Fragment based synthesis of peptides such as ll37

Abstract

The present invention relates to the fragment-based synthesis of peptides, in particular the peptide LL37. In specific the present invention relates to a method for fragmented solid phase synthesis of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, comprising providing a first fragment of LL37, or a variant thereof, coupled to a first resin solid support by a cleavable linker, providing at least one further side chain protected fragment of LL37 coupled to a second resin solid support, wherein the fragment-resin linker is acid labile, cleaving the further side chain protected fragment(s) from the second solid support without deprotecting the side chains, and sequentially coupling the first and further fragments to produce a peptide of SEQ ID NO: 1, or a variant thereof.

Description

[0001] FRAGMENT BASED SYNTHESIS OF PEPTIDES SUCH AS LL37

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the fragment-based synthesis of peptides, in particular the peptide LL37.

[0004] BACKGROUND OF THE INVENTION

[0005] Over recent years advances have been seen in solid phase peptide synthesis, with several strategies proposed to overcome the problem of reliable synthesis of peptides longer than 30 amino acids. Due to the constant material loss from repetitive couplings with a less than 100% efficiency and formation of secondary structure in the longer peptides, producing long peptides that are more than 20 amino acids long may present a technical challenge as well as a challenge due to the amount of consumables being lost during the synthesis for the longer peptides.

[0006] With the ever-increasing demand for peptides in clinical settings, the optimization of synthesis strategies is welcomed both from a financial perspective but also from a more technical perspective.

[0007] Accordingly, new ways to overcome these issues are welcomed in the field of peptide synthesis.

[0008] In humans, CAMP encodes the peptide precursor CAP-18 (18 kDa), which is processed by proteinase 3-mediated extracellular cleavage into the active form LL-37, which contains 37 amino acids.

[0009] LL37 is an antimicrobial peptide, which has been shown to have antimicrobial activity against multiple Gram-positive and Gram-negative human pathogens. LL37 belongs to the cathelicidin family and is released as a mature peptide by stimulated neutrophil granulocytes. LL37 plays an important role in the first line of defence against infections and is toxic to both bacterial and normal cells.

[0010] It has been reported that LL37 is effective against certain bacterial infections even at nanomolar concentrations. In addition to its ability to be anti-biofilm and antimicrobial, LL37

[0011] Inspicos / 13 / 11 / 2025 / 15:45 plays an important role in regulating the balance of pro- and anti-inflammatory molecules and appears to play a role in balancing inflammation with healing.

[0012] Since LL37 is a peptides containing more than 37 amino acids which mainly folds into a helical structure with a slightly amphipathic fold, meaning that the charges of the peptide is mainly positioned on one side of the helix, while the hydrophobic residues are mainly located on the opposite side of the helix, resulting in a peptides which may insert into membranes and form pores with a charged pore (see Sancho-Vaello et al., "The structure of the antimicrobial human cathelicidin LL-37 shows oligomerization and channel formation in the presence of membrane mimics", Scientific Reports volume 10, Article number: 17356 (2020)).

[0013] The folding and oligomerization of LL37 complicates the large-scale linear synthesis and makes upscaling of linear synthesis of LL37 challenging.

[0014] OBJECT OF THE INVENTION

[0015] It is an object of embodiments of the invention to provide improved methods for the synthetic production of peptides, in particular improved methods for production of the peptide LL37.

[0016] SUMMARY OF THE INVENTION

[0017] It has been found by the present inventor(s) that fragment-based synthesis of peptides, in particular the peptide LL37 is a suitable alternative to linear synthesis, as it reduces the time spend on synthesis, the raw material requirements, the overall cost of synthesis. Additionally, the fragment based synthesis provides a more scalable synthesis.

[0018] In its broadest aspects, the present disclosure relates to a method for solid phase synthesis of a polypeptide, comprising

[0019] a. providing a first side chain protected peptide fragment (i), coupled to a first resin solid support via a cleavable linker,

[0020] b. providing at least one further side chain protected peptide fragment coupled to a second resin solid support via a cleavable linker, wherein the linker is TFA or HFIP labile, at a TFA concentration of 5% TFA or less, or at a concentration of 50% HFIP or less,

[0021] Insp icos / 13 / 11 / 2025 / 15 : 45 c. cleaving the further peptide fragment(s) from the second solid support with TFA or HFIP, and

[0022] d. sequentially coupling the first and further fragments to produce a polypeptide, e. optionally, cleaving the polypeptide from the first resin solid support,

[0023] f. optionally, exchanging the cleavage salt with an acetate salt to obtain an acetate form of the polypeptide, and

[0024] g. optionally, purifying the polypeptide.

[0025] In further aspects, the present disclosure relates to a method for fragmented solid phase synthesis of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, comprising

[0026] a. providing a first side chain protected fragment of LL37, or a variant thereof, coupled to a first resin solid support by a cleavable linker,

[0027] b. providing at least one further side chain protected fragment of LL37 coupled to a second resin solid support via a linker, wherein linker is acid labile at a TFA concentration of 5% TFA or less, or 50% HFIP or less,

[0028] c. cleaving the further side chain protected fragment(s) from the second solid support with TFA and / or HFIP, without deprotecting the side chains, and

[0029] d. sequentially coupling the first and further fragments to produce a peptide of SEQ ID NO: 1, or a variant thereof.

[0030] In a further aspect, the present disclosure relates to a method for fragmented synthesis of an acetate form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, comprising

[0031] a) providing a first side chain protected fragment (i) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 2 coupled to a first resin solid support via a Wang linker,

[0032] b) providing a further side chain protected fragment (ii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 3, 5 or 7, which is coupled to a resin solid support via a CTC linker,

[0033] c) providing a further side chain protected fragment (iii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 4, 6 or 8, which is coupled to a resin solid support via a CTC linker,

[0034] d) cleaving the fragments (ii) and (iii) from the solid support using 2% TFA in DCM, or 30% HFIP in DCM,

[0035] e) sequentially coupling the first fragment (i) with fragment (ii), to obtain fragment (iv), followed by coupling of fragment (iii) to fragment (vi) to obtain a peptide of SEQ ID NO: 1, or a variant thereof, coupled to a first resin solid support,

[0036] Inspicos / 13 / 11 / 2025 / 15:45 f) cleaving the peptide obtained in step e) from the linker using at least 70% TFA, to obtain a TFA salt form of the peptide of SEQ ID NO: 1, or a variant thereof,

[0037] g) exchanging the TFA salt with an acetate salt to obtain an acetate salt form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, and

[0038] h) optionally, purifying the LL37 peptide, or a variant thereof.

[0039] In a further aspect, the present disclosure also relates to a method for fragmented synthesis of an acetate form of an LL37 peptide of SEQ ID NO: 1 comprising

[0040] a) providing a first side chain protected fragment (i) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 2, or a variant thereof, coupled to a first resin solid support via a Wang linker,

[0041] b) providing a further side chain protected fragment (ii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 3, which is coupled to a resin solid support via a CTC linker,

[0042] c) providing a further side chain protected fragment (iii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 4, which is coupled to a resin solid support via a CTC linker,

[0043] d) cleaving the fragments (ii) and (iii) from the solid support using about 2% TFA, e) coupling the first fragment (i) with fragment (ii), to obtain fragment (iv), followed by coupling of fragment (iii) to fragment (vi) to obtain a peptide of SEQ ID NO: 1, coupled to the first resin solid support,

[0044] f) cleaving the solid support linked peptide obtained in step e) from the linker using at least 50% TFA, preferably at least 70% TFA, to obtain a TFA salt form of the peptide of SEQ ID NO: 1,

[0045] g) exchanging the TFA salt with an acetate salt to obtain an acetate salt form of an LL37 peptide of SEQ ID NO: 1, and

[0046] h) optionally purifying the LL37 peptide, or a variant thereof.

[0047] In additional aspects, the present disclosure also relates to peptides produced according to the methods of the disclosure.

[0048] The present disclosure also relates to the use peptides produced according to the methods of the disclosure as medicaments.

[0049] Inspicos / 13 / 11 / 2025 / 15:45 LEGENDS TO THE FIGURE

[0050] Fig. 1: Primary sequence of Cathelicidin LL-37 (A), and fragment overview for tested synthesis routes (B-E).

[0051] Fig. 2: General solid phase peptide synthesis cycle using Wang / CTC Resin.

[0052] Fig. 3: Structure of the (A) Fragment A and (B) Fragment D peptidyl Wang linker resin.

[0053] Fig. 4: Synthetic scheme for (A) Fragment B and (B) Fragment C using CTC linker resin.

[0054] Fig. 5: Synthetic scheme for (A) Fragment E and (B) Fragment H using CTC linker resin.

[0055] Fig. 6: Synthetic scheme for (A) Fragment F and (B) Fragment G using CTC linker resin.

[0056] Fig. 7: Synthetic scheme for Fragment I using CTC linker resin.

[0057] Fig. 8: Synthetic scheme for Cathelicidin LL-37 using Frag A, B & C.

[0058] Fig. 9: Synthetic scheme for Cathelicidin LL-37 using Frag D, E & C.

[0059] Fig. 10: Synthetic scheme for Cathelicidin LL-37 using Frag D, F & G.

[0060] Fig. 11: Synthetic scheme for Cathelicidin LL-37 using Frag D, H & I

[0061] DETAILED DISCLOSURE OF THE INVENTION

[0062] Specific embodiments of the invention

[0063] The present disclosure relates to the fragment-based synthesis of peptides, in particular the peptide Cathelicidin LL-37, using different linker resins with different cleavage properties, allowing fragment-based synthesis of peptides, which allows for a faster, more flexible and more efficient synthesis of peptides longer than 30 amino acids.

[0064] In a first aspect the present disclosure relates to a method for fragmented solid phase synthesis of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, comprising

[0065] Inspicos / 13 / 11 / 2025 / 15:45 a) providing a first side chain protected fragment of LL37, or a variant thereof, coupled to a first resin solid support by a cleavable linker,

[0066] b) providing at least one further side chain protected fragment of LL37 coupled to a second resin solid support via a linker, wherein the fragment-resin linker is acid labile at a TFA (trifluoroacetic acid) concentration of 5% TFA or less, or 50% HFIP (Hexafluoroisopropanol) or less,

[0067] c) cleaving the further side chain protected fragment(s) from the second solid support with TFA and / or HFIP, without deprotecting the side chains, and

[0068] d) sequentially coupling the first and further fragments to produce a peptide of SEQ ID NO: 1, or a variant thereof.

[0069] As used herein, "fragment-resin linker", "linker resin" and "resin linker" all relates to the linker used between the solid support (often a bead of functionalized crosslinked polystyrene) and the C-terminal of the peptide. The linker is used as the anchoring point of the peptide to the solid support and provides the option to perform washouts of unreacted chemicals between synthesis steps and to exchange the reaction media during synthesis.

[0070] LL-37

[0071] LL37 is the functional peptide obtained from posttranslational processing of the expression product from the human CAMP gene (ncbi gene ID: 820). CAMP's expression product (see e.g., uniport identifier Q1KLY3) contains a signal peptide (residues 1-30) followed by a Cathelin-like domain (CLD) (residues 31-131), followed by the antibacterial peptide FALL-39 (132-170) or antibacterial peptide LL37 (residues 134-170). FALL-39 is obtained by proteolytical cleavage of the CAMP expression product by cathepsin CTSG and neutrophil elastase ELANE, while LL37 is obtained by proteolytic cleavage of the CAMP expression product by proteinase PRTN3. Other processing pathways leads to additional antimicrobial peptide products being produced upon processing of the CAMP expression product, such as LL23 which covers residues 134-156 of the CAMP expression product, and LL29 which cover residues 134-162 of CAMP expression product.

[0072] LL37 is an antimicrobial peptide, which has been shown to have antimicrobial activity against multiple Gram-positive and Gram-negative human pathogens. LL37 belongs to the cathelicidin family and is released as a mature peptide by stimulated neutrophil granulocytes. LL37 plays an important role in the first line of defence against infections and is toxic to both bacterial and normal cells.

[0073] Insp icos / 13 / 11 / 2025 / 15 : 45 It has been reported that LL37 is effective against certain bacterial infections event at nanomolar concentrations. In addition to its ability to be anti-biofilm and antimicrobial, LL37 plays an important role in regulating the balance of pro- and anti-inflammatory molecules and appears to play a role in balancing inflammation with healing. Accordingly, in some embodiments, the LL37 peptide produced by methods described herein may also be for use as a medicament.

[0074] Generally, in solution LL37 adopts an amphipathic helical structure, with one side along the length of the helix being primarily hydrophobic, and the other side of the helix carrying the charged sidechains. LL37 have been shown to form oligomers, primary tetramers and to form current conducting pores in lipid membranes.

[0075] "LL37" denotes the peptide having the sequence SEQ ID NO: 1 (LLGDFFRKSK EKIGKEFKRI VQRIKDFLRN LVPRTES).

[0076] Variants of LL37 is preferably proteogenic variants but may also include non-proteogenic and post-translationally modified variants. Examples of post-translationally modified variants are e.g., side chain phosphorylation, acetylation, or acylation. Examples of non-proteogenic amino acids are e.g., selenomethionine, selenocysteine, pyrrolysine, ornithine, D-amino acids, N-methyl alanine and o-aminoisobutyric acid.

[0077] Accordingly, a variant of LL37 may comprise one or more mutations at one or more residue positions that differ from the sequence of SEQ ID NO: 1. It is preferred residue 12 is K, residues 13 and 20 are I and residue 21 is V, with reference to SEQ ID NO: 1. Further, for some synthesis paradigms, it may be preferred that residue is 9 is S, residue 10 is K, residue 20 is I and residue 21 is V, with reference to SEQ ID NO: 1. For other synthesis paradigms, it may be preferred that residue is 10 is K, residue 11 is E, residue 20 is I and residue 21 is V, with reference to SEQ ID NO: 1.

[0078] A variant of LL37 may also be FALL39 of SEQ ID NO: 11 (H-FALLGDFFRK SKEKIGKEFK RIVQRIKDFL RNLVPRTES-OH). FALL39 may also be produced by the methods described herein, using a fragment-based synthesis, e.g., using fragments D (SEQ ID NO: 2), H (SEQ ID NO: 7) and a fragment consisting or comprising the amino acids sequence of SEQ ID NO: 12 (H-FALLGDFFRK SKEK-OH).

[0079] Inspicos / 13 / 11 / 2025 / 15:45 Solid phase peptide synthesis

[0080] Solid phase peptide synthesis (SPPS) is a well-known method for producing peptides. The Example of the present disclosure provides the details of the synthesis of particular nonlimiting peptides, using well known chemical reagents. The Examples provided herein provides exemplary methods for synthesizing peptides, but it is evident to the skilled person that other chemical agents may also be suited for the synthesis. Thereby, the methods of synthesis disclosed herein are not to be seen as limited in terms of the chemical reagents disclosed herein.

[0081] The peptide fragments produced herein are generally produced using Fmoc based synthesis, according to Fig. 2, which entails repetitive rounds of deprotection and coupling. Usually when using Fmoc based synthesis, the deprotection of the orthogonally Fmoc protected amino acid is done using piperidine, such as 20% piperidine in N,N-dimethylformamide (DMF), while the coupling is usually performed using one or more coupling reagents, such as e.g., HOBt-H2O (3 equivalents) and DIC (3 equivalents) as coupling reagents. The process and reagents used for SPPS is well known to the skilled person, and examples of the reagents and specific steps used for SPPS are provided herein in Examples 1 and 2.

[0082] SPPS is usually performed on a solid support, be it a membrane e.g., cellulose or beads e.g., polystyrene beads. Often SPPS is conducted on a polymeric resin, such as e.g., functionalized cross-linked polystyrene beads, which are functionalized with one or more linkers, enabling cleavage of the intended peptide form the solid support. An example of such is e.g., 1% crosslinked polystyrene beads functionalized with the TFA labile p-benzyloxybenzyl alcohol linker, also referred to herein as a Wang resin. Depending on the linker used, the cleavage may be an acid cleavage e.g., using TFA as cleavage agent, but cleavage may also be conducted as an enzymatic cleavage, oxidative cleavage, or cleavage with other suitable chemicals, such as e.g., piperidine, methanol or borohydride.

[0083] Preferably the peptides produced in the present disclosure are prepared using one or more resins as solid support.

[0084] For a fragment-based synthesis it is preferable that the linker used for the different fragments have different chemical properties, in the sense that selective cleavage of the individual fragments from the solid support is possible without significant deprotection of the side chains.

[0085] Inspicos / 13 / 11 / 2025 / 15:45 The Examples presented herein exemplifies the use of a p-benzyloxybenzyl alcohol linker resin (Wang resin) for the C-terminal fragment, and a 2-Chlorotrityl Chloride (CTC) linker Resin for the further fragments.

[0086] In cases where C-terminal modification of the desired peptide is required, alternatives to the Wang resin may be used e.g., a Rink-linker, a Rink Amide linker, a Sieber amide linker, or a DHP HM linker, as depicted in table 1.

[0087] Table 1. Selected linker resins

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[0089] <

[0090] <

[0091] >

[0092] <

[0093]

[0094] Inspicos / 13 / 11 / 2025 / 15:45 >

[0095]

[0096] For the fragment synthesis, it is preferable that the linker used in the further fragments is an acid labile linker which produces a carboxylic acid upon cleavage with an acid, such linkers are e.g., CTC or HMPB-MBHA. The effect of using an acid labile linker is that it allows for selective deprotection of the C-terminal, enabling subsequent coupling with a primary amine of another peptide, preferably the N-terminal of the previous fragment.

[0097] In the present disclosure the term "acid labile linker" means that the linker is cleavable in the presence of less than 5% TFA or less than 50% HFIP. For instance, the CTC linker and HMPB-MBHA linker are both cleavable at a TFA concentration of less than 2% in DCM (dichloromethane).

[0098] Accordingly, in embodiments, the method further comprises a step e) comprising cleaving the coupled peptide from the first solid support using TFA to obtain a TFA salt of an LL37 peptide of SEQ ID NO: 1, or a variant thereof.

[0099] In embodiments, the first resin comprises a Wang linker, CTC linker, Rink Amide linker, Sieber amide linker, or a DHP HM linker, preferably the first resin comprises a Wang linker.

[0100] In further embodiment, it is preferable that the second resin comprises a CTC linker or a HMPB-MBHA linker, preferably a CTC linker.

[0101] In that regard, it is preferable that the cleavage in step d) is performed using no more than 3% TFA, preferably 2% TFA, or no more than 50% HFIP, preferably no more than 30% HFPI.

[0102] Inspicos / 13 / 11 / 2025 / 15:45 In some instances, when the method includes coupling of more than two fragments it may be preferable that the second and third, or further fragments have different linker cleavage properties e.g., such that the synthesis may be performed by coupling of the second and third fragments before coupling to the first fragment. Such a synthesis may comprise a first fragment is linked to a e.g., a HMBA-AM linker resin, the second and / or third fragment to a, CTC or HMPB-MBHA linker resin.

[0103] Salt exchange

[0104] It is commonly recognised that TFA salts as such are not preferable when peptides are to be used for in vivo studies or in a clinical setting. Often for in vivo studies, TFA can lead to unwanted side effects which are not related to the peptide as such, but is due to the TFA as such. Accordingly, it is beneficial to prepare the peptides as a different salt, e.g., by salt exchange.

[0105] Methods for performing salt exchange are well known to the skilled person, and some exemplary methods for performing TFA to acetate salt exchange are provided in the examples of the present disclosure.

[0106] The TFA may be exchanged with any suitable salt, such as e.g., acetate or sodium, given that the salt is well tolerated by in vivo, and that it does not in itself lead to significant side effects upon administration.

[0107] Accordingly, in embodiments, the method further comprises a step f) comprising exchanging the TFA salt with an acetate salt to obtain an acetate form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, and optionally a step f) comprising purifying the LL37 peptide to a purity of at least 95% w / w. In further embodiments, the method further comprises a step f) comprising exchanging the TFA salt with an sodium salt to obtain an sodium form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, and optionally a step f) comprising purifying the LL37 peptide to a purity of at least 95% w / w.

[0108] Fragment based synthesis

[0109] Fragment based synthesis relates to the synthesis of longer peptides, / .e., peptides containing more than 30 amino acid residues, which is done in a fragment-based manner compared to a linear synthesis, meaning that individual fragments of the longer peptides are synthesized individually and then coupled together to generate the full-length peptide. The requirement for fragment-based synthesis, is firstly that the relevant coupling points can be

[0110] Inspicos / 13 / 11 / 2025 / 15:45 specifically deprotected and used for coupling sites, and secondly that the sites used for coupling are efficient coupling sites. While the first can be designed using different linker strategies as described above, the identification of the particular coupling sites in the peptides often requires the initiation of tedious research programmes to identify the efficient coupling sites. On the other hand, linear synthesis of peptides of >30 amino acids can be challenging e.g., due to secondary structure formation or aggregation, and in many cases the synthesis of such longer peptides must be abandoned. Furthermore, the less than 100% efficient coupling of each amino acid also entails that the longer the peptide being produced, the less product is obtainable after each coupling, to a point where even very efficient couplings results in very low amounts of the product being produced when more than 30 couplings are performed, making such a synthesis very resource intensive and costly.

[0111] Accordingly, production of peptides by fragment-based synthesis is beneficial since it allows for a cost and resource saving synthesis, but also a time reducing synthesis, since the fragments may be produced in parallel. Additional benefits of fragments-based synthesis are also described elsewhere herein.

[0112] Peptides produced by fragment-based synthesis are generally easily recognisable post synthesis when compared to linearly synthesised peptides, since unintentional fragments resulting from the synthesis will have different masses compared to the unintended fragments obtained from linear synthesis, due to recombination of more different fragments in the fragment-based synthesis. Such fragments may be predicted by various means, and may be verified using e.g., LC-MS or LC-MS / MS. Therefore, the disclosure in some embodiments relates to an LL37 peptide produced by a method as disclosed herein, preferably in an LL37 peptide in an acetate form.

[0113] As used herein the term "fragment coupling" related to the coupling of one or more fragments in a coupling reaction, preferably between a carboxyl of one peptide and a primary amine on a different peptide. Often such coupling is assisted by one or more chemical reagents, such as e.g., COMU (l-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate') and DIPEA (N,N-Diisopropylethylamine) , PyBOP (benzotriazol-l-yloxytripyrrolidinophosphonium hexafluorophosphate) and DIPEA, HATU (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium) and DIPEA, Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) and DIC (diisopropylcarbodiimide , HATU, HOAt (1-Hydroxy-7-aza-lH-benzotriazole) and DIPEA, or DIC and HOBt (l-Hydroxy-7-aza-lH-benzotriazole) in different equivalents. The coupling is further often assisted by heating to a non-hydrolytic temperature.

[0114] Insp icos / 13 / 11 / 2025 / 15 : 45 As can be seen from the examples, some fragments have a better coupling efficiency than others, and the coupling reagent employed may also have an impact on the coupling efficiency.

[0115] The optimal strategy identified in Example 1 was the coupling of Fragment D (H-VQRIKDFLRN LVPRTES-OH, SEQ ID NO: 2), H (H-IGKEKFRI-OH, SEQ ID NO: 7) and I (H-LLGDFFRK-OH, SEQ ID NO: 8), while couplings were also seen for D (H-VQRIKDFLRNLVPRTES-OH, SEQ ID NO: 2),E (H-KEKIGKEFKRI-OH, SEQ ID NO: 3) and C (H-LLGDFFRKS-OH, SEQ ID NO: 4), and D (H-VQRIKDFLRNLVPRTES-OH, SEQ ID NO: 2), F (H-EKIGKEFKRI-OH, SEQ ID NO: 5) and G (H-LLGDFFRKSK-OH, SEQ ID NO: 6).

[0116] Furthermore, the optimal coupling reagent for the above couplings were found to be a mixture of HOBt and DIPEA. Accordingly, in embodiments, the coupling of the fragments in step d) is performed in the presence of HOBt.H2O, preferably, a mixture of HOBt.H2O and DIPEA.

[0117] The coupling reagents are often used at different equivalents relative the amount of product being coupled. Usually, when HOBt is used, it is used in 5-10 equivalents, preferably 6-8 equivalents. Additionally, DIPEA is often also used in the coupling reaction, and usually also in 5-10 equivalents, preferably 6-8 equivalents. In particular embodiments, HOBt.H2O and DIPEA are both used in 6-8 equivalent. Other suitable coupling agents are shown in Example 1 and 2, along with the respective equivalents of each coupling agent used.

[0118] Therefore, it is preferable that two fragments (ii) and (iii) are provided under step b) of the method. In embodiments, the method comprises coupling of three fragments (i), (ii) and (iii).

[0119] Additionally, in a synthesis where three fragments are used, it is preferable that the first fragment (i), comprises an N-terminal V, and the further fragment (ii) comprises a C-terminal I.

[0120] For the further coupling it is preferable that the further fragment (ii) comprises a C-terminal I, and an N-terminal K, E or I, while the further fragment (iii) comprises a C-terminal S or K. When the further fragment (ii) comprises an N-terminal K it is preferred that the further fragment (iii) comprise a C-terminal S. When the further fragment (ii) comprises an N-terminal E or I it is preferred that the further fragment (iii) comprise a C-terminal K.

[0121] Accordingly, in preferred embodiments, the first fragment is a peptide according to SEQ ID NO: 2 (H-VQRIKDFLRNLVPRTES-OH). In additional embodiments, (i) may comprise or consists of a sequence according to SEQ ID NO: 7 (H-IGKEFKRI-OH). Furthermore, (ii) may

[0122] Inspicos / 13 / 11 / 2025 / 15:45 comprise or consists of a sequence according to SEQ ID NO: 8 (H-LLGDFFRKSKEK-OH). In additional embodiments, (i) may comprise or consists of a sequence according to SEQ ID NO: 3 (H-KEKIGKEFKRI-OH). Further, (ii) may comprise or consists of a sequence according to SEQ ID NO: 4 (H-LLGDFFRKS-OH). in additional embodiments, (i) may comprise or consists of a sequence according to SEQ ID NO: 5 (H-EKIGKEFKRI-OH). Further, (ii) may comprise or consists of a sequence according to SEQ ID NO: 6 (H-LLGDFFRKSK-OH).

[0123] Accordingly, the methos in some instances entails a synthesis scheme as described in Figs. 8-11. In particular the synthesis may entail joining of three fragments, denoted (i), (ii) and (iii), where, fragments (i) and (ii) are coupled to provide a fragment (iv), which is subsequently coupled to (iii) to produce fragment (v), which is the full length peptide. In some embodiments, (iii) is cleaved form the linker resin and coupled to (ii) before cleavage of (ii) from the linker resin and coupling of (ii) to (i). In some embodiments, (ii) and (iii) are coupled to produce (vi), followed by cleavage using about 2% TFA or about 30% HFIP, and coupling to (i) to produce (v).

[0124] Alternatively, the synthesis may entail joining of two fragments, denoted (a) and (b), where fragments (b) and (b) are coupled to provide a fragment (c) which is the full length peptide. In other alternatives, the synthesis may entail coupling of at least two fragments, such as 2, 3, 4, 5, 6, 7, 8 9 or 10 fragments. In embodiments, the fragments are between 4-30 amino acids each, preferably between 6-20 amino acids each.

[0125] Exemplary fragments for LL37 synthesis are shown in table 2 below.

[0126] Table 2. Exemplary fragments for LL37 synthesis.

[0127]

[0128] Accordingly, in particular embodiments the present disclosure provides a method for fragmented synthesis of an acetate form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, comprising

[0129] Inspicos / 13 / 11 / 2025 / 15:45 a) providing a first side chain protected fragment (i) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 2 coupled to a first resin solid support via a Wang linker,

[0130] b) providing a further side chain protected fragment (ii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 3, 5 or 7, which is coupled to a resin solid support via a CTC linker,

[0131] c) providing a further side chain proetected fragment (iii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 4, 6 or 8, which is coupled to a resin solid support via a CTC linker,

[0132] d) cleaving the fragments (ii) and (iii) from the solid support using 2% TFA in DCM, or 30% HFIP in DCM,

[0133] e) sequentially coupling the first fragment (i) with fragment (ii), to obtain fragment (iv), followed by coupling of fragment (iii) to fragment (vi) to obtain a peptide of SEQ ID NO: 1, or a variant thereof, coupled to a first resin solid support,

[0134] f) cleaving the peptide obtained in step e) from the linker using at least 70% TFA, to obtain a TFA salt form of the peptide of SEQ ID NO: 1, or a variant thereof, g) exchanging the TFA salt with an acetate salt to obtain an acetate salt form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, and

[0135] h) optionally, purifying the LL37 peptide, or a variant thereof.

[0136] Moreover, the method is more specifically a method for fragmented synthesis of an acetate form of an LL37 peptide of SEQ ID NO: 1 comprising

[0137] a) providing a first side chain protected fragment (i) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 2, or a variant thereof, coupled to a first resin solid support via a Wang linker,

[0138] b) providing a further side chain protected fragment (ii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 3, which is coupled to a resin solid support via a CTC linker,

[0139] c) providing a further side chain protected fragment (iii) of LL37 comprising or consisting of an amino acid sequence of SEQ ID NO: 4, which is coupled to a resin solid support via a CTC linker,

[0140] d) cleaving the fragments (ii) and (iii) from the solid support using about 2% TFA, e) coupling the first fragment (i) with fragment (ii), to obtain fragment (iv), followed by coupling of fragment (iii) to fragment (vi) to obtain a peptide of SEQ ID NO: 1, coupled to the first resin solid support,

[0141] f) cleaving the solid support linked peptide obtained in step e) from the linker using at least 50% TFA, preferably at least 70% TFA, to obtain a TFA salt form of the peptide of SEQ ID NO: 1,

[0142] Inspicos / 13 / 11 / 2025 / 15:45 g) exchanging the TFA salt with an acetate salt to obtain an acetate salt form of an LL37 peptide of SEQ ID NO: 1, and

[0143] h) optionally purifying the LL37 peptide, or a variant thereof.

[0144] Method for fragment-based synthesis of peptides

[0145] The fragment-based synthesis provides a flexibility in the variant peptides which may be produced in a synthesis. For instance, in a fragmented synthesis, the synthesis may be designed such that a region of interest is used as a variable region, i.e., if e.g., a peptide of interest contains between 30-100 amino acids, and a region of interest, in which sequence variation is desired, then the peptide may be synthesized in two fragments, one with the wild-type sequence and one containing the sequence variants. In example, the peptide could be a peptide containing 50 residues, where residues 25-35 are variable regions. In such a case, the fragment approach may be used to prepare a first fragment e.g., residues 1-24, which is linked to a resin, and then a second fragment 25-36, followed by a third fragment containing residues 27-50. The second fragment may be synthesized in different variants, but with a constant N- and C-terminal, thereby increasing the likelihood that the coupling efficiency is maintained. Accordingly, such fragmented strategy may be preferred over having to synthesize each of the full-length variant peptides individually, thus making it easier to scale up the production of the full-length peptide variants. For the synthesis to remain efficient it may however be preferable that the length of the fragments is in the range 4-30 residues.

[0146] The present disclosure therefore also presents a method for solid phase synthesis of a polypeptide, comprising

[0147] a. providing a first peptide (normally side chain protected) fragment (i), coupled to a first resin solid support via a cleavable linker,

[0148] b. providing at least one further peptide (normally side chain protected) fragment coupled to a second resin solid support via a linker, wherein the fragment-resin linker is TFA or HFIP labile, at a TFA concentration of 5% TFA or less, or at a concentration of 50% HFIP or less,

[0149] c. cleaving the further peptide fragment(s) from the second solid support with TFA or HFIP, and

[0150] d. sequentially coupling the first and further fragments to produce a polypeptide.

[0151] For coupling reactions to occur between the N-terminal of one peptide and the C-terminal of another peptide, it is, as known by the skilled person, required that the N- and C-termini are

[0152] Inspicos / 13 / 11 / 2025 / 15:45 deprotected, such that the NH group and the COOH group may couple and form an amine bond.

[0153] Accordingly, the methods therefore often also comprise a step of deprotection of the N-terminal of the fragments before coupling.

[0154] For such a synthesis to be feasible an orthogonal use of protection groups is often required. Examples of orthogonal chemistries are e.g., piperidine labile protection groups such as e.g., Fmoc, combined with acid labile protection groups, with Boc, tBu, Trt and Pbf, being the most common. Often a residue is often protected by an Fmoc group on the main chain amino group, while reactive sidechains are often protected with Boc, tBu, Trt or Pbf, depending on the sidechain reactivity. In such a synthesis, Boc, or another acid labile protection group is often used as the preferred protection group on the main chain amine group on the final amino acid in the synthesis, such that it may be deprotected in the final global deprotection.

[0155] The use of orthogonal protection groups enables the use of local deprotection and global deprotection. As such, local deprotection is specific deprotection of the N-terminal, or desired reactive side chains, and global deprotection is the, often, final deprotection step, which provides the final synthesized peptide in a combined deprotection and cleavage reaction. As used herein the term "Global deprotection" refer to a deprotection of all reactive groups of the synthesized peptide and cleaves the peptide from the solid support. In embodiments the global deprotection is performed using at least 70% TFA, such as at least 75%, 80%, 85%, 90% 92.5% or such as at least 95%. In other embodiments, the global deprotection is performed using reagent K, which contains TFA, water, phenol, thioanisole and EDT in the ratios 82.5:5:5:5:2.5, respectively. In other embodiments, the global deprotection is performed using a mixture of TFA, TIPS (Triisopropylsilane), DODT (3,6-dioxa-l,8-octanedithiol) and Water in the ratios 92.5:2.5:2.5:2.5, respectively. The skilled person will know additional suitable cocktails for global deprotection and cleavage.

[0156] As used herein the term "local deprotection" refer to a deprotection of specific or selected reactive groups of the synthesized peptide, such as e.g., the N-terminal, or specific reactive side chains.

[0157] Pharmaceutical uses of acetate forms of LL37

[0158] In important embodiments of the present invention are provided an acetate form of LL37. This acetate form is particularly useful as a medicament, which finds use in the compositions

[0159] Inspicos / 13 / 11 / 2025 / 15:45 suitable for treatment of periodontal disease, which are disclosed in detail in European patent application No: 24168328.3 filed on 3 April 2024.

[0160] Hence, in one embodiment, the present invention provides for a method of method for treatment, including prophylactic treatment, of periodontal disease, the method comprising injection of an effective amount of a composition comprising LL37 (in particular the acetate form) as prepared herein into one or more periodontal pockets in the oral cavity of a subject in need thereof, wherein the composition further comprises a gelling agent / mixture and wherein the composition increases its viscosity at the time of injection from an initial value of at most 1000 mPa.s to an increased value of at least 4 Pa.s in the periodontal pocket to attain a semi-solid form, whereby LL37 exhibits protracted release from the composition so as to exert antimicrobial and anti-inflammatory effects in the periodontal pocket(s) for a period of at least 24 hours.

[0161] Said composition comprises - according to European patent application No: 24168328.3 - as an active ingredient peptide LL37 in admixture with a gelling agent or gelling mixture, wherein said gelling agent or gelling mixture exhibits increased viscosity upon introduction into the environment of a periodontal pocket, wherein said increased viscosity is triggered by any one of 1) an increase of the composition in temperature to about 37°C as present in a periodontal pocket, or 2) by contact of the composition with water as present in a periodontal pocked, or 3) by contact of the composition with calcium ions,

[0162] and wherein said increased viscosity causes protracted release of LL37 from the composition.

[0163] The disclosure in European patent application No: 24168328.3 provides all necessary details for putting this use of LL37 into practice.

[0164] EXAMPLE 1

[0165] Fragment based synthesis of LL37

[0166] Aim

[0167] The aim of the present example is to provide suitable synthesis strategies for synthesis of LL37 by fragment based solid phase peptide synthesis. Generally, synthesis of LL37 with the fragment approach involves elements from standard Fmoc-based solid phase peptide synthesis, followed by global deprotection and purification by RP-HPLC. However, certain

[0168] Inspicos / 13 / 11 / 2025 / 15:45 modifications have been introduced, such as the use of CTC resin as the solid phase for production of 2 / 3 fragments of LL37. The synthesis paradigms are also described in Fig. 1.

[0169] Methods

[0170] Each fragment was synthesized on the appropriate solid support, and the feasibility of coupling the individual fragments was assessed to identify the most effective approach for synthesizing the target Cathelicidin LL-37.

[0171] Four different fragment routes were tested, as described in Table 3.

[0172] Table 3. Synthesis routes used in the present example

[0173]

[0174] General solid phase peptide synthesis protocol

[0175] The synthesis of the C-terminal amino acid attached (mother fragment) peptide fragment was conducted using Wang resin, while the other fragments were synthesized on CTC resin. The peptide fragments derived from CTC resin can be effectively removed using a mild cleavage method with either 30% HFIP in dichloromethane (DCM) or 2% TFA in DCM. This approach allows for the recovery of the synthesized peptide in its protected form from the CTC solid support.

[0176] Wang resin used for the C-terminal amino acid and CTC resin used for the other fragments The standard solid-phase peptide synthesis (SPPS) protocol begins with swelling the Wang / CTC resin in DMF for 1-2 hours. Once the resin is adequately swollen, the first orthogonally protected Fmoc-amino acid is attached / coupled to the resin.

[0177] Inspicos / 13 / 11 / 2025 / 15:45 Loading of 1stAmino acid (AA1) over Wang resin

[0178] The swollen Wang resin was treated with a solution of orthogonally protected Fmoc amino acid (Fmoc-AAl-OH, 3 Equiv.) and HOBt-H2O (3 Equiv.) in DMF (10 volumes relative to resin weight). DIC (3 Equiv.) and DMAP (0.3 Equiv.) were then added to the mixture. The reaction was maintained under gentle shaking on an orbital shaker or stirred manually in a solidphase peptide synthesis glass reactor for 12 hours. After this period, the attachment / loading of the first amino acid was evaluated using the UV method (Method provided in the section 4.1.1).

[0179] Note: If the attachment is sufficient, the reaction mixture was washed with DMF (3 x 10 volumes) and then subjected to capping with a solution of acetic anhydride, DIPEA, and DCM (1:2:7). If the attachment is insufficient, the coupling step was repeated before proceeding with the capping.

[0180] Loading of 1stAmino acid (AA1) over CTC resin

[0181] The swollen CTC resin was treated with a solution of orthogonally protected Fmoc amino acid (Fmoc-AAl-OH, 1 Equiv.) in DMF (10 volumes relative to resin weight). DIPEA (3 Equiv.) was then added to the mixture. The reaction was maintained under gentle shaking on an orbital shaker or stirred manually in a solid-phase peptide synthesis glass reactor for 6 hours. After this period, the attachment / loading of the first amino acid was evaluated using the UV method.

[0182] If the attachment was sufficient, the reaction mixture was washed with DMF (3 x 10 volumes) and then subjected to capping with 10% DIPEA in methanol (10V relative to the resin weight). If the attachment was insufficient, the coupling step was repeated before proceeding with the capping.

[0183] Fmoc de-protection and couplings

[0184] After loading the first amino acid and capping, the Fmoc-amino acid-loaded resin was treated with 20% piperidine in DMF (2 x 10 V) and stirred for 30 minutes to deprotect the Fmoc group. The reaction mixture was then filtered, and the resin was washed sequentially with DMF (1 x 10 V), IPA (1 x 10 V), DCM (2 x 10 V), and finally with DMF (2 x 10 V), whereafter the coupling reactions were conducted using HOBt-H2O (3 equivalents) and DIC (3 equivalents) as coupling reagents.

[0185] Inspicos / 13 / 11 / 2025 / 15:45 The Fmoc-deprotected peptidyl resins were subjected to the subsequent coupling and deprotection steps according to the peptide sequence. A detailed general synthetic scheme is illustrated in Fig. 2.

[0186] The synthesis of the different fragments was conducted such that the Wang linked fragments (A and D) could remain linked to the resin during coupling of the further fragments, while the CTC linked fragments could be cleaved from the resin using 2% TFA or 30% HFIP in DMF, without risking global deprotection, thereby allowing for subsequent specific coupling reactions.

[0187] Additionally, for the C-terminal fragments (A and D), the N-terminal was deprotected using 20% Piperidine in DMF before coupling to the further fragments (E, F or H).

[0188] Accordingly, following coupling of the last amino acid in each of the further fragment, no Fmoc deprotection was performed, and the CTC conjugated fragments were cleaved from the resin, without Fmoc deprotection, leaving the Fmoc protected group intact on the further fragments.

[0189] Fragment coupling

[0190] To obtain the intermediate coupling products AB, DE, DF and DH a first round of fragment coupling was performed.

[0191] Firstly, the further fragments B, E, F or H was dissolved in NMP and treated with HOBt-F O and DIC as described in table 4. The reaction mixture was then treated with the Fmoc-deprotected peptidyl resin (A or D) and stirred at 25 ± 5°C for 12 h. The progress of the reaction was monitored using the KAISER test, which indicated negative results, confirming successful coupling.

[0192] Following this, the reaction mixture was filtered and washed with DCM (3 x 30 mL) and DMF (3 x 30 mL) before proceeding with Fmoc deprotection using 20% piperidine in DMF (2 x 10 mL) for 15 minutes. The reaction mass was filtered and washed with DMF (1 x 15 mL), IPA (1 x 15 mL), DMF (3 x 15 mL), DCM (3 x 15 mL), and finally with MTBE (2 x 15 mL), after which the Fmoc deprotected peptidyl resin was dried under vacuum.

[0193] Following the fragment coupling, a portion of the fragments AB, DE, DF and DH underwent global deprotection using a cleavage cocktail (0.5 mL) for 3 h. The reaction mixture was filtered, and the filtrate was precipitated using MTBE (3 mL), and analysed using HPLC.

[0194] Inspicos / 13 / 11 / 2025 / 15:45 To obtain the full-length coupling products DEC, DFG and DHI a further round of fragment coupling was performed, combining the fragments DE, DF or DH with the N-terminal fragments C, H or I, respectively.

[0195] The further coupling was done using fragments C, G, or I which were dissolved in NMP and treated with HOBt-FhO and DIC as described in table 4. The reaction mixture was then treated with the Fmoc-deprotected peptidyl resin (DE, DF or DH) and stirred at 25 ± 5°C for 12 h. The progress of the reaction was monitored using the KAISER test, which indicated negative results, confirming successful coupling. Following this, the reaction mixture was filtered and washed with DMF (3 x 15 mL), DCM (3 x 15 mL), and finally with MTBE (2 x 15 mL), after which the peptidyl resin was dried under vacuum.

[0196] Table 4. Reaction conditions for the coupling of fragments

[0197]

[0198] Following the fragment coupling, the crude cathelicidin LL-37 derived from fragments DEC, DFG and DHI underwent global deprotection using a cleavage cocktail (92.5:2.5:2.5:2.5 of

[0199] Inspicos / 13 / 11 / 2025 / 15:45 TFA, TIPS, DODT and Water) for 3 h. The reaction mixture was filtered, and the filtrate was precipitated using MTBE (3 mL), and analysed using HPLC.

[0200] Purification of crude cathelicidin LL-37 peptides

[0201] The crude cathelicidin LL-37, derived from both linear and fragment strategies, was purified using the AKTA Pure 150 system with a 50 x 250 mm ID XBridge column (stationary phase: C18, 10 pm). The details of the gradient method employed, are provided in Table 5, respectively.

[0202] Table 5: Gradient system used for the purification of Cathelicidin LL-37

[0203]

[0204] Salt conversion of purified peptides

[0205] The purified cathelicidin LL-37 in TFA buffer fractions was further washed using ammonium acetate as a washing gradient as indicated in table 6. After salt conversion, the final purified fractions were lyophilized, resulting in the desired cathelicidin LL-37 product as an acetate salt.

[0206] Table 6: Acetate Conversion for Cathelicidin LL-37

[0207]

[0208] Inspicos / 13 / 11 / 2025 / 15:45 Results

[0209] The present example shows that the target peptide, Cathelicidin LL-37 could be successfully synthesized using the fragment strategies outlined above, with the optimal strategy being the synthesis using the fragments D, H and I. In this approach, the mother fragment, designed with 17 amino acids (SEQ ID NO: 2), was derived from Wang resin, while the other two fragments, comprising 8 and 12 amino acids (SEQ ID NOs: 7 and 8), respectively, were derived from CTC resin.

[0210] Besides the optimal strategy, both the strategies using fragments D, E and C and fragments D, F and G also provided a successful strategy for fragment based synthesis of Cathelicidin LL-37.

[0211] The example also show that not all coupling sites are suitable for use in the fragment based synthesis, such as e.g., the coupling sites used in fragments A and B, which did not result in any coupling of the fragments with the tested coupling reagents.

[0212] In all three cases the fragments were coupled sequentially, leading to the successful formation of the Cathelicidin LL-37, demonstrating the effectiveness of the fragment-based synthesis strategy.

[0213] EXAMPLE 2

[0214] Comparison between fragment-based synthesis and linear synthesis strategies

[0215] Aim

[0216] The aim of the present example is to compare linear and fragment-based synthesis of LL37.

[0217] Methods

[0218] Fragment-base synthesis

[0219] The fragment-based synthesis was conducted as described in Example 1, using fragments D, H and I, also described in Tables 2, 4 and 5.

[0220] Inspicos / 13 / 11 / 2025 / 15:45 Linear synthesis

[0221] The linear synthesis of cathelicidin LL-37 started with Wang resin. The resin underwent capping to prevent further reactions at unreacted sites, followed by a series of Fmoc deprotection steps and couplings with orthogonally protected Fmoc amino acids.

[0222] The peptidyl resin was subjected to global deprotection to release the free peptide. The crude product was then precipitated and dried, yielding 70 g of crude cathelicidin LL-37 with a purity of 61%. This synthesis effectively demonstrated the viability of the linear strategy for producing cathelicidin LL-37.

[0223] Purification

[0224] The crude cathelicidin LL-37, derived from both linear and fragment strategies, was purified using the AKTA Pure 150 system with a 50 x 250 mm ID XBridge column (stationary phase: C18, 10 pm). The details of the gradient method employed, are provided in Tables 7, respectively.

[0225] Table 7: Gradient system used for the purification of Cathelicidin LL-37

[0226]

[0227] Salt conversion of purified peptides

[0228] The purified cathelicidin LL-37 in TFA buffer fractions was further washed using ammonium acetate as a washing gradient as indicated in table 8. After salt conversion, the final purified fractions were lyophilized, resulting in the desired cathelicidin LL-37 product as an acetate salt.

[0229] Inspicos / 13 / 11 / 2025 / 15:45 Table 8: Acetate Conversion for Cathelicidin LL-37

[0230]

[0231] Results

[0232] The comparative results for the synthesis of cathelicidin LL-37 derived from both linear and three fragment strategies are detailed in Table 9. This table summarizes key metrics such as percentage recovery of the pure product from its respective crude product, purity, and synthesis time for each approach, allowing for an insightful comparison of their efficiencies.

[0233] Table 9: Comparison data of Cathelicidin LL-37 (linear Vs fragment strategy)

[0234]

[0235] Inspicos / 13 / 11 / 2025 / 15:45

[0236] &

[0237]

[0238] The analytical test result for the Cathelicidin LL-37 synthesized from the linear and fragment strategy are provided in Table 10.

[0239] Table 10: Comparison of analytical test results (linear Vs fragment strategy)

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[0245]

[0246] Inspicos / 13 / 11 / 2025 / 15:45 The linear strategy for synthesizing cathelicidin LL-37, while straightforward, yielded a specific level of purity typical of conventional peptide synthesis methods. In contrast, the fragment strategy, which involves the synthesis of smaller peptide fragments and their subsequent coupling, demonstrated advantages in scalability and flexibility in sequence design. The comparative results indicate that both strategies achieve similar yield and purity levels; however, the fragment technology resulted in a single maximum purity of less than 0.5% area, while the linear approach showed 0.69%. Additionally, the fragment based synthesis also allows for a slightly higher recovery from the crude product at 10.5% for the fragment based synthesis, compared to 9.71% for the linear synthesis. Notably, the fragment strategy required less time overall compared to the linear synthesis, making it a more efficient option for peptide production.

[0247] Inspicos / 13 / 11 / 2025 / 15:45

Claims

1. 292.CLAIMS1. A method for fragmented solid phase synthesis of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, comprising4.a. providing a first side chain protected fragment of LL37, or a variant thereof, coupled to a first resin solid support by a cleavable linker,5.b. providing at least one further side chain protected fragment of LL37 coupled to a second resin solid support via a linker, wherein the linker is acid labile at a TFA concentration of 5% TFA or less, or 50% HFIP or less,6.c. cleaving the further side chain protected fragment(s) from the second solid support with TFA and / or HFIP, without deprotecting the side chains, and7.d. sequentially coupling the first and further fragments to produce a peptide of SEQ ID NO: 1, or a variant thereof.

2. The method according to claim 1, wherein the method further comprises a step e) comprising cleaving the coupled peptide from the first solid support using TFA to obtain a TFA salt of an LL37 peptide of SEQ ID NO: 1, or a variant thereof.

3. The method according to claim 2, wherein the method further comprises a step f) comprising exchanging the TFA salt with an acetate salt to obtain an acetate form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, and optionally a step f) comprising purifying the LL37 peptide to a purity of at least 95% w / w.

4. The method according to any one of the preceding claims, wherein the first fragment is a peptide of amino acid sequence SEQ ID NO: 2 (H-VQRIKDFLRNLVPRTES-OH).

5. The method according to any one of the preceding claims, wherein two fragments (ii) and (iii) are provided under step b).

6. The method according to claim 5, wherein (ii) comprises an N-terminal K, E or I and a C-terminal I.

7. The method according to claim 5 or 6, wherein (iii) comprises a C-terminal S or K.

8. The method according to any one of claims 5-7, wherein (ii) comprises or consists of amino acid sequence SEQ ID NO: 7 (H-IGKEFKRI-OH), and (iii) comprises or consists of amino acid sequence SEQ ID NO: 8 (H-LLGDFFRKSKEK-OH).15.Inspicos / 13 / 11 / 2025 / 15:45 309. The method according to any one of claims 5-7, wherein (ii) comprises or consists of amino acid sequence SEQ ID NO: 3 (H-KEKIGKEFKRI-OH), and (iii) comprises or consists of amino acid sequence SEQ ID NO: 4 (H-LLGDFFRKS-OH).

10. The method according to any one of claims 5-7, wherein (ii) comprises or consists of amino acid sequence SEQ ID NO: 5 (H-EKIGKEFKRI-OH), and (iii) comprises or consists of amino acid sequence SEQ ID NO: 6 (H-LLGDFFRKSK-OH).

11. The method according to any one of the preceding claims, wherein the coupling of the fragments in step d) is performed in the presence of HOBt.H2O.

12. The method according to any one of the preceding claims, wherein the first resin comprises a Wang linker, CTC linker, Rink Amide linker, Sieber amide linker, or a DHP HM linker.

13. The method according to any one of the preceding claims, wherein the second resin comprises a CTC linker or a HMPB-MBHA linker.

14. The method according to any one of the preceding claims, wherein the cleavage in step d) is performed using no more than 3% TFA, preferably 2% TFA, or no more than 50% HFIP, preferably no more than 30% HFPI.

15. A method for fragmented synthesis of an acetate form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, comprising23.a. providing a first fragment (i) of LL37 comprising or consisting of amino acid sequence SEQ ID NO: 2 coupled to a first resin solid support via a Wang linker,24.b. providing a further fragment (ii) of LL37 comprising or consisting of amino acid sequence SEQ ID NO: 3, 5 or 7, which is coupled to a resin solid support via a CTC linker,25.c. providing a further fragment (iii) of LL37 comprising or consisting of amino acid sequence SEQ ID NO: 4, 6 or 8, which is coupled to a resin solid support via a CTC linker,26.d. cleaving the fragments (ii) and (iii) from the solid support using 2% TFA in DCM, or 30% HFIP in DCM,27.e. sequentially coupling the first fragment (i) with fragment (ii), to obtain fragment (iv), followed by coupling of fragment (iii) to fragment (vi) to obtain a peptide of SEQ ID NO: 1, or a variant thereof, coupled to a first resin solid support,28.f. cleaving the peptide obtained in step e) from the linker using at least 70% TFA, to obtain a TFA salt form of the peptide of SEQ ID NO: 1, or a variant thereof,29.Inspicos / 13 / 11 / 2025 / 15:45 g. exchanging the TFA salt with an acetate salt to obtain an acetate salt form of an LL37 peptide of SEQ ID NO: 1, or a variant thereof, and30.h. optionally, purifying the LL37 peptide, or a variant thereof.

16. A method for fragmented synthesis of an acetate form of an LL37 peptide of SEQ ID NO: 1 comprising32.a. providing a first fragment (i) of LL37 comprising or consisting of amino acid sequence SEQ ID NO: 2, or a variant thereof, coupled to a first resin solid support via a Wang linker,33.b. providing a further fragment (ii) of LL37 comprising or consisting of amino acid sequence SEQ ID NO: 3, which is coupled to a resin solid support via a CTC linker, c. providing a further fragment (iii) of LL37 comprising or consisting of amino acid sequence SEQ ID NO: 4, which is coupled to a resin solid support via a CTC linker, d. cleaving the fragments (ii) and (iii) from the solid support using about 2% TFA, e. coupling the first fragment (i) with fragment (ii), to obtain fragment (iv), followed by coupling of fragment (iii) to fragment (vi) to obtain a peptide of SEQ ID NO: 1, coupled to the first resin solid support,34.f. cleaving the solid support linked peptide obtained in step e) from the linker using at least 50% TFA, preferably at least 70% TFA, to obtain a TFA salt form of the peptide of SEQ ID NO: 1,35.g. exchanging the TFA salt with an acetate salt to obtain an acetate salt form of an LL37 peptide of SEQ ID NO: 1, and36.h. optionally purifying the LL37 peptide, or a variant thereof.

17. An acetate form of an LL37 peptide produced according to any one of the preceding claims.

18. An acetate form of an LL37 peptide produced according to any one of claims 1-16, for use as a medicament.

19. The acetate form according to claim 18, for use in a method of treatment of periodontal disease, the method comprising injection of an effective amount of a composition comprising said acetate form into one or more periodontal pockets in the oral cavity of a subject in need thereof, wherein the composition further comprises a gelling agent / mixture and wherein the composition increases its viscosity at the time of injection from an initial value of at most 1000 mPa.s to an increased value of at least 4 Pa.s in the periodontal pocket to attain a semi-solid form, whereby the acetate form exhibits protracted release from the composition so as to exert antimicrobial and anti-inflammatory effects in the periodontal pocket(s) for a period of at least 24 hours.40.Insp icos / 13 / 11 / 2025 / 15 : 45

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