Antibody- like binding molecules and uses thereof

Abstract

The present invention relates to proteomimetic agents with reversible antibody-like binding properties, in particular for use in the fields of immunology and endocrinology and methods of preparations and uses thereof.

Description

[0001] 1

[0002] ANTIBODY- LIKE BINDING MOLECULES & USES THEREOF

[0003] Field of the Invention

[0004] The present invention relates to proteomimetic agents with reversible antibody-like bindingproperties, in particular for use in medicine for example in the fields of immunology,5 endocrinology, oncology, infectious diseases.

[0005] Background of the Invention

[0006] Tight and specific binding to a protein interface is quintessential to biomedical research and a well-established strategy for therapeutic intervention. For the most part, this is achieved with monoclonal antibodies (MAb), but there is a great demand for smaller protein scaffolds that10exhibit simple conjugation chemistry and better tissue penetration by virtue of their smaller size. Efforts in this direction have led to the development of synthetic antibody fragments,including single chain variable fragments (Worn et al., 2001, J. Mol. Biol. 305, 989-1010,https: / / doi.org:10.1006 / jmbi.2000.4265) (scFv) and nanobodies, the antigen-binding domainsderived from single chain antibodies (Muyldermans et al., 2013, Annu. Rev. Biochem.82, 775-15 797, https: / / doi.org:10.1146 / annurev-biochem-063011-092449; Steeland et al., 2016, Drug Discov. Today 21, 1076-1113, https: / / doi.org:10.1016 / j.drudis.2016.04.003).

[0007] Nanobodies are dramatically smaller than regular antibodies (12-15 kDa vs 150-160 kDa) andincreasingly used in research, with several candidates in clinical development. In parallel, alternative protein scaffolds have been investigated as platforms for antibody mimicry20(Gebauer et al., 2009, Curr. Opin. Chem. Biol. 13, 245-255 (2009).

[0008] https: / / doi.org:10.1016 / j.cbpa.2009.04.627). Prominent examples of affinity proteins includeaffibodies (Nord et al., 1995, Protein Eng. 8, 601-608,https: / / doi.org:DOI10.1093 / protein / 8.6.601), affimer (Tiede et al., 2017, Elife 6,https: / / doi.org:10.7554 / eLife.24903), avimerm (Silverman. et al., 2005, Nat. Biotechnol.23,25 1556-1561, https: / / doi.org:10.1038 / nbt1166), DARPins (Binz et al., 2003,. J. Mol. Biol. 332,489-503, https: / / doi.org:10.1016 / S0022-2836(03)00896-9; Binz et al., 2004, Nat. Biotechnol.

[0009] 22, 575-582, https: / / doi.org:10.1038 / nbt962) and monobodies (Koide et al., 1998, .J. Mol. Biol.

[0010] 284, 1141-1151, https: / / doi.org:DOI 10.1006 / jmbi.1998.2238).

[0011] Among these, affibodies stand out as being the smallest in class (58 residues, < 10 kDa).

[0012] 30Affibodies make use of a three-helix bundle derived from the bacterial Z domain of protein (Nord et al., 1997, Nat. Biotechnol.15, 772-777, https: / / doi.org:10.1038 / nbt0897-772; Stahl et al., 2017, Trends Biotechnol.35, 691-712, https: / / doi.org:10.1016 / j.tibtech.2017.04.007; Frejd et al., 2017, Exp. Mol. Med.49, e306, https: / / doi.org:10.1038 / emm.2017.35). 2

[0013] Combinatorial libraries randomizing 13 residues located at the interface between the two interacting helices and the target have been used to identify tight binders to diverse proteintargets. Notably, affibodies targeting HER2 (Orlova et al., 2006, Cancer Re.s 66, 4339-4348,https: / / doi.org:10.1158 / 0008-5472.Can-05-3521; Eigenbrot et al., 2010, P. Nat. Acad. Sci.

[0014] 5 USA 107, 15039-15044, https: / / doi.org:10.1073 / pnas.1005025107; Baum et al., 2010, J. Nucl.

[0015] Med. 51, 892-897, https: / / doi.org:10.2967 / jnumed.109.073239 and Hu et al., 2022, Front.Oncol. 12, 917439, HER3 (Kronqvist et al., 2011, Protein Eng. Des. Sel. 24, 385-396),PDGFRβ (Lindborg et al., 2011, J. Mol. Biol. 407, 298-315), EGFR (Ozcan et al., 2006, P.Nat. Acad. Sci. USA 103, 5735-5740), VEGF (Fedorova et al., 2011, Chem. Biol.18, 839-845,10 https: / / doi.org:10.1016 / j.chembiol.2011.05.011), IGF (Liu et al., 2022, Pharmaceutics 14,1475, https: / / doi.org:10.3390 / pharmaceutics14071475), TNF-⍺ (Jonsson et al., 2009,Biotechnol. Appl. Bioc.54, 93-103, https: / / doi.org:10.1042 / Ba20090085) and PD-L1 (Grindel,et al., 2022, ACS Chem. Biol. 17, 1543-1555, https: / / doi.org:10.1021 / acschembio.2c00218) have been identified using phage display or ribosomal display, suggesting that the affibody platform is15 general and applicable to a broad array of targets. More recently, the affibody scaffold has been used toengineer an optogenetic switch that requires the affibody to discriminate between two conformations ofa light responsive protein, further highlighting the power of this plaform (Kuwasaki et al., 2022, Nat.Biotechnol.40, 1672-1679, https: / / doi.org:10.1038 / s41587-022-01351-w).

[0016] In addition to the three-helix bundle derived from protein A’s Z domain, other three-helix20 bundles have been explored as general platforms for antibody mimicry. Alphabodies (Desmetet al., 2014, Nat. Commun. 5, https: / / doi.org:ARTN 523710.1038 / ncomms6237) make use ofcomputationally designed three-helix bundles. The discovery of a potent IL-23 antagonist from the alphabody scaffold further illustrates the generality of a platform leveraged on a three-helix bundle. In parallel, Baker and coworkers have leveraged advances in the computational25 approach to identify de novo miniproteins with exceptionally high affinity for select targets(Chevalier et al., 2017, Nature 550, 74-79, https: / / doi.org:10.1038 / nature23912; Silva. et al.,2019, Nature 565, 186-191, https: / / doi.org:10.1038 / s41586-018-0830-7). Among the choicesof miniprotein scaffolds, the three-helix bundle with a similar overall architecture to affibodies yet utilizing a different primary sequence, afforded high-affinity binders to the receptor binding30 domain (RBD) of the SARS-CoV-2 spike (Cao et al., 2020, Science 370, 426-431,https: / / doi.org:10.1126 / science.abd9909). Inhibition of the RBD-ACE2 interaction by these denovo miniproteins was shown to efficiently prevent viral entry into cells (Wang et al., 2020,Cell 181, 894-904, https: / / doi.org:10.1016 / j.cell.2020.03.045). More recent work reported thedevelopment of TLR3 agonists (Adams et al., 2024, BioRxiv, 2024.2004.2017.589973, 3

[0017] https: / / doi.org:10.1101 / 2024.04.17.589973). The third helix of the three-helix bundle does not take part in the binding interface and only serves a structural role in the scaffold. Consequently,efforts have been directed at truncating affibodies to a two-helix coiled coil (Braisted et al.,1996, P. Nat. Acad. Sci. USA 93, 5688-5692, https: / / doi.org:10.1073 / pnas.93.12.5688).

[0018] 5 However, simple truncation of the third helix often results in poorly folded structures that require further stabilization by i) covalent cross-linking across the two helices, ii) the introduction of point mutations at the interface of the two helices, and iii) optimization of theinter-helical hinge region. Synthetically tractable two-helix PNA-peptide conjugates led to thediscovery of a tight binder to spike’s RBD (Imiolek et Winssinger, 2022, ChemBioChem,10 https: / / doi.org:10.1002 / cbic.202200561) wherein PNA appended at the C- and N-terminus ofthe peptide was used to promote the coiled coil interaction, with an additional disulfide bondbetween the two helices to stabilize the desired conformation. However, this design proved notto be general, and measurable binding to other targets could not be achieved, notably HER2.Currently, there exists an evident need for synthetic molecules with antibody-like binding15 properties with on-demand switching of binding properties for use in prognostic and predictivemedicine but also as therapeutics.

[0019] Summary of the Invention

[0020] The present invention is directed to the unexpected finding that it is possible to design targetagents comprising two alpha helices in a coiled-coil arrangement which are assembled in a 20 particular manner via PNA hybridization wherein PNA moieties are located on each of the alpha-helices on the face of those helices not having a binding affinity to a target rather than at the end of the helices. This design advantageously provides a means of adjusting thephysicochemical properties of our assembly through modification of the PNA backbone (e.g.,pegPNA, arg-PNA) without compromising binding.

[0021] 25The inventors have in particular designed target agents in the form of alpha-helix self- assembled proteomimetic (ASAPs) for Human epidermal growth factor receptor 2 (Her2). Overexpression of this tyrosine kinase receptor is a prognostic and predictive biomarker in breast and gastric / gastroesophageal cancer. Her2 is also present in other cancers like ovary, endometrium, bladder, lung, colon, and head and neck. Therefore, its inactivation has potential 30 therapeutic applications. The inventors also describe the inhibition of viral replication when mimicking LCB-1 with target agent of the invention.

[0022] An object of this invention is to provide proteomimetic agents with reversible antibody-likebinding properties. 4

[0023] It is advantageous to provide target agents easy to assemble synthesize using templated nativechemical ligation (T-NCL) or templated CuAAC (T-CuAAC) and combinations (NCL withTCuAAC or T-NCL with CuAAC).

[0024] It is advantageous to provide target agents that have binding properties that can be turned on5 and off, akin to allosteric control in proteins.

[0025] An important advantage of this allosteric control (on-demande on-off activity) is that it providesa generic mechanism for an antidote. It also allows the binding activity to be regulated by nucleic acid circuitry (Watson et al., https: / / doi.org / 10.1021 / jacs.0c12970; Watson et al https: / / doi.org / 10.1016 / j.cbpa.2021.102104).

[0026] 10 It is an object of the invention to provide new target agents useful for intervention where affinityproteins are efficacious.

[0027] Objects of this invention have been achieved by providing a target agent according to claim 1. Disclosed herein, according to a first aspect of the invention, is a target agent comprising a coiled-coil arrangement consisting of two alpha helices wherein:

[0028] 15 - the two alpha-helices are from about 11 to 25 amino acids in length;

[0029] - the two alpha-helices are connected together from one of their ends through a hinge;- each alpha-helix has a helix face comprising a region essentially parallel to the alpha-helix axis having a binding affinity to a target;

[0030] - each alpha-helix has on its face which is opposite to its face having a binding affinity20to the said target, at least one peptide nucleic acid (PNA) moiety connected to an amino acid of the said alpha-helix;

[0031] wherein

[0032] - the said at least one PNA moiety of the first alpha helix is connected to an amino acidof the said first alpha-helix located within a distance of 6-15 amino-acid to the hinge;25 - the said at least one PNA moiety of the second alpha helix is connected to an aminoacid of the said second alpha-helix which is within a distance of about 8-15Å from an amino acid of the first helix to which the PNA moiety of the first helix is connected to;- the said at least one PNA moieties of each helix comprise 3 to 20 atoms are at leastpartially complementary to each other to be able to hybridize.

[0033] 30 Another aspect of the invention provides a pharmaceutical composition comprising at least onecompound according to the invention, as well pharmaceutically acceptable salts thereof and apharmaceutically acceptable carrier, diluent or excipient thereof. 5

[0034] A first aspect of the invention provides agents of the invention as well pharmaceutically acceptable salts thereof for use as therapeutics and theragnostics.

[0035] Another aspect of the invention resides in a use of an agent according to the invention for thepreparation of a pharmaceutical composition, in particular for the treatment of a cancer, in5 particular breast cancer or a viral infection, in particular SARS infections such as SARS CoV2 infections.

[0036] Another aspect of the invention relates to a pharmaceutical composition comprising at least one compound according to the invention in combination at least one agent useful inimmunotherapy and / or in the treatment of cancer or a viral infection.

[0037] 10 Another aspect of the invention is a method for preventing in a subject suffering from a cancer,in particular breast cancer or a viral infection, in particular SARS infections such as SARSCoV2 infections or at risk of suffering from such disorders, said method comprisingadministering a compound according to the invention or a pharmaceutical formulation thereofin a subject in need thereof.

[0038] 15 Another aspect of the invention is a method of imaging a subject, said method comprising administering to the subject a compound of radiolabelled compound according to the inventionor a radiopharmaceutical composition thereof, and imaging the said subject.

[0039] Another aspect of the invention is a process for the preparation of an agent according to theinvention as defined below.

[0040] 20Further objects and advantageous aspects of the invention will be apparent from the claims and / or from the following detailed description of embodiments of the invention with referenceto the annexed drawings. The compounds described herein have an activity that parallelsaffinity proteins such as monoclonal antibodies or affobodies but are prepared synthetically and thus are not limited to connonical proteogenic amino acids.

[0041] 25 Brief description of the drawings

[0042] Figure 1 schematizes the structure of a target agent according to the invention (A) and itsformation for example through templated NCL (B) wherein the target agent can be synthesizedby templated native chemical ligation (T-NCL), using the hybridization between a peptidenucleic acid (PNA) moiety present on each alpha-helix to bring, a thioester group located on30 the first helix and near its C-terminus (e.g. an aminoadipic acid (Aad) with a thioester inposition in position 11 (SPh= thiophenyl) from the first helix) and a HS group of a cysteine atthe N-terminus (e.g. at position 12) of a second fragment containing the second helix, the hinge 6

[0043] and a small portion (3 amino acids) of the first helix in close proximity (high effectiveconcentrations) following hybridization.

[0044] Figure 2 represents the design of a target agent from the invention as described in Example 1.a. Sequence of ZHER2:342 with the three helices in different shades of greys (SEQ ID NO: 1). b.

[0045] 5 Structure of the HER2-ZHER2:342 complex with highlighted interaction surface (PDB3MZW). c. Bottom view of the three-helix bundle with spheres representing the position ofsidechains interacting with the target; d. Front view of the three-helix bundle (top) andcorresponding helical wheel (bottom) representation of ZHER2 with the aminoacids in positionsa,e and b interacting with the target e. Schematic representation of a truncated two-helix10 ZHER2 with modified positions E15Aad, F30K selected for PNA conjugation; f. sequences ofthe target agents from the invention based on the starting sequences.

[0046] Figure 3 illustrates the modulation of the activity of a target agent of the invention as describedin Example 2. A target agent of the invention of SEQ ID NO: 9 wherein the PNA moietieshave been replaced as respectively by on helix 1: a PNA moiety (4) comprising: a N-terminal 15 non-hybridizing T (1) on the thioester fragment pairing PNA strand GCC (2) (for pairing with the PNA moiety of helix 2) and a 4 PNA overhang comprising a diamino purine (D) (3) (A) and on helix 2, a PNA moiety (7) comprising a N-terminal SerSer spacer (5) to adjust spacing of unpaire PNA on the pairing PNA strand CGG (6) (for pairing with the PNA moiety of helix1) is designed (A). B: a displacing PNA moiety (SEQ ID NO: 23) is added to the designed20 target agent resulting in pairing of displacing PNA (8) with the PNA moiety of helix 1 (4) dueto the unpairing of the PNAs from each helices (C).

[0047] Figure 4 provides the representations of the two RBD-binding helices of three-helix bundleLCB-1 sequence as described in Khatri et al., 2022, Nat. Chem. Biol. 18, 1046,https: / / doi.org:10.1038 / s41589-022-01060-0 in helical wheel (top) and in 3D representation25 (bottom) with highlighted positions I12 and L31 selected for PNA conjugation and SAPs (A)and the sequences of target agents of the invention based thereon as described in Example 3(B).

[0048] Example 5 provides a schematic representation of copper-catalyzed-azide-alkynecycloaddition CuAAC using the same sequences as in Example 3 with substituting G in the30sequence of the hinge for propargyl as described in Example 4.

[0049] Detailed Description of the invention

[0050] The term “coiled coil arrangement” refers to a 3D arrangement between two alpha helices, eachhelix comprising one face with hydrophobic residues which can involve into hydrophobic 7

[0051] interactions with the face comprising hydrophobic residues of the other helix. The coiled coilarrangement can be parallel or antiparallel. According to a particular aspect, the coiled-coil arrangement is an antiparallel coiled coil arrangement.

[0052] The term “hinge” refers to a linking moiety maintaining the alpha helices in coiled coil 5 arrangement. The hinge is connected to one of the ends of each alpha helices. The hinge can be of peptidic or non-peptidic nature and has a length from about 6 to about 20 atoms.

[0053] According to a particular aspect, a hinge can be peptidic and comprises a sequence of about 4to about 12 aminoacids such as DPNL (SEQ ID: 10), LPNL (SEQ ID: 11), LGHA (SEQ ID:12) or a variant thereof as described herein. The hinge can be connected to one of the helix10 through a 2-aminoisobutyric acid moiety in order to enhance the alpha helicity of this helix.

[0054] According to one aspect, the hinge can be connected to the C-term of the first alpha helix and to the N-term of the second alpha helix to maintain the alpha helices in an antiparallel coiledcoil arrangement. According to one aspect, the hinge can be non-peptidic such asa polyelthylene glycol (PEG) chain and / or contain elements that facilitate a ligation chemistry15 (e.g. click chemistry) or preorganize the two helices (e.g. 1,2 substitudes cyclopentyl orcyclohexyl, aromatic and heteroaromatic rings with ortho and meta substituents). Further,substitution of an amide bond in the hinge sequences for a triazole are well tolerated.

[0055] The term “PNA moiety” refers to a molecular moiety comprising a peptide nucleic acidsequence of from about 3 to 10 nucleic acids and can be of the same or different length on each20helix. The PNA moieties of each helix comprise 3 to 20 atoms (separating the first PNA residue and the alpha carbon of the amino acid which is part of the helix). The PNA sequence of onehelix only needs to be able to hybridize to a portion of the opposing PNA of the other helix andnot necessarily over its entire length. According to a particular embodiment, theoligonucleotide sequences which can be used as PNA moiety can be PNA sequences or known25 analogs (preferably D strereochemsitry to avoid hybridization and cross talk with endogenous oligonucleotides), DNA or modified RNA for metabolic stability (preferably L strereochemsitry to avoid hybridization and cross talk with endogenous oligonucleotides),other oligonucleotide such as locked nucelic acid or other well-known xenonucleic acids orPNA / DNA chimeras. The PNA moiety may further comprise modifications on the backbone30to optimize physicochemical properties (Saarbach et al.,https: / / doi.org / 10.1016 / j.cbpa.2019.06.006). The PNA moiety can be conjugated to an aminoacid from the face of the alpha-helix which is opposite to the helix face having a binding affinityto the target of interest through amide bond reaction or various ligation chemistry strategies,for example by click reactions such as CuAAC, strain promoted azide-alkyne cycloaddition 8

[0056] (SPAAC) or other click cycloadditions such as tetrazene- strained alkyne , cysteine addition toaldehyde, nitrile, Michale acceptor, substitution reaction based on the unique reactivity of thiol and benzyl, allylic or actyl leaving groups.

[0057] The amino acids of the hinge or the helix can be D or L or a combination thereof but should 5 not compromise the formation of the alpha-helices (no D / L mixture within the same helix).

[0058] The term “hydrophobic residue” according to the invention refers to any natural or unnaturalamino acid known to interact favorably by hydrophobic interactions such as Valine, Leucine,Isoleucine, Methionine, Phenylalanine and tyrosine or tryptophan. Examples of unnaturalamino acids include cyclobutyl alanine (Cba) which is close in properties to Val, cyclohexyl10alanine (Cha), halogenated or otherwise substituted aromatic amino acids (Phe, Tyr, Trp), alternative heterocyclic rings such as pyridyl alanine.

[0059] The term “any natural or unnatural amino acid” according to the invention refers to thecanonical amino acids and their modifications (alternative side chains, alternative stereochemistry).

[0060] 15 The term “cationic residue” refers to any natural or unnatural amino acid which has a cation onits side chain or a basic amine on the side chain that is protonated at physiological pH (i.e. cationic).

[0061] The term “anionic residue” refers to any natural or unnatural amino acid that has a an anion onits side chaine or a carboxylic acid on the side chain that is deprotonotated at physiological pH20(i.e. anionic).

[0062] The term “neutral residue with an hydrophilic side chain” refers to any natural or unnaturalamino acid with a side chain containing heteroatoms but no charges such as Apargine,Glutamine, Serine, threonine.

[0063] The term “an amino acid as a point of attachment on its the alpha carbon for the PNA moiety”25 refers to any amino acid with a suitable functional group for the PNA such as a Lysine residue to link the C-ter of PNA via an amide bond or a Homoglutamic acid to link the N-ter of PNA via an amide bond.

[0064] The term “variant” is used herein, is a peptide or a polypeptide substantially homologous to the referenced peptide sequence, but which has an amino acid sequence different from that of the30 referenced. Such variants may be synthetically generated, for example, by modifying one ormore of the above polypeptide sequences of the invention described herein using any of a number of techniques well known in the art. Substantially homologous means a variant amino acid sequence which is identical to the referenced peptide sequence with the exception of the deletion, insertion and / or substitution of a few amino acids, e.g.1, 2, 3, 4, or 5 amino acids 9

[0065] amongst the defined non-variable amino acids of the referenced polypeptide sequences. The identity or homology of two amino acid sequences or of two nucleic acid sequences can bedetermined by visual inspection and / or mathematical calculation, or more easily by comparingsequence information using known computer program used for sequence comparison such as5 Clustal package version 2.1. According to a particular embodiment, a variant of a target agentof the invention has at least 80% of sequence homology with each of the helices, the hinge can be replaced by a hinge according to the invention, while the overall 3D structure of the coiled- coil arrangement of the two helices is preserved, namely wherein at least one PNA moiety of the first alpha helix is connected to an amino acid of the said first alpha-helix located within a10distance of 6-15 amino-acid to the hinge and at least one PNA moiety of the second alpha helix is connected to an amino acid of the said second alpha-helix which is within a distance of about 8-15Å from an amino acid of the first helix to which the PNA moiety of the first helix is connected to.

[0066] A conservative, or homologous, variant may comprise a sequence having at least one15 conservatively substituted amino acid. A conservative (or homologous) variant may include asequence comprising one or more conservatively substituted amino acids. For example, quaternary amino acid analogs (e.g., Ala → Aib) may be incorporated to enhance proteolytic stability and enforce helicity. Another example is the use of amino acid surrogates. Numerous surrogates of arginine have been described, in which the three-carbon chain between the α-20carbon and the guanidinium group is replaced by aromatic or heteroaromatic linkers. Similarly, a leucine residue oriented away from the binding interface may be replaced with a surrogate in which the terminal isopropyl groups are cyclized with heteroatoms (e.g., piperidin-4-yl- alanine). Additional examples include substitution of lysine with ornithine or alkylated derivatives thereof, or substitution of tyrosine and tryptophan with halogenated analogs.

[0067] 25 The term “large hydrophobic residues” refers to amino acids such as I, L, F and analogs thereofsuch as Cyclohexyl alanine, Cyclobutyl alanine, p-Chlorophenyl alanine, etc..

[0068] As used herein, “treatment” and “treating” and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and / or may be30therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease.

[0069] The term “efficacy” of a treatment according to the invention can be measured based on changes in the course of a disease in response to a use or a method according to the invention. 10

[0070] The term "subject" as used herein, refers to a human or a non-human mammal, such as non-human primate (e.g. chimpanzees and other apes and monkey species), a farm animal (e.g.cattle, sheep, pigs, goats and horses), a domestic mammal (e.g. dogs and cats), or a laboratoryanimal (e.g. rodents, such as mice, rats and guinea pigs).

[0071] 5 The term “efficacy” of a treatment according to the invention can be measured based onchanges in the course of disease in response to a use or a method according to the invention.For example, the efficacy of a treatment according to the invention can be measured by its impact on signs or symptoms of illness. A response is achieved when the subject experiences partial or total alleviation, or reduction of unwanted symptoms of illness. According to a10 particular embodiment, the efficacy can be measured through the assessment of tumor volume.

[0072] The term “pharmaceutical formulation” refers to preparations which are in such a form as to permit biological activity of the active ingredient(s) to be unequivocally effective and which contain no additional component which would be toxic to subjects to which the said formulation would be administered.

[0073] 15 Compounds of the invention

[0074] Referring to the figures, in particular Figure 1A, is provided an illustration of a target agentcomprising a coiled-coil arrangement consisting of two alpha helices wherein:

[0075] - the two alpha-helices 1a, 1b are from about 11 to 25 amino acids in length;

[0076] - the two alpha-helices are connected together from one of their ends through a hinge 2;20 - each alpha-helix has a helix face 3a, 3b comprising a region essentially parallel to thealpha-helix axis having a binding affinity to a target;

[0077] - each alpha-helix has on its face which is opposite to its face having a binding affinityto the said target, at least one peptide nucleic acid (PNA) moiety connected to an aminoacid of the said alpha-helix;

[0078] 25 wherein

[0079] - the said at least one PNA moiety of the first alpha helix is connected to an amino acidof the said first alpha-helix located within a distance of 6-15 amino-acid to the hinge;- the said at least one PNA moiety of the second alpha helix is connected to an aminoacid of the said second alpha-helix which is within a distance d of about 8-15Å from an30amino acid of the first helix to which the PNA moiety of the first helix is connected to;

[0080] - the said at least one PNA moieties of each helix comprise 3 to 20 atoms are at leastpartially complementary to each other to be able to hybridize. 11

[0081] According to another particular aspect, the said at least one peptide nucleic acid (PNA) moiety present on the helix face which is opposite to the helix face having a binding affinity to the said target is connected to an amino acid from the said face of the alpha-helix and forms a molecular chain pointing in a direction essentially perpendicular to the said helix axis.

[0082] 5 According to one particular aspect, each alpha-helix has on its face which is opposite to its facehaving a binding affinity to the said target, one peptide nucleic acid (PNA) moiety connected to an amino acid of the said alpha-helix.

[0083] According to one particular aspect, each alpha helix is from about 12 to 20 amino acids inlength (e.g. from 14 to 18 amino acids).

[0084] 10 According to another particular aspect, the hinge has a length of 6 to 20 atoms.

[0085] According to another particular aspect, the hinge is a peptidic sequence.

[0086] According to another particular aspect, the PNA moieties of each helix comprise 5 to 7 atoms. According to another particular aspect, each alpha-helix has a helix face comprising a regionessentially parallel to the alpha-helix axis having a binding affinity to a target entity, for15 example the target entity is a cell surface oncogenic biomarker or an extracellular protein of thearpeutic interest. Examples of target entities include but is not limited to EGFR, HER2, HER3, PDL1, PD1, CD3, IL17, Factor X and albumin.

[0087] According to another further particular aspect, the target is a target protein selected from Her2and SARS- CoV’s spike.

[0088] 20 According to another particular aspect, the hinge is selected from DPNL (SEQ ID: 10) andLGHA (SEQ ID: 12) or a variant thereof, in particular where an amide bond is substituted bya triazole.

[0089] According to one particular aspect, the first alpha helix has the following sequence:

[0090] Xc111Xc112Xc113Xc114Xc115Xc116Xc117RXc119Xc1110Xc1111Xc1112AXc1114 (SEQ ID NO: 13, first25 consensus first helix),

[0091] wherein Xc111 is an hydrophobic natural or unnatural amino acid such as Phenylalanine or isabsent; Xc112 is selected from any natural or unnatural amino acid or is absent; Xc115, Xc116,Xc119and Xc1110are independently selected from any natural or unnatural amino acid; Xc113is a cationic natural or unnatural amino acid such as Lysine; Xc114is an anionic natural or30unnatural amino acid such as glutamic acid; Xc117 is a neutral natural or unnatural amino acidwith an hydrophilic side chain such as aspargine; Xc1111 is an amino acid with a functional 12

[0092] group on its side chain as a point of attachment to the PNA moiety such as a a glutamic acidwhere the side chain acid can be linked to the PNA by amide bond formation or an aminoadipic acid (Aad), also known as homoglutamic acid (hE)which can be linked in the same fashion;

[0093] Xc1112 is any amino acid with a thiol group such as a cysteine or a homocysteine; Xc1114 are5 independently selected large hydrophobic natural or unnatural amino acids such as I, L, F andanalogs thereof and wherein residues Xc112, X 115, Xc116, Xc119, Xc1110 (underlined) are presenton the helix face having a binding affinity to a target of interest.

[0094] According to a further particular aspect, the first alpha helix has the a sequence of SEQ ID: 13wherein the hydrophobic natural or unnatural amino acid are selected from Valine, Leucine,10Isoleucine, Methionine, substituted or unsubstituted Phenylalanine, substituted or unsubstitutedTyrosine, substituted or unsubstituted Tryptophane, Cba, Cha and pyridyl analanine.

[0095] According to a further particular aspect, the first alpha helix of SEQ ID NO: 13 has thefollowing sequence:

[0096] FXc112KEXc115Xc116NRYWXc1111CAL (SEQ ID NO: 14, subspecific first helix)15 wherein Xc112, Xc115, Xc116 and Xc1111 are as defined herein, or is an homologous variant thereof.

[0097] According to another further particular aspect, the first alpha helix is a homologous variant ofSEQ ID NO: 14 wherein said variant comprises at least one of those mutations in SEQ IDNO: 14: wherein F can be substituted Phenylalanine such as halogenated Phenylalanine, K canbe substituted by Ornithine or methylated lysine or ornithine, E can be substituted by an amino20acid with other type of acidic group such as a phosphonate or sulfonate in lieu of thecarboxylate, N can be substituted by heterocyclic surrogate of the amide side chains or an urea,Y or W could be halogenated or hydroxyl substrituted aromatic variant or other heterocycles,C can be substituted by any amino acid with a thiol group, L can be substituted by a cyclobutylalanine, cyclohexyl alanine, t-butyl alaine or halogenated variants thereof.

[0098] 25 According to a further particular aspect, the first alpha helix is of SEQ ID NO: 14 wherein:

[0099] Xc111 and Xc112 area absent, Xc115, is Cha; Xc116 is Arginine, Xc1111 is Aad.

[0100] According to one particular aspect, the first alpha helix has the following sequence:

[0101] Xc211Xc212Xc213Xc214Xc215Xc216Xc217Xc218Xc219Xc2110Xc2111Xc2112Xc2113Xc2114Xc2115Xc2116Xc2117

[0102] Xc2118(SEQ ID NO: 15, second consensus first helix),

[0103] 30 Wherein Xc211 is an anionic natural or unnatural amino acid such as aspartic acid; Xc212 andXc218 are independently selected cationic natural or unnatural amino acids such as Lysine; Xc213, 13

[0104] Xc214, Xc216, Xc217, Xc2110, Xc2111, Xc2113, Xc2114, Xc2117 and Xc2118 are independently selected fromany natural or unnatural amino acid; Xc215, Xc219, Xc2115 and Xc2116 are independently selectedlarge hydrophobic natural or unnatural amino acids such as I, L, F and analogs thereof; Xc2112is an amino acid with a functional group on its side chain as a point of attachment to the PNA5 moiety such as a a glutamic acid where the side chain acid can be linked to the PNA by amidebond formation or an aminoadipic acid which can be linked in the same fashion; wherein aminoacids Xc213, X214, Xc216, X217, Xc2110, Xc2111, Xc2113, Xc2114, Xc2117 and Xc2118 (underlined) arepresent on the helix face having a binding affinity to a target of interest.

[0105] According to another further particular aspect, the first alpha helix of SEQ ID NO: 15 has the10following sequence:

[0106] DKXc213Xc214IXc216Xc217KIXc2110Xc2111Xc2112Xc2113Xc2114LLXc2117Xc2118(SEQ ID NO: 16, subspecific 2 first helix)

[0107] wherein Xc213, Xc214, Xc216, Xc2110Xc2111Xc2112Xc2113Xc2114, Xc2117 and Xc2118 are as definedherein, or is an homologous variant thereof.

[0108] 15 According to another further particular aspect, the first alpha helix is a homologous variant ofSEQ ID NO: 16 wherein said variant comprises at least one of those mutations in SEQ IDNO: 16 wherein K can be substituted by Ornithine or methylated lysine or ornithine, D can besubstituted by another amino acid with an acid side chain such as E or unnatural amino acids,I can be substituted by large hydrophobic residue such as Cba (Carboranylalanine), Cha20(Cyclohexylglycine) or other analogs, L can be substituted by large hydrophobic residue such as Cba, Cha or other analogs.

[0109] According to another further particular aspect, the first alpha helix of SEQ ID NO: 15 has the following sequence:

[0110] DKEWILQKIYEXc2112MRLLDE (SEQ ID NO: 17, specific first helix, consensus 2),25 wherein Xc2112 is as defined herein, or is an homologous variant thereof.

[0111] According to another further particular aspect, the first alpha helix is a homologous variant ofSEQ ID NO: 17 wherein said variant comprises at least one of those mutations in SEQ IDNO: 17 wherein K can be substituted by Ornithine or methylated lysine or ornithine, D can besubstituted by another amino acid with an acid side chain such as E or unnatural amino acids,30I can be substituted by by large hydrophobic residue such as Cba, Cha or other analogs, L can be substituted by substituted by large hydrophobic residue such as Cba, Cha or other analogs, 14

[0112] M can be susbsituted by homocysteine (Hcy) or methylated homocysteine (HcyMe) and Xc2112is Aad.

[0113] According to one particular aspect, the second alpha helix has the following sequence:

[0114] XC121XC122XC123XC124XC125XC126AXC128XC129XC1210XC1211XC1212XC1213XC1214XC1215 (SEQ ID5 NO: 18 first consensus second helix),

[0115] wherein XC121, XC124 and XC1211 are independently selected neutral natural or unnatural aminoacids with heteroatoms such as threonine, asparagine, glutamine or variants thereof; XC122,XC123, XC125, XC126, XC129, XC1210 and XC1213 are independently selected from any natural orunnatural amino acid; Xc128 is an amino with a functional group on its side chain as a point of10 attachment to the PNA moiety such as lysine (Lys) or diaminopropionic acid (Dap) where theside chain amino group is linked to the PNA via an amide bond; XC1212is a large hydrophobicnatural or unnatural amino acid such as I, L, F and analogs thereof; XC1214 and XC1215 is ananionic natural or unnatural amino acid, wherein amino acids XC122, XC123, XC125, XC126, XC129, XC1210 and XC1213 (underlined) are present on the helix face having a binding affinity to a15 target of interest and wherein XC1214 and XC1215 can be independently present or absent.

[0116] According to a further particular aspect, the second alpha helix of SEQ ID NO: 18 has thefollowing sequence:

[0117] TNQQKRAXC128IRSIYEE (SEQ ID NO:19, subspecific second helix),wherein XC128 is defined herein, or is an homologous variant thereof.

[0118] 20According to another further particular aspect, the first alpha helix is a homologous variant ofSEQ ID NO: 18 wherein said variant comprises at least one of those mutations in SEQ IDNO: 18 wherein K can be substituted by Ornithine or methylated lysine or ornithine, E can besubstituted by an amino acid with other type of acidic group such as a phosphonate or sulfonatein lieu of the carboxylate, N can be substituted by heterocyclic surrogate of the amide side25 chains or an urea, Y could be halogenated or hydroxyl substrituted aromatic variant or otherheterocycles such as pyridine or benzofuran.

[0119] According to another further particular aspect, the first alpha helix is a homologous variant ofSEQ ID NO: 18 wherein said variant comprises the modifications in SEQ ID NO: 18 whereinG (XC1214) and E (XC1215) are added at the end of the C-term of SEQ ID NO: 18.

[0120] 30 According to one particular aspect, the second alpha helix has the following sequence: 15

[0121] XC221AXC223XC224XC225XC226XC227XC228XC229XC2210XC2211XC2212XC2213Xc2214XC2215XC2216

[0122] (SEQ ID NO: 20 second consensus second helix),

[0123] wherein XC221, XC228, XC2212 are independently selected anionic natural or unnatural aminoacids such as aspartic acid or glutamic acid; XC223, XC224, XC227, XC2210, XC2211 and Xc2214 are5 independently selected from any natural or unnatural amino acid; XC225, XC2215and XC2216areindependently selected cationic natural or unnatural amino acids such as arginine and lysine;

[0124] XC226 is an hydrophobic residue natural or unnatural amino acid; XC229 is an amino acid with afunctional group on its side chain as a point of attachment to the PNA moiety such as lysine(Lys) or diaminopropionic acid (Dap) where the side chain amino group is linked to the PNA10 via an amide bond; XC2213 is a large hydrophobic natural or unnatural amino acid such as I, L,F and analogs thereof; and wherein amino acids XC223, XC224, XC227, XC2210, XC2211 and Xc2214(underlined) are present on the helix face having a binding affinity to a target of interest.According to a further particular aspect, the second alpha helix of SEQ ID NO: 20 has thefollowing sequence:

[0125] 15 EAXC223XC224RVXC227DXC229XC2210XC2211EFXc2214KK (SEQ ID NO: 21, subspecificsecond helix),

[0126] wherein Xc223, Xc224, Xc227, Xc229, Xc2210, Xc2211, and Xc2214 are as defined herein, or is anhomologous variant thereof.

[0127] According to another further particular aspect, the first alpha helix is a homologous variant of20 SEQ ID NO: 21 wherein said variant comprises at least one of those mutations in SEQ IDNO: 21: wherein F can be substituted by Phenylalanine such as halogenated Phenylalanine, Kcan be substituted by Ornithine or methylated lysine, D can be substituted by another aminoacid with an acid side chain such as E or unnatural amino acids, E can be substituted by anamino acid with other type of acidic group such as a phosphonate or sulfonate in lieu of the 25 carboxylate.

[0128] According to a further particular aspect, the second alpha helix of SEQ ID NO: 20 has thefollowing sequence:

[0129] EASMRVSDXC229IYEFMKK (SEQ ID NO: 22, specific second helix, second consensus),wherein Xc229 is as defined herein or is an homologous variant thereof.

[0130] 30According to another further particular aspect, the first alpha helix is a homologous variant ofSEQ ID NO: 22 wherein said variant comprises at least one of those mutations in SEQ IDNO: 22: wherein F can be substituted Phenylalanine such as halogenated Phenylalanine, K can 16

[0131] be substituted by Ornithine or methylated lysine, D can be substituted by another amino acidwith an acid side chain such as E or unnatural amino acids, E can be substituted by an amino acid with other type of acidic group such as a phosphonate or sulfonate in lieu of the carboxylate.

[0132] 5 According to one particular aspect, the target agent is characterized by a first alpha helix ofSEQ ID No: 13 and a second alpha helix of SEQ ID No: 18 in a coiled-coil arrangementwherein Xc1114 from the first helix is connected to the Xc121 from the second helix through ahinge and Xc1111 from the first helix is connected to a first peptide nucleic acid (PNA) moietyand Xc228from the second helix is connected to a second peptide nucleic acid (PNA) moiety10wherein the said first and second PNA moieties are at least partially complementary to each other to be able to hybridize.

[0133] According to another particular aspect, the target agent is characterized by a first alpha helix ofSEQ ID No: 15 and a second alpha helix of SEQ ID No: 20 in a coiled-coil arrangementwherein Xc2118 from the first helix is connected to the Xc221 from the second helix through a 15 hinge and Xc2112from the first helix is connected to a first peptide nucleic acid (PNA) moiety and Xc229 from the second helix is connected to a second peptide nucleic acid (PNA) moiety wherein the said first and second PNA moieties are at least partially complementary to each other to be able to hybridize.

[0134] According to another particular aspect, the PNA moiety may further comprise an active agent20(e.g. cytotoxic agent for tardegeted drug delivery, nucleic acids for antisense therapy) or a labeling moiety such as dye or radio nuclei for imaging application (ex. PET) or photodynamic therapy or localized radiation therapy. An example of a fluorescent analog is provided inExample 1. An example of DOTA analog for radiolabeling is provided in Example 5.

[0135] According to another particular aspect, PNA moieties have respectively a sequence of CCG25 and GGC.

[0136] According to one particular aspect, the target agent has a sequence selected from SEQ ID NO:5 and SEQ ID NO: 9 and variants thereof.

[0137] According to one particular aspect, the target agent has a sequence selected from SEQ ID NO:9 or a variant thereof, wherein said variant can have at least 80% of sequence homology with30SEQ ID NO: 9 in each of the helices, the hinge being replaced by a hinge according to the invention, while the overall 3D structure of the coiled-coil arrangement of the two helices is preserved, namely wherein at least one PNA moiety of the first alpha helix is connected to an 17

[0138] amino acid of the said first alpha-helix located within a distance of 6-15 amino-acid to the hinge and at least one PNA moiety of the second alpha helix is connected to an amino acid of the said second alpha-helix which is within a distance of about 8-15Å from an amino acid of the first helix to which the PNA moiety of the first helix is connected to.

[0139] 5 According to one particular aspect, the target agent has a sequence selected from SEQ IDNO: 23 and SEQ ID NO: 24 and variants thereof.

[0140] Synthesis of compounds of the invention

[0141] The compounds can be prepared by standard techniques as described herein. In particular, first a thioester can be introduced on the C-term of helix 1 and then a N-ter Cys can be introduced10on a peptidic fragment comprising the hinge and helix 2.

[0142] Compositions

[0143] Pharmaceutical compositions of the invention can contain one or more compounds accordingto the invention and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0144] Compositions of this invention may also be formulated for parenteral administration including, 15 but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents. Further materials as well as formulation processing techniques and the like are set out inRemington: The Science & Practice of Pharmacy, 23rd Edition, 2020, Ed. Adeboye Adejare,20Academic Press, which is incorporated herein by reference.

[0145] According to a particular aspect, is provided a pharmaceutical composition comprising at leastone compound according to the invention and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0146] According to a particular aspect, is provided a radiopharmaceutical composition comprising a25 radiolabelled compound according to the invention, as well pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier, diluent or excipient thereof.

[0147] The radiopharmaceutical composition can further comprise one or more radiolysis stabilizers,one or more free metal chelators, and / or one or more pH stabilizers.

[0148] The invention provides compounds of the invention, compositions thereof and methods using30 the same useful in the prevention and / or treatment of a medical disorder, in particular cancer,immunological disorders or infectious diseases. 18

[0149] Mode of administration

[0150] Compounds and compositions of this invention may be administered or delivered in anymanner including, but not limited to subcutaneous injection.

[0151] The dosage administered, as single or multiple doses, to an individual will vary depending upon5 a variety of factors, including pharmacokinetic properties, subject conditions andcharacteristics (sex, age, body weight, health, and size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.

[0152] Combination

[0153] According to one embodiment of the invention, the compounds according to the invention and10pharmaceutical formulations thereof can be administered alone or in combination with a co-agent useful in the prevention and / or treatment of a disease.

[0154] According to one aspect, compounds of the invention can be administered in combination withat least one therapeutic molecule.

[0155] The invention encompasses the administration of a compound of the invention wherein the 15 compound is administered to a subject prior to, simultaneously or sequentially with atherapeutic regimen or at least one co-agent. The compound according to the invention that isadministered simultaneously with said at least one co-agent can be administered in the same ordifferent compositions and in the same or different routes of administration.

[0156] Subjects

[0157] 20 In an embodiment, subjects according to the invention are suffering from a disorder.

[0158] In a further particular embodiment, subjects according to the invention are subjects suffering from immunological or endocrinal disorders.

[0159] Use according to the invention

[0160] In a particular embodiment, the invention provides compounds, methods, uses and 25 compositions useful in the treatment of immunological and endocrinal disorders such as psoriasis.

[0161] According to a particular aspect, in the target agents, the PNA moieties (PNA, DNA, XNA)direct the folding of the peptides into an active conformation. Hybridization with a competingoligonucleotide (antidote) would disrupt the folding leading to the antiparallel coiled coil30 conformation and lead to weaker binding to the target . 19

[0162] In a particular embodiment, the invention provides compounds, methods, uses andcompositions useful for the in vivo targeted delivery of substances. Many research applicationsare also possible, including reversible immunofluorescence.

[0163] According to a particular aspect, is provided a radiopharmaceutical composition according to5 the invention for use in imaging a target tissue or in radiation therapy.

[0164] References cited herein are hereby incorporated by reference in their entirety. Such modifications are intended to fall within the scope of the appended claims. The invention having been described, the following examples are presented by way of illustration, and not limitation.

[0165] 10EXAMPLES

[0166] Example 1: Synthesis of compounds of the invention targeting HER2

[0167] A well-known sequence targeting HER2 (SEQ ID NO: 1) (ZHER2:342 (Orlova et al., 2006,supra; Eigenbrot et al., 2010, supra), Fig.2a), a well-established biomarker in cancer and an important target for therapeutics was used as a strating point for the design of the alpha helices15 of target of the invention. This structure clearly shows that the third helix (yellow) of the three-helix bundle is not engaging in complex formation. The important residues involved in targetinteraction are located at positions a, e, b of the helical heptad (blue and red helices, Fig.2cand d). However, the inter-helical interface with the third helix contains a number ofhydrophobic residues that are substantial for the three-helix packing.

[0168] 20A truncated ZHER2:342 sequence (5-37) restricted to the sequence of the target binding helices (SEQ ID NO: 2) was synthesized but had no measurable affinity by SPR to HER2 (Fig.2a), indicating that this truncated peptide does not recapitulate the folding of ZHER2which confirmed that truncation of the third helix leads to a dramatic loss of affinity (Webster et al., 2009,ChemBioChem, 10, 1293-1296). However, mutations were introduced which restored affinity25 to 5.8 nM (Fig. 2a, 2-helix ZHER2,mut (SEQ ID NO: 3, wherein letters in bold denote themutations)).

[0169] However, in order to be able to introduce a peptide nucleic acid (PNA) moiety by templatednative chemical ligation (T-NCL) of the peptide fragments (helix 1 and helix 2 with the hinge), a cytein needs to be present on the first helix next to the hinge to allow the synthesis of a whole30 thioester fragment starting from the PNA C-terminus without the need to synthesize andincorporate a dedicated peptide thioester precursor subsequent to C to N peptide synthesis (as illustrated on Fig.1B). The best tolerated cysteine mutations were at positions 17 (N17C in SEQ ID NO: 3) and 12 (A12C in SEQ ID NO: 3) leading to a reduction in affinity of the half 20

[0170] ot the third of the 2-helix ZHER2,mut, respectively due to impaired interaction between thehelices due to the introduction of the cyteine. The tolerated cysteine mutation, A12C (KD = 19nM) being located directly next to the envisioned PNA-anchoring E11 was choosen and targetsagent of the invention (SEQ ID NO: 4, 5 and 8) comprising a PNA moiety at position 11 on 5 the first helix and a complementary PNA moiety at position 36 was formed as described below using aminoadipic acid (Aad) and ornithine (O) (FmocAadOAll) as side-chain PNA anchor since it would not be prone to cyclization during SPPS:

[0171] a) Introduction for the thioether part on the C-term of helix 1

[0172] The peptide is prepared by standard solid phase peptide synthesis (SPPS) on a Rink amide resin10 using Fmoc strategy. The PNA group is synthetized first. Fmoc-Aad-O-All is then coupled atthe N-ter of the PNA via the side chain carboxylic acid of Aad and peptide synthesis is continued according to standard Fmoc-based SPPS. At the end of the peptide synthesis, the allyl group is removed under standard condition (Pd(0)L2 and a nucleophile, where L can be a variety of ligand including PPh3 and the nucleophile can be a stannane, silane or aniline). The 15 resulting free carboxylic acid is converted to a thio ester using either a cabodiimide and thiophenol or a disulfide of thiophenol and PPh3. The final product is cleaved from the resinusing a TFA cocktail as know in the art of peptide synthesis. The synthesis of PNA-peptideconjugates is well established (https: / / doi.org / 10.1038 / s41587-024-02209-z; https: / / doi.org / 10.1002 / cbic.202200561; https: / / doi.org / 10.1038 / s41557-021-00829-5).

[0173] 20 b) Incorporation of the N-ter Cys on the fragment comprising the hinge and helix 2The N-ter Cys fragment is also prepapred by SPPS as previously reported PNA-peptide conjugates using and FmocLys(Mtt)OH or FmocDap(Mtt)OH at the position where the PNA will be introduced. The peptide synthesis is terminated with BocCys(OH) such that the Mttgroup can be removed (HFIP-DCM) and the PNA synthesis performed on the free amine. The25 final product is cleaved from the resin using a TFA cocktail.

[0174] The same chemistry was used to generate small variations in the distance between the helix and the PNA, aiming for similar distances between helix backbone and both PNA strands (i.e. Aadin lieu of Glu, 2,3 diamino propionic acid (Dap) in lieu of Lys and adapting with Gly / PEG30spacer pairs. For example, the use of Aad on helix positions the N-ter of the PNA five atoms away for the alpha carbon of helix 1 and is best aligned with a Lys on helix 2 which position the C-ter of the complementary PNA 6 atom away from the alpha carbon of helix 2. If a Dap 21

[0175] was used in lieu of the of Lys, the C-ter of the PNA would only be 3 atoms away from the alpha carbon of helix. However and Dap-Gly linker would restore the six atom connectivity.

[0176]

[0177] The different combinations were ligated via T-NCL to obtain various target agent of the 5 invention such as of SEQ ID: 6.

[0178] The target agent of SEQ ID NO: 5 was found to have a comparable affinity (KD = 5.0 nM) tothe 2-helix ZHER,mut (5.8 nM) while the same sequence without the PNA moiety (i.e. SEQID NO: 3 with A13C mutation had a low affinity with a KD = 96 nM, suggesting that the PNAhybridization compensates for the lack of electrostatic interaction between E11 and K26 10 deriving from the cytein mutation.

[0179] Comparison between target agent of the invention such as of SEQ ID NO: 7 (KD = 3.3 nM)and 8 (KD = 4.7 nM) also indicates that PNA hybridization overcomes the detrimental effect ofthe A12C mutation, and there is no more penalty in affinity for the presence of the cysteine asobserved for the mutation A12C in SEQ ID 8 (SEQ ID NO: 7) compared to the effect of this15 mutation in absence of PNA moiety (KD = 19 nM). The best compound in this series (SEQ ID NO: 4) has an affinity of 2.1 nM and benefits of a M to Nle (Norleurcine) mutation in SEQID NO: 7 which was introduced to avoid possible methionine oxidation. Collectively, thesedata support the fact that hybridization of the PNA moieties constrains the conformation of the two helices into a conformation that recapitulates the Z domain.

[0180] 20 The contribution of the PNA moiety in the favorable alignment of the two helices is further evident from the fact that small distance variations in the anchoring moiety between peptidehelix and the PNA used to form the PNA group lead to substantial differences in affinity. Forexample, using a 5-atom and 6-atom PNA moiety, measured from the alpha carbon of helix 1and 2 and the N-ter and C-ter of the PNA, respectively, was found to yield a 10-fold25 improvement in affinity compared to the use of an 8-atom and 14-atom PNA moiety in theotherwise identical compound.

[0181] A comparison of reaction kinetics for the T-NCL with matching PNA moieties of the agent ofSEQ ID NO: 4 (GCCG and CGGC) vs the same agent but with mismatched PNA (GCCG andATTA) was carried out. The rate of the perfect match T-NCL performed at 100 μM is more 30 than 20 times faster (>80% conversion in 30 min) than the mismatched one at the same substrate 22

[0182] concentrations, supporting that the kinetic of the formation of the target agent of the invention is influenced by the matching between the PNA moieties of the helices.

[0183] The proteolytic stability of the target agent of the invention of SEQ ID NO:4 vs the native two-helix peptide as tested against trypsin as a representative digestive enzyme showed that the5 target agent of the invention of SEQ ID NO:4: exhibited a half-life greater than twice as longcompared to its non-stabilized congener.

[0184] The target agent of the invention of SEQ ID NO: 9 bearing a cyclohexylalanine at the originalMet5, features an affinity of 580 pM, with a dissociation half-life of 9.6 min (koff = 1.2 x 10-3s). This is comparable to values reported for the Fab of Herceptin (KD= 500 pM, koff= 4 x 10-10 4s).

[0185] A fluorescently labeled version of this compound, prepared as the parent compound but addinga Cy3 through regular amid coupling at the N-ter of the PNA of helix 2 (2.aΔGE-Cy3) (SEQID NO: 9) was found to strongly stain a Her2+ cell line (SK-BR3) but not a Her2- cell line(HeLa Kyoto).

[0186] 15 Example 2: Regulation of the affinity of target agents of the invention

[0187] The affinity of a target agent of the invention can be regulated on demand through displacementby competition. In order to illustrate this possibility, it was designed a target agent of theinvention of SEQ ID NO: 9 wherein the PNA moieties have been replaced as follows and asillustrated in Fig.3A:

[0188] 20 - On helix 1, the PNA moiety (4) comprised: a N-terminal non-hybridizing T (1) on thethioester fragment pairing PNA strand GCC (2) (for pairing with the PNA moiety ofhelix 2) and a 4 PNA overhang comprising a diamino purine (D) (3) and- On helix 2, the PNA moiety (7) comprised: a N-terminal SerSer spacer (5) to adjustespacing of unpaire PNA on the pairing PNA strand CGG (6) (for pairing with the PNA 25 moiety of helix 1).

[0189] Diamino purine (D) hybridizes with three hydrogen bonds to thymine and forms a strongerduplex (Haaima et al., 1997, Nuc. Acids Res. 25, 4639-4643https: / / doi.org:10.1093 / nar / 25.22.46). In the present design, PNA strand displacement will benefit from an additional T-D pairing of the PNA moiety of helix 1 and the displacing PNA30and the strand displacement equilibrium will be further favored by the presence of adiaminopurine in the PNA overhang. Indeed, the D–T pairing is stronger than A-T or G-C,helping with displacement. 23

[0190] A diamino purine containing PNA fragment was used as displacing PNA moiety Fig.3B) andthe addition of 2 equivalents of PNA of this fragment for strand displacement resulted allowedthe pairing of displacing PNA with the PNA moiety of helix 1 (4) and in a ca.40-fold loss of affinity (0.66 nM → 26 nM, Fig.3C) due to the unpairing of the PNAs from each helices. This5 data supports the fact that the activity of a target agent of the invention can be regulated on-demand through a toehold-mediated displacement mechanism.

[0191] 10Example 3: Synthesis of compounds of the invention targeting receptor-binding domain (RBD) of SARS-CoV-2’s spike protein

[0192] Design, combinatorial pairing and selection against spike’s RBD

[0193] To test whether the same strategy could be transposed to another three-helix miniprotein, thefollowing target agent was designed. LCB-1 is a computationally designed binder of the15 receptor-binding domain (RBD) of SARS-CoV-2’s spike protein (Cao et al., 2020, Science370, 426-431, https: / / doi.org:10.1126 / science.abd9909).

[0194] Truncation of the third helix from LCB-1 (Fig. 4), resulting in a 2-helix LCB-1 (residues 1-38) was previously shown to result in greatly reduced affinity towards RBD, while areengineered turn (LGHA→LaDB) resulted in better affinity in vitro and restored in vivo20efficacy due to peptide dimer formation that was sought to induce RBD receptor dimerization (Khatri et al., 2022, Nat. Chem. Biol.18, 1046, https: / / doi.org:10.1038 / s41589-022-01060-0). Two mutations on the non-binding interface (I12E and L31E) of this 2-helix peptide resultedin a monomeric state of the peptide and were well tolerated with respect to in vitro binding.Analysis of the structure of the two-helix LCB1 (Fig.4) revealed I12 and L31 to be at a 10Å25 distance, making them suitable positions to conjugate the PNA moieties. To assess the impactof point mutations, a truncated 2-helix LCB-1 (residues 1-38) and the corresponding I12E,L31E mutant (2-helix LCB1-2E) were synthesized. The affinities to RBD as measured by SPRwere 4.7 nM, and 1.9 nM, respectively.

[0195] Then, the impact of an M13C mutation required for the aimed T-NCL assembly of the two30 helices was tested in the I12E, L31E mutant and showed a dramatic loss of affinity (>1000nM), consistent with M13 being critical for either interhelical packing and / or interaction withthe protein (position a in the heptad, Fig.4, top). Based on the observation that PNA-enforcedfolding could compensate for unfavorable mutations in the coiled coil formation of the two- 24

[0196] helix peptides in Example 1 for HER2, the same synthetic strategy was used with 2-helix LCB- 1 with I12Aad and L31K mutations to conjugate the PNAs and M13C to enable T-NCL.SAPRBD-1 had an affinity of 27 nM, which compared to 2-helix LCB12EM13C, furthersupports the assertion that the PNA hybridization stabilizes the binding conformation and 5 overcomes much of the penalty of the M13C mutation.

[0197] Target agents of the invention were explored exhibiting affinities of 5.2 nM (SEQ ID NO: 23),and 70 pM (SEQ ID NO: 24), respectively. The latter target agents of the invention displayedan antiviral activity (2.8 nM) in a virus entry inhibition assay (Bekliz et al., 2022, Nat.Commun.13, https: / / doi.org:ARTN 384010.1038 / s41467-022-31556-1).

[0198] 10For comparison, in the same virus entry inhibition assay, LCB1 and the mutated truncated 2- helix LCB1-2Eshowed and IC50of 0.6 nM and 32.1 nM respectively.

[0199] Collectively, the data supports the beneficial role of the PNA moieties in structuring the coiled coil architecture affording a functional outcome close the three-helix bundle of the miniproteins. By comparison to clinically approved MAbs, tixagevimab (AZD8895) and15 cilgavimab (AZD1061) have an affinity of 2.8 and 13 pM respectively (Zost et al., 2020, Nature584, 443, https: / / doi.org:10.1038 / s41586-020-2548-6; Dockerill, et al., 2024, Nat. Biotechnol., https: / / doi.org:10.1038 / s41587-024-02209-z).

[0200] Examples 4: Synthesis of compounds of the invention with modified hinge region to enable ligation of both helices by CuAAC as exemplified with a compound targeting receptor-20binding domain (RBD) of SARS-CoV-2’s spike protein

[0201] To test wether other functionalities would be tolerated in the hinge region, the substitution ofan amide bond for a triazole was tested in a hinge sequence. Triazole are know known tosurrogate of amides (Horne, et al. 2004, J. Am. Chem. Soc., 126, 47, 15366–15367,https: / / doi.org / 10.1021 / ja0450408) and offer the possibility of copper-catalyzed-azide-alkyne 25 cycloaddition CuAAC (Tornoe et al. J. Org. Chem. 2002, 67, 9, 3057–3064, https: / / doi.org / 10.1021 / jo011148j) between the two helices. Using the same sequences as inExample 3 (SEQ ID NO: 24: DKEWILQKIY EXXRLLDELG HAEASMRVSD XIYEFMKK,hinge shown in bold – see a schematic representation on Fig.5). The peptides were preparedas decribed in Example 1 with substituting G in the sequence of the hinge for propargyl amine30and using N3-His(Tr)-OH as the as the N-term residue to prepare helix 2. Mixing the two helicesin water:DMSO (5:1) at 20 µM and copper (10 µM), tris(3-hydroxypropyltriazolylmethyl)amine (TBTA, 15 µM) and sodium ascorbate (200 µM) affordsthe desired product via a templated CuAAC (T-CuAAC) with complete consumption of the 25

[0202] starting material suggesting a near quantitative reaction. Affinity measurements by SPR andcomparison under the same conditions as used in Example 3 show an affinity of 20 pM for the compound with the triazole linkage instead of the amide (70 pM KD).

[0203] Examples 5: Synthesis of compounds for radionuclei labelling exemplified with the 5 synthesis of DOTA conjugates for targeting HER2-positive cells with radionuclei

[0204] A compound binding HER2-positive cells (SEQ ID NO: 9) was prepared by coupling DOTA(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid) at the C-term of the PNA linked tohelix 1. Specifically, the synthesis of the thioester fragment was initiated with Fmoc-Lys(Mtt)-OH and continued with the PNA sythtesis followed by and peptide synthesis as in examples 1.

[0205] 10Prior to the formation of the thioester, the Mtt group on the side chain of lysine was removedand the resulting amine was conjugated to DOTA as previously reported (Koustoulidou et al.,2022, Pharmaceuticals, 15(9), Article 1155, https: / / doi.org / 10.3390 / ph15091155). Theremaining synthetic steps follow the same workflow as in example 1. Complexation of gallium using the compound at 70 µM, GaCl3 at 100 µM in 0.1 N HCl at 90°C for 10 min resulted in 15 complete conversion to the gallium-complexed compound. Affinity measurement using the same conditions as used in Example 1 showed the same affinity of the compound-DOTA-Ga conjugate as the parent compound lacking DOTA. Given the known instability of peptidesequences containing an Asp-Pro combination and its presence in the hinge (DPNL, SEQ IDNO: 10), the same chemistry was performed to prepare the same compound with a variant of20the SEQ ID NO: 10 hinge sequence wherein the D is susbituted by a E. Both compoundsshowed comparable affinity by SPR (ca. 500 pM) but the compound with the hinge sequenceof SEQ ID NO: 10 showed 15% cleavage after 1h at 90°C, 1 N HCl, whereas the compoundwith the variant of the SEQ ID NO: 10 was found to be stable under the same conditions.Therefore, those data support that target agents of the invention can advantageously comprise 25 a labeling moiety such as dye or radio nuclei for imaging applications (ex. PET) orphotodynamic therapy or localized radiation therapy. Given that Her-2 is a well establishedbiomarker in cancer and a therapeutic taget, including of antibody-drug-conjugates, the tagetagents could be used for targeted delivery of radionucleic which for imaging and therapeutic benefits or chromophore for photodynamic therapy.

[0206] 30 Examples 6: Synthesis of compounds of the invention which target PD-L1

[0207] Using the same chemistry as in Example 1, the following target agents for PD-L1 were prepared: 26

[0208] YAKERNKRAYhE(PNA)CLYDPNLTNAQKWAK(PNA)IWKIDDEGE (SEQ ID NO: 27) and YAKERNKRAYhE(PNA)CAYDPNLTNAQKZAK(PNA)IWKIDDEGE (SEQ ID NO:28) wherein -Z stands for naphthylalanine) - were found to inhibit PD-L1 binding to PD-1 witha Ki of 72 nM and 94 nM respectively. These sequences comprise:

[0209] 5 - as helix 1 the following sequence: YAKERNKRAYhE(PNA)CL(or A)Y (SEQ ID NO:

[0210] 25);

[0211] - as helix 2 the following sequence: TNAQKW(or Z)AK(PNA)IWKIDDEGE SEQ IDNO: 26)

[0212] - SEQ ID NO: 10 as hinge;

[0213] 10 - As PNA moiety on the first helix: a Glutamic acid modified with a PNA moietyGCCGArg;

[0214] - As PNA moiety on the second helix: a Lysine modified with a PNA moiety CGGCArg;wherein the underlined residues are present on the helix face having a binding affinity to PD- L1 target.

[0215] 15 Example 7: Design of a compound targeting IL-23R

[0216] Using the workflow described in the description, the following target agent’ structure can be designed based on Berger et al., 2024, Cell, 187, 4305-4317 https: / / doi.org / 10.1016 / j.cell.2024.05.052:PLWQVFYhE(PNA)LNTCIKRT-triazol- HPTCKKK(PNA)AKALREELKKG which is obtained as described in Example 4 from the20CuAAC of the first helix where the C-term is a propargyl amide of threonine and the second helix below where the N-term is a histidine with an azide instead of the alpha-amino group for templated CuAAC (T-CuAAC). This sequence comprises:

[0217] - as helix 1 the following sequence: PLWQVFYhE(PNA)LNTCIKRT (SEQ ID NO: 29);- as helix 2 the following sequence: TCKKK(PNA)AKALREELKKG (SEQ ID NO: 30)25 - as hinge, the structure -triazol-HP; 27

[0218] -

[0219]

[0220] - as PNAmoiety on the first helix: a homoglutamic acidacid modified with a PNA moietyGCCGArg;

[0221] - as PNAmoiety on the second helix: a Lysine modified with a PNA moiety CGGCArg;5wherein the underlined residues are present on the helix face having a binding affinity to IL- R3 target.

[0222] The results demonstrate that affinity proteins composed of a three-helix bundle can be mimicked by target agents as self-assembled proteomimetics (SAPs) composed of PNA- peptide conjugates that are readily accessible by SPPS. The PNA facilitates the ligation of 10 fragments and protects against proteolytic digestion. In addition, the hybridization-enforced structuring of the target agents offers a simple mechanism to allosterically regulate its affinityby toehold mediated strand displacement as illustrated in Example 2. This may be important todevelop therapeutics targeting mechanism prone to adverse reactions. 28

[0223] LIST OF SEQUENCES SEQ ID NO: 1:

[0224] Artificial sequence ZHER2:342 - receptor tyrosine-protein kinase erbB-2 isoform b [Homo5 sapiens] (not from the invention) VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQA PK SEQ ID NO: 2:

[0225] Artificial sequence ZHer2:342,4-37 (not from the invention)

[0226] 10FNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDD

[0227] SEQ ID NO: 3:

[0228] Artificial sequence 2-helix ZHer2,mut FSKEMRNRYWEAALDPNLTNQQKRAKIRSIYEEGE SEQ ID NO: 4:

[0229] 15 Artificial sequence SAPHer2-5 FSKEXaa1RNRYWXaa2CALDPNLTNQQKRAXaa3IRSIYEEGE

[0230] Wherein Xaa1 is Nle (Norleucine), Xaa2 is aminoadipic acid modified with a PNA moietyGCCGArg and Xaa3 is a Lysine modified with a PNA moiety CGGCArg

[0231] SEQ ID NO: 5:

[0232] 20Artificial sequence SAPHer2-1 FSKEMRNRYWXaa1AALDPNLTNQQKRAXaa2IRSIYEEGE

[0233] wherein Xaa1 is Glutamic acid modified with a PNA moiety GCCGArg and Xaa2 is a Lysine modified with a PNA moiety CGGCArg

[0234] SEQ ID NO: 6:

[0235] 25 Artificial sequence SAPHer2-2 FSKEMRNRYWXaa1AALDPNLTNQQKRAXaa2IRSIYEEGE

[0236] Wherein Xaa1 is Glutamic acid modified with a PNA moiety Gly GCCGArg and Xaa2 is a is2,3-diaminopropionic acid (Dap) modified with a PNA moiety Peg+ CGGCArg .

[0237] 30 29

[0238] SEQ ID NO: 7:

[0239] Artificial sequence SAPHer2-4 FSKEMRNRYWXaa1CALDPNLTNQQKRAXaa2IRSIYEEGE

[0240] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg and Xaa2 is a Lysine5 modified with a PNA moiety CGGCArg

[0241] SEQ ID NO: 8:

[0242] Artificial sequence SAPHer2-6 FSKEMRNRYWXaa1AALDPNLTNQQKRAXaa2IRSIYEEGE

[0243] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg and Xaa2 is a Lysine10modified with a PNA moiety CGGCArg

[0244] SEQ ID NO: 9:

[0245] Artificial sequence 2.a ΔGE KEXaa1RNRYWXaa2CALDPNLTNQQKRAXaa3IRSIYEE

[0246] Wherein Xaa1 is a cyclohexylglycine, Xaa2 is aminoadipic acid modified with a PNA moiety15 of GCCG and Xaa3 is a Lysine modified with a PNA moiety CGGCArg

[0247] SEQ ID NO: 10

[0248] Artificial sequence hinge 1

[0249] DPNL SEQ ID NO: 11

[0250] 20Artificial sequence hinge 2

[0251] LPNL SEQ ID NO: 12

[0252] Artificial sequence hinge 3

[0253] LGHA

[0254] 25SEQ ID NO: 13

[0255] Artificial sequence, first consensus of first helix Xc111Xc112Xc113Xc114Xc115Xc116Xc117RXc119Xc1110Xc1111Xc1112AXc1114

[0256] wherein Xc111 is an hydrophobic natural or unnatural amino acid such as Phenylalanine or isabsent; Xc112 is selected from any natural or unnatural amino acid or is absent; Xc115, Xc116,30 Xc119and Xc1110are independently selected from any natural or unnatural amino acid; Xc113is a cationic natural or unnatural amino acid such as Lysine; Xc114 is an anionic natural or 30

[0257] unnatural amino acid such as glutamic acid; Xc117is a neutral natural or unnatural amino acidwith an hydrophilic side chain such as aspargine; Xc1111 is an amino acid with a functionalgroup on its side chain as a point of attachment to the PNA linking moiety such as a a glutamicacid where the side chain acid can be linked to the PNA by amide bond formation or an 5 aminoadipic acid which can be linked in the same fashion; Xc1112is any amino acid with a thiolgroup such as a cysteine or a homocysteine; Xc1114 are independently selected largehydrophobic natural or unnatural amino acids such as I, L, F and analogs thereof and whereinresidues Xc112, X 115, Xc116, X 119, Xc1110 (underlined) are present on the helix face having abinding affinity to a target of interest.

[0258] 10SEQ ID NO: 14

[0259] Artificial sequence, subspecific first helix

[0260] FXc112KEXc115Xc116NRYWXc1111CAL

[0261] wherein Xc112, Xc115, Xc116 and Xc1111 are as defined herein.

[0262] SEQ ID NO: 15

[0263] 15 Artificial sequence, second consensus first helix Xc211Xc212Xc213Xc214Xc215Xc216Xc217Xc218Xc219Xc2110Xc2111Xc2112Xc2113Xc2114Xc2115Xc2116Xc2117

[0264] Xc2118

[0265] Wherein Xc211 is an anionic natural or unnatural amino acid; Xc212 and Xc218 are independentlyselected cationic natural or unnatural amino acids; Xc213, Xc214, Xc216, Xc217, Xc2110, Xc2111, Xc2113,20 Xc2114, Xc2117 and Xc2118 are independently selected from any natural or unnatural amino acid;

[0266] Xc215, Xc219,Xc2115 andXc2116are independently selected large hydrophobic natural or unnaturalamino acids such as I, L, F and analogs thereof; Xc2112 is an amino acid with a functional groupon its side chain as a point of attachment to the PNA moiety such as a a glutamic acid wherethe side chain acid can be linked to the PNA by amide bond formation or an aminoadipic acid25 which can be linked in the same fashion; wherein amino acids Xc213, X214, Xc216, X217, Xc2110,Xc2111, Xc2113, Xc2114, , Xc2117 and Xc2118 (underlined) are present on the helix face having abinding affinity to a target of interest.

[0267] 30 31

[0268] SEQ ID NO: 16

[0269] Artificial sequence, subspecific 2 first helix DKXc213Xc214IXc216Xc217KIXc2110Xc2111Xc2112Xc2113Xc2114LLXc2117Xc2118 wherein Xc213, Xc114, Xc216, Xc2110Xc2111Xc2112Xc2113Xc2114, Xc2117 and Xc2118 are as defined 5 herein.

[0270] SEQ ID NO: 17

[0271] Artificial sequence, specific first helix, consensus 2

[0272] DKEWILQKIYEXc2112MRLLDE

[0273] wherein Xc2112is as defined herein.

[0274] 10SEQ ID NO: 18

[0275] Artificial sequence, first consensus second helix XC121XC122XC123XC124XC125XC126AXC128XC129XC1210XC1211XC1212XC1213XC1214XC1215wherein XC121, XC124 and XC1211 are independently selected neutral natural or unnatural aminoacids with heteroatoms such as threonine, asparagine, glutamine or variants thereof; XC122,15 XC123, XC125, XC126, XC129, XC1210 and XC1213 are independently selected from any natural orunnatural amino acid; Xc128 is an amino with a functional group on its side chain as a point ofattachment to the PNA moiety such as lysine (Lys) or diaminopropionic acid (Dap) where the side chain amino group is linked to the PNA via an amide bond; XC1212is a large hydrophobicnatural or unnatural amino acid such as I, L, F and analogs thereof; XC1214 and XC1215 is an20anionic natural or unnatural amino acid and wherein amino acids XC122, XC123, XC125, XC126,XC129, XC1210 and XC1213 (underlined) are present on the helix face having a binding affinity toa target of interest and wherein XC1214 and XC1215 can be independently present or absent.

[0276] SEQ ID NO: 19

[0277] Artificial sequence, subspecific second helix

[0278] 25 TNQQKRAXC128IRSIYEE

[0279] wherein XC128 is defined herein.

[0280] SEQ ID NO: 20

[0281] Artificial sequence, second consensus second helix

[0282] XC221AXC223XC224XC225XC226XC227XC228XC229XC2210XC2211XC2212XC2213Xc2214XC2215XC2216 32

[0283] wherein XC221, XC228, XC2212 are independently selected anionic natural or unnatural aminoacids XC223, XC224, XC227, XC2210, XC2211 and Xc2214 are independently selected from any naturalor unnatural amino acid; XC225, XC2215 and XC2216 are independently selected cationic natural or unnatural amino acids; XC226 is an hydrophobic residue natural or unnatural amino acid; XC2295 is an amino acid with a functional group on its side chain as a point of attachment to the PNAmoiety such as lysine (Lys) or diaminopropionic acid (Dap) where the side chain amino group is linked to the PNA via an amide bond; XC2213 is a large hydrophobic natural or unnaturalamino acid such as I, L, F and analogs thereof; and wherein amino acids XC223, XC224, XC227,XC2210, XC2211 and Xc2214 (underlined) are present on the helix face having a binding affinity to10a target of interest.

[0284] SEQ ID NO: 21

[0285] Artificial sequence, subspecific second helix EAXC223XC224RVXC227DXC229XC2210XC2211EFXc2214KK

[0286] wherein Xc223, Xc224, Xc227, Xc229, Xc2210, Xc2211, and Xc2214 are as defined herein.

[0287] 15 SEQ ID NO: 22

[0288] Artificial sequence, specific second helix

[0289] EASMRVSDXC229IYEFMKK

[0290] wherein Xc229is as defined herein.

[0291] SEQ ID NO: 23

[0292] 20 Artificial sequence, SAPRBD-4DKEWILQKIYEXaa1Xaa2RLLDELGHAEASMRVSDXaa3IYEFMKK

[0293] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg, Xaa2 is a homocysteine and Xaa3 is a Lysine modified with a PNA moiety CGGCArg

[0294] SEQ ID NO: 24

[0295] 25 Artificial sequence, SAPRBD-5 DKEWILQKIYEXaa1Xaa2RLLDELGHAEASMRVSDXaa3IYEFMKK

[0296] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg, Xaa2 is a S-methylated homocysteine Hcy(Me) and Xaa3 is a Lysine modified with a PNA moietyCGGCArg

[0297] 30 33

[0298] SEQ ID NO: 25

[0299] Artificial sequence, first helix

[0300] YAKERNKRAYXaaCXaa2Y

[0301] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg and Xaa2 is a 5 Leucine or an Alanine

[0302] SEQ ID NO: 26

[0303] Artificial sequence, second helix

[0304] TNAQKXaa1AXaa2IWKIDDEGE

[0305] Wherein Xaa1 is tryptophane or naphthylalanine; Xaa2 is a Lysine modified with a PNA moiety10GCCGArg

[0306] SEQ ID NO: 27

[0307] Artificial sequence which targets PD-L1 YAKERNKRAYXaa1CLYDPNLTNAQKWAXaa2IWKIDDEGE

[0308] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg; Xaa2 is a Lysine15 modified with a PNA moiety GCCGArg

[0309] SEQ ID NO: 28

[0310] Artificial sequence which targets PD-L1 YAKERNKRAYXaa1CAYDPNLTNAQKWAXaa2IWKIDDEGE

[0311] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg; Xaa2 is a Lysine20modified with a PNA moiety GCCGArg

[0312] SEQ ID NO: 29

[0313] Artificial sequence, first helix

[0314] PLWQVFYXaa1LNTCIKRT

[0315] Wherein Xaa1 is aminoadipic acid modified with a PNA moiety GCCGArg.

[0316] 25 SEQ ID NO: 30

[0317] Artificial sequence, second helix

[0318] TCKKKXaa1AKALREELKKG

[0319] Wherein Xaa1 is a Lysine modified with a PNA moiety GCCGArg

[0320] 30

Claims

34Claims1. A target agent comprising a coiled-coil arrangement consisting of two alpha heliceswherein:- the two alpha-helices are from about 11 to 25 amino acids in length;5 - the two alpha-helices are connected together from one of their ends through ahinge;- each alpha-helix has a helix face comprising a region essentially parallel to thealpha-helix axis having a binding affinity to a target;- each alpha-helix has on its face which is opposite to its face having a binding10affinity to the said target, at least one peptide nucleic acid (PNA) moiety connected to an amino acid of the said alpha-helix;wherein- the said at least one PNA moiety of the first alpha helix is connected to an aminoacid of the said first alpha-helix located within a distance of 6-15 amino-acid to 15 the hinge;- the said at least one PNA moiety of the second alpha helix is connected to anamino acid of the said second alpha-helix which is within a distance of about 8- 15Å from an amino acid of the first helix to which the PNA moiety of the firsthelix is connected to;20 - the said at least one PNA moieties of each helix comprise 3 to 20 atoms are atleast partially complementary to each other to be able to hybridize.

2. A compound to claim 1, wherein each alpha helix is from about 12 to 20 amino acidsin length.

3. A compound according to claim 1 or 2, wherein the hinge has a length of 6 to 20 atoms.25 4. A compound according to claim 3, wherein the hinge is selected from DPNL (SEQ ID:10) and LGHA (SEQ ID: 12) or a variant thereof, in particular where an amide bondis substituted by a triazole.

5. A compound according to any one of claims 1 to 4, wherein the first alpha helix has thefollowing sequence:30Xc111Xc112Xc113Xc114Xc115Xc116Xc117RXc119Xc1110Xc1111Xc1112AXc1114 (SEQ ID NO: 13, first consensus first helix),35wherein Xc111 is an hydrophobic natural or unnatural amino acid such as Phenylalanineor is absent; Xc112is selected from any natural or unnatural amino acid or is absent;Xc115, Xc116, Xc119 and Xc1110 are independently selected from any natural or unnaturalamino acid; Xc113 is a cationic natural or unnatural amino acid such as Lysine; Xc114 is 5 an anionic natural or unnatural amino acid such as glutamic acid; Xc117is a neutralnatural or unnatural amino acid with an hydrophilic side chain such as aspargine; Xc1111is an amino acid with a functional group on its side chain as a point of attachment to thePNA moiety such as a a glutamic acid where the side chain acid can be linked to the PNA by amide bond formation or an aminoadipic acid which can be linked in the same10fashion; Xc1112 is any amino acid with a thiol group such as a cysteine or ahomocysteine; Xc1114 are independently selected large hydrophobic natural or unnaturalamino acids such as I, L, F and analogs thereof and wherein residues Xc112, X 115, Xc116,X 119, Xc1110 (underlined) are present on the helix face having a binding affinity to atarget of interest.15 6. A compound according to claim 5, wherein the first alpha helix of SEQ ID NO: 13 hasthe following sequence:FXc112KEXc115Xc116NRYWXc1111CAL (SEQ ID NO: 14, subspecific first helix) wherein Xc112, Xc115, Xc116and Xc1111are as defined in claim 5, or is a homologous variant thereof.20 7. A compound according to any one of claims 1 to 4, wherein the first alpha helix has thefollowing sequence:Xc211Xc212Xc213Xc214Xc215Xc216Xc217Xc218Xc219Xc2110Xc2111Xc2112Xc2113Xc2114Xc2115Xc2116Xc2117 Xc2118 (SEQ ID NO: 15, second consensus first helix),Wherein Xc211 is an anionic natural or unnatural amino acid such as aspartic acid; Xc21225 and Xc218 are independingly selected cationic natural or unnatural amino acids such asLysine; Xc213, Xc214, Xc216, Xc217, Xc2110, Xc2111, Xc2113, Xc2114, Xc2117 and Xc2118 areindependently selected from any natural or unnatural amino acid; Xc215, Xc219, Xc2115 andXc2116are independently selected large hydrophobic natural or unnatural amino acidssuch as I, L, F and analogs thereof; Xc2112 is an amino acid with a functional group on30 its side chain as a point of attachment to the PNA moiety such as a a glutamic acidwhere the side chain acid can be linked to the PNA by amide bond formation or an aminoadipic acid which can be linked in the same fashion; wherein amino acids Xc213,36X214, Xc216, X217, Xc2110, Xc2111, Xc2113, Xc2114, , Xc2117 and Xc2118 (underlined) are presenton the helix face having a binding affinity to a target of interest.

8. A compound according to claim 7, the first alpha helix of SEQ ID NO: 15 has thefollowing sequence: DKEWILQKIYEXc2112MRLLDE (SEQ ID NO: 17, specific5 first helix, consensus 2), wherein Xc2112 is as defined in claim 7, or is a homologousvariant thereof.

9. A compound according to any one of claims 1 to 4, wherein the second alpha helix hasthe following sequenceXC121XC122XC123XC124XC125XC126AXC128XC129XC1210XC1211XC1212XC1213XC1214XC121510 (SEQ ID NO: 18 first consensus second helix),wherein XC121, XC124 and XC1211 are independently selected neutral natural or unnaturalamino acids with heteroatoms such as thronine, asparagine, glutamine or variantsthereof; XC122, XC123, XC125, XC126, XC129, XC1210 and XC1213 are independently selectedfrom any natural or unnatural amino acid; Xc128 is an amino with a functional group on15 its side chain as a point of attachment to the PNA moiety such as lysine (Lys) ordiaminopropionic acid (Dap) where the side chain amino group is linked to the PNAvia an amide bond; XC1212 is a large hydrophobic natural or unnatural amino acid suchas I, L, F and analogs thereof; XC1214and XC1215is an anionic natural or unnatural aminoacid, wherein amino acids XC122, XC123, XC125, XC126, XC129 , XC1210 and XC121320(underlined) are present on the helix face having a binding affinity to a target of interest and wherein XC1214 andXC1215can be independently present or absent.

10. A compound according to claim 10, wherein the second alpha helix of SEQ ID NO: 18has the following sequence:TNQQKRAXC128IRSIYEE (SEQ ID NO:19, subspecific second helix), wherein25 XC128 is defined in claim 10, or is an homologous variant thereof11. A compound according to any one of claims 1 to 4, wherein the second alpha helix hasthe following sequence :XC221AXC223XC224XC225XC226XC227XC228XC229XC2210XC2211XC2212XC2213Xc2214XC2215XC2216 (SEQ ID NO: 20 second consensus second helix),30 wherein XC221, XC228, XC2212 are independently selected anionic natural or unnaturalamino acids such as aspartic acid or glutamic acid; XC223, XC224, XC227, XC2210, XC221137and Xc2214 are independently selected from any natural or unnatural amino acid; XC225,XC2215and XC2216are independently selected cationic natural or unnatural amino acids such as arginine and lysine; XC226 is an hydrophobic residue natural or unnatural aminoacid; XC229 is an amino acid with a functional group on its side chain as a point of5 attachment to the PNA moiety such as lysine (Lys) or diaminopropionic acid (Dap) where the side chain amino group is linked to the PNA via an amide bond; XC2213 is alarge hydrophobic natural or unnatural amino acid such as I, L, F and analogs thereof;and wherein amino acids XC223, XC224, XC227, XC2210, XC2211 and Xc2214 (underlined) arepresent on the helix face having a binding affinity to a target of interest.10 12. A compound according to claim 11, wherein the second alpha helix of SEQ ID NO: 20has the following sequence:wherein Xc223, Xc224, Xc227, Xc229, Xc2210,Xc2211,and Xc2214are as defined in claim 13.EAXC223XC224RVXC227DXC229XC2210XC2211EFXc2214KK (SEQ ID NO: 21,subspecific second helix), wherein Xc223, Xc224, Xc227, Xc229, Xc2210, Xc2211, and Xc221415 are as defined in claim 13, or is a homologous variant thereof.

13. A compound according to claim 13 or 14, whetein the second alpha helix of SEQ IDNO: 13 has the following sequence:EASMRVSD XC229IYEFMKK (SEQ ID NO: 22, specific second helix, secondconsensus) wherein Xc229 is as defined in claim 13 or is an homologous variant20thereof.

14. A compound having a sequence selected from the following group: SEQ ID NO: 5,SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 24 and variants thereof.

15. A compound according to any one of claims 1 to 14 further comprising a labelingmoiety such as dye or radio nuclei for imaging application (ex. PET) or photodynamic 25 therapy or localized radiation therapy.

16. A compound according to any one of claims 1 to 15 for use as a medicament.

17. A pharmaceutical composition comprising at least one target compound according toany one of claims 1 to 15, as well pharmaceutically acceptable salts thereof and apharmaceutically acceptable carrier, diluent or excipient thereof.3818. A radiopharmaceutical composition comprising a radiolabelled compound according toclaim 15, as well pharmaceutically acceptable salts thereof and a pharmaceuticallyacceptable carrier, diluent or excipient thereof.

19. A pharmaceutical composition according to claim 17 or 18 further comprising at least5 one agent useful in immunotherapy and / or in the treatment of cancer or a viral infection.

20. A compound according to any one of claims 1 to 15 as well pharmaceutically acceptablesalts thereof for use as therapeutics and theragnostics.

21. A compound according to any one of claims 1 to 4 as well pharmaceutically acceptablesalts thereof for use as in the treatment of a cancer, in particular breast cancer or a viral10infection, in particular SARS infections such as SARS CoV2 infections.

22. A method for preventing in a subject suffering from a cancer, in particular breast canceror a viral infection, in particular SARS infections such as SARS CoV2 infections or at risk of suffering from such disorders, said method comprising administering a compound according to any one of claims 1 to 4 or a pharmaceutical formulation thereof 15 in a subject in need thereof.

23. A radiopharmaceutical composition according to claim 18 for use in imaging a targettissue or in radiation therapy.

24. A method of imaging a subject, said method comprising administering to the subject acompound of radiolabelled compound according to claim 15 or a radiopharmaceutical20 composition according to claim 18, and imaging the said subject.

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