: 33: KVFAGIPTVSKSVWSKLQSIGIRQH,