Radiosensitising peptide complex

Abstract

The present invention relates to a supramolecular peptide complex comprising several peptide oligomerisation domains capable of forming multivalent complexes, bonded to at least one antigen-binding molecule and to at least one chelating group binding at least one metal cation of a heavy metal (M) having an oxidation state (M2+, M3+, M4+).

Description

[0001] Radiosensitizing peptide complex

[0002] The present invention relates to novel supramolecular peptide complexes intended to increase the effectiveness of radiotherapy. More specifically, the invention relates to the use of supramolecular peptide complexes as radiosensitizers.

[0003] In the ever-evolving field of cancer treatment, radiotherapy remains at the forefront of cancer treatment. As a result, significant work has been done to improve the therapeutic window of external beam radiotherapy.

[0004] Radiosensitizing agents have emerged as a promising avenue to locally enhance the therapeutic impact of radiation in tumor cells. These radiosensitizers rely on manipulating the physical interactions between ionizing radiation, particularly X-rays, and radiosensitizing agents comprising heavy metals. This approach involves the use of high atomic number (Z) metals, which have the ability to enhance electron production through photoelectric effects and Auger electron emission when exposed to radiation. This amplified number of electrons, in turn, generates secondary electrons through physical processes, including Compton scattering and photoelectric absorption, ultimately leading to the production of reactive oxygen species (ROS).This amplification of ionization leads to radiation-induced damage directly to cancer cells when a radiosensitizing agent is present in a tumor. Overall, these radiosensitizers significantly improve the therapeutic ratio of radiotherapy, making it a compelling area of ​​research in the quest to improve the impact of cancer treatment. Notable developments in radiosensitizers include Nanobiotix's hafnium oxide nanoparticles (WO2014114732) and NH Theraguix's silica-based gadolinium nanoparticles (WO2021019268), both currently undergoing clinical evaluation and which have shown promise in increasing the radiation dose delivered to patients.

[0005] Yet, the desire to explore more complex radiosensitizing agents is increasing, for example, by combining various high-atomic number metals, such as gadolinium and bismuth in metallic form, to exploit the complementary advantages they offer. Nevertheless, previous Monte Carlo simulation studies have demonstrated that localized dose escalation is directly related to the number of nanoparticles internalized per cell and not to the atomic number of the metal composing the nanoparticle. However, such a result is difficult to validate biologically, as complex synthesis processes and potential interference between multiple metal components have posed challenges to directly compare the radiosensitization properties of different metals within the same nanoparticle framework.

[0006] This application proposes a novel approach to fill this gap in the understanding of radiosensitization.

[0007] According to the present invention, supramolecular heterodimeric peptides functionalized on the one hand with molecules binding to at least one antigen to improve tumor targeting, and on the other hand with heavy metals are therefore advantageously used as radiosensitizing agents in radiotherapy treatment.

[0008] Summary of the invention

[0009] According to a first aspect, the invention relates to a supramolecular peptide complex comprising several peptide oligomerization domains capable of forming multivalent complexes, linked to at least one antigen-binding molecule (i.e. an antibody or an antibody fragment) and to at least one chelating group binding at least one metal cation of a heavy metal (M) in the form (M 2+ , M 3+ , M 4+ ).

[0010] According to a second aspect, the invention relates to a composition comprising a plurality of supramolecular peptide complexes as detailed herein, and a pharmaceutically acceptable carrier.

[0011] In a third aspect, the invention relates to a composition comprising a plurality of supramolecular peptide complexes as detailed herein as a radiosensitizing agent and a composition comprising an effective amount of supramolecular peptide complexes as detailed herein for use in the treatment of a tumor or as a contrast agent. Detailed description of the invention

[0012] According to a first aspect, the invention relates to a supramolecular tetrameric peptide complex comprising:

[0013] (a) a first structure represented by the following formula (I):

[0014] (I) (A)n-(L1)x-P1-(L2)x-(A')n' preferably A-L1-P1-L2 or A-L1- 1 , in which

[0015] A and A are an antibody or antibody fragment,

[0016] P1 is a first peptide comprising at least one oligomerization domain, preferably tetramerization domain,

[0017] L1 and L2 are spacers that independently represent a carbon chain or a peptide, with n being an integer of 1 or 2, and n' being an integer of 0 or 1 or 2 x and x' independently represent a number of 0 or 1,

[0018] (b) a second structure represented by the following formula (II):

[0019] (i) P2-L3-[B@(M)] m in which

[0020] B is a chelating group binding at least one metal cation of a heavy metal (M) having an oxidation state (M 2+ , M 3+ , M 4+ ) having an atomic number Z greater than or equal to 40, for example an atomic number Z between 40 and 213.

[0021] P2 is a second peptide comprising at least one oligomerization domain, preferably tetramerization domain,

[0022] L3 is a spacer, which preferably comprises a group selected from a [1,2,3]-triazole ring, isoxazole, isoxazoline, oxadiazoles, a pyrazole, a dihydropyrazine, an amide, a maleimide, a hydrazone and an oxime, with m being an integer of 1, 2, 3 or 4, preferably 1 or 2 @ represents a non-covalent type bond between the chelating group and the metal cation of a heavy metal (M) having an oxidation state (M 2+ , M 3+ , M 4+ ) wherein the first and second peptides P1 and P2 associate and form primary homodimers and / or primary heterodimers, which associate and form “dimers of dimers”.

[0023] According to a general aspect of the invention, the first and second peptides P1 and P2 may be any peptide sequence capable of associating into stable supramolecular multivalent complexes such as tetrameric complexes, dimer of dimers.

[0024] 1. Peptide comprising at least one oligomerization domain

[0025] Examples of peptides containing oligomerization domains that may be used in the present invention include, but are not limited to, the tetramerization domain of p53, p63, p73, the pentameric domains of bacterial protein toxins such as cholera toxin, or Shiga enterotoxin.

[0026] According to the present invention, the supramolecular tetrameric peptide complexes comprise oligomerization peptide domains which are not all identical insofar as the invention relates to heteromultimeric supramolecular complexes which are tetramers of heterodimers, consisting either of a homodimer of a first peptide associated with a homodimer of a second peptide, or of two heterodimers consisting of the first and second peptides; said peptides each comprising at least one oligomerization domain, preferably a tetramerization domain.

[0027] According to the present invention, the supramolecular tetrameric peptide complex is a "dimer of dimers" consisting of four peptides comprising at least one oligomerization domain.

[0028] According to a particular embodiment of the invention, the first and second peptides P1 and P2, comprising at least one oligomerization domain, combine to form homodimers, respectively a primary dimer P1 @@ P1 and a primary dimer P2 @@ P2.

[0029] According to another particular embodiment of the invention, the first and second peptides P1 and P2 each comprising at least one oligomerization domain, preferably tetramerization domain, combine to form a primary heterodimer P1 @@ P2. According to a particular embodiment of the invention, two primary dimers combine to form one or more “dimer(s) of dimers” of formulae:

[0030] @@ represents non-covalent bonds between the α-helical tetramerization domains of peptides P1 and P2.

[0031] According to the invention, “dimers of dimers” correspond to supramolecular peptide complexes of the “tetramer heterodimer” type.

[0032] According to the invention, an oligomerization domain is a peptide domain which allows association into a primary dimer then into dimers of dimers corresponding to a quaternary structure.

[0033] According to a particular embodiment of the invention, the oligomerization domains of peptides P1 and P2 comprise a p-sheet-like structure followed by an α-helix. The supramolecular tetrameric peptide complex of the invention is a symmetrical dimer of primary dimers formed by helix-helix contacts. The tetramer is stabilized not only by hydrophobic interactions but also by a number of salt bridges, involving basic and acidic amino acid residues such as lysine and glutamic acid.

[0034] According to a particular embodiment of the invention, the conjugation of the oligomerization domains of peptides P1 and P2 to molecules of varied molecular masses and activities, such as an antibody or an antibody fragment such as a VHH or chelating groups binding a heavy metal (M) does not impact the modularity and the efficiency of the heteromerization of said peptides P1 and P2.

[0035] In a particular embodiment, peptides P1 and P2 comprise a tetramerization domain from a protein such as p53, p63 or p73.

[0036] Preferably, peptides P1 and P2 comprise a tetramerization domain chosen from the group consisting of peptide sequences comprising: - the tetramerization domain of the human p53 protein (SEQ ID NO: 1)

[0037] GEYFTLQIRGRERFEMFRELNEALELKDAQA

[0038] - the tetramerization domain of the mutant protein p53E343K (SEQ ID NO: 2)

[0039] GEYFTLQIRGRERFEMFRKLNEALELKDAQA

[0040] - the tetramerization domain of the mutant protein p53E346K (SEQ ID NO: 3)

[0041] GEYFTLQIRGRERFEMFRELNKALELKDAQA

[0042] - the tetramerization domain of the mutant protein p53E343K / E346K (SEQ ID NO: 4)

[0043] GEYFTLQIRGRERFEMFRKLNKALELKDAQA

[0044] - the tetramerization domain of the mutant protein p53K351 E (SEQ ID NO: 5)

[0045] GEYFTLQIRGRERFEMFRELNEALELEDAQA

[0046] - the tetramerization domain of p63 (SEQ ID NO: 6);

[0047] DELLYLPVRGRETYEMLLKIKESLELMQYLP

[0048] - the tetramerization domain of p73 (SEQ ID NO: 7);

[0049] EDTYYLQVRGRENFEILMKLKESLELMELVP

[0050] - the tetramerization domain of the Xenopus laevis p53 protein (SEQ ID NO:

[0051] 8);

[0052] EEIFTLRIKGRSRYEMIKKLNDALELQESLD

[0053] - the tetramerization domain of the rainbow trout p53 protein (SEQ ID NO:

[0054] 9)

[0055] DEIYTLQIRGKEKYEMLKKFNDSLELSELVP

[0056] The invention also includes mimetic (or peptidomimetic) peptides, analogs, or homologs of SEQ ID NO: 1 to 9, which associate and form primary homodimers and / or primary heterodimers, which associate and form "dimers of dimers".

[0057] According to the invention, peptides P1 and P2 are identical or different, provided that they associate and form a stable supramolecular tetrameric peptide complex. In a particular embodiment, peptides P1 and P2 are identical, that is to say that their sequences correspond to the same tetramerization domain among the sequences SEQ ID NO: 1 to 9 or their mimetic (or peptidomimetic), analogous, or homologous peptides.

[0058] In a particular embodiment, the peptides P1 and P2 are identical and correspond to the sequence SEQ ID NO: 1 or their mimetic (or peptidomimetic), analogous, or homologous peptides.

[0059] The structure of multivalent peptide assemblies, e.g., the p53 tetramer, has been well studied using X-ray crystallography and NMR techniques. As a result, a person skilled in the art is able to design peptide mimetics, analogs, homologues, or derivatives that would not affect (and might even enhance) the formation of a stable heteromultimeric complex. For example, each monomer (i.e., each P1 or P2 peptide comprising a tetramerization domain) in the context of the human p53 tetrameric domain (p53tet) adopts an identical structure, namely an N-terminal p-sheet (residues 326-333) followed by a C-terminal α-helix (residues 335-354).Two peptides (monomers) associate in an antiparallel manner via p-sheet contacts as well as hydrophobic interactions involving residues of an α-helix to form a “primary dimer” (4 Stable Human p53 Heterotetramer Based on Constructive Charge Interactions within the Tetramerization Domain; Richard D. Brokx et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 4, Issue of January 24, pp. 2327-2332, 2003).

[0060] Two primary dimers then associate via an interface derived from residues located in their α-helical domains to form a 'dimer of dimers', called a p53 tetramer. Amino acid mutations located at this interface have highlighted the importance of hydrophobic residues leading to tetramer formation as well as stable p53 dimers. For example, Davison et al. {Characterization of the oligomerization defects of two p53 mutants found in families with Li-Fraumeni and Li-Fraumeni-like syndrome', Oncogene (1998) showed that the substitution of proline for lysine at position 344 abolishes its tetramer-forming activity.

[0061] Furthermore, point mutation studies in the p53 tetramerization domain sequences suggest that the presence of salt bridges involving Lys351 and Glu343 and / or Glu346 contributes to four or more ionic interactions located at the interface of the p53 tetrameric complex promoting stabilization and self-association of primary dimers.

[0062] Thus according to the present invention, sequences of the tetramerization domain of p53, p63 and p73 may encompass such substitutions resulting in the preservation or strengthening of residue interactions which are essential in the proper formation of the tetrameric complex of peptides each comprising at least one oligomerization, preferably tetramerization, domain.

[0063] The term “analogous,” as used herein, means a peptide having an amino acid sequence of a reference sequence except for one or more amino acid substitutions, insertions, and / or deletions. The amino acid substitutions may be conserved or non-conserved in nature. Amino acid residues that are “conservative variants” or “conservative substitutions” for corresponding residues in a reference sequence are those that are physically or functionally similar to the corresponding reference residues, e.g., that have similar size, shape, electrical charge, hydrophobicity, hydrophilicity, polarity, reactive chemical properties, including the ability to form covalent or hydrogen bonds, and other properties. Particularly advantageous conservative variants are those that meet the criteria defined for an “accepted point mutation.”Conservative amino acid variants usually include substitutions in the following groups: I. glycine, alanine, valine, isoleucine, leucine II. aspartic acid, glutamic acid, asparagine, glutamine III. serine, threonine IV. lysine arginine V. phenylalanine, tyrosine.

[0064] In certain aspects of the present disclosure or embodiments of the present invention, the domain, peptide or peptidomimetic of the present invention comprises the domains, peptides or peptidomimetics with modified sequences containing substitutions of functionally equivalent amino acid residues, relative to the domains, peptides or peptidomimetics mentioned above. For example, one or more amino acid residues within the sequence may be replaced by another amino acid of similar polarity (having similar physicochemical properties) that acts as a functional equivalent, resulting in a silent alteration. The substitution of an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.For example, positively charged (basic) amino acids include arginine, lysine, and histidine (as well as homoarginine and ornithine). Nonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophan, and methionine. Uncharged polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Negatively charged (acidic) amino acids include glutamic acid and aspartic acid. The amino acid glycine can be included in either the nonpolar amino acid family or the uncharged polar (neutral) amino acid family. Substitutions made within an amino acid family are generally considered conservative substitutions.

[0065] The term "homologous" as used herein means a peptide having an amino acid sequence having at least 70%, preferably 80-90%, even more preferably 95-99% identity to a reference amino acid sequence, i.e., the sequences of p53, p63 or p73.

[0066] The expression "95 to 99% identity" means "95, 96, 97, 98, or 99% identity."

[0067] Analysis of individual amino acid contributions to the stabilization of the helical interface of tetramers shows a cluster of hydrophobic residues (M340, L344, A347, L348, and L350) that stabilize all architectures, while the contributions of charged amino acids vary significantly between supramolecular complexes. Energetic data reveal that mutations favor the formation of heterotetramers over (K3)4 or (E3)4 homotetramers (Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells, Marc Vigneron et al., Bioconjugate Chemistry 2019 30 (6), 1734-1744).

[0068] According to the present invention, the homologous and / or analogous peptides of the sequences of P1 and P2, preferably p53, p63 or p73, retain the amino acids which contribute to the stabilization of their helical interface and to their ionic interactions.

[0069] According to an advantageous embodiment of the invention, the first and second peptides P1 and P2 are different and represent either the mutant p53E343K / E346K (SEQ ID NO: 4), or the mutant p53E351 K (SEQ ID NO: 5), or mimetic peptides, analogs, or homologues thereof, and associate into one or more heterodimeric tetramers. According to an advantageous embodiment of the invention, the first and second peptides P1 and P2 are different and represent either the mutant p53E343K / E346K (SEQ ID NO: 4), or the mutant p53E351 K (SEQ ID NO: 5), or mimetic peptides, analogs, or homologues thereof. P1 and P2 associate and form primary homodimers and / or primary heterodimers, which associate and form "dimers of dimers" i.e. heterodimeric tetramers of formulas (III):

[0070] @@ represents non-covalent bonds between the α-helical tetramerization domains of peptides P1 and P2.

[0071] According to an advantageous embodiment of the invention, the first structure of the supramolecular tetrameric peptide complex is represented by the following formulas:

[0072] A-L1- (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-L2 (h) preferably A-L1- (SEQ ID NO: 4 or SEQ ID NO: 5)-L2.

[0073] According to an advantageous embodiment of the invention, the second structure of the supramolecular tetrameric peptide complex is represented by the following formulas:

[0074] (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9) - L3-[B@(M)] m(Ih) preferably (SEQ ID NO: 4 or 5)-L3-[B@(M)] m or (SEQ ID NO: 4 or SEQ ID NO: 5)-L3-B@(M), or mimetic peptides, analogs, or homologues thereof According to another advantageous embodiment of the invention, the first structure and the second structure of the supramolecular tetrameric peptide complex are represented by the following formulas:

[0075] (h) A-L1- (SEQ ID NO: 4)-L2 and

[0076] (Ih) (SEQ ID NO: 5)-L3-[B@(M)] m Or

[0077] (SEQ ID NO: 5)-L3-B@(M) or

[0078] (h) A-L1- (SEQ ID NO: 5)-L2 and

[0079] (Ih) (SEQ ID NO: 4)-L3-[B@(M)] m Or

[0080] (SEQ ID NO: 4)-L3-B@(M) or mimetic peptides, analogs, or homologues thereof.

[0081] 2. Antibodies and antibody fragments

[0082] According to the present invention, the term "antibody" refers to immunoglobulin molecules, i.e. molecules which contain at least one antigen-binding site (i.e. epitope) and bind immunospecifically to said antigen.

[0083] As such, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments as well as antibody variants (including derivatives) and antibody fragments.

[0084] The antibody can be a monoclonal or polyclonal antibody.

[0085] The term "monoclonal antibody" according to the present invention refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies constituting the population bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody, these variants being generally present in minor amounts. Unlike polyclonal antibody preparations which generally comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant of the antigen.

[0086] The term "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.

[0087] Specific examples of monoclonal antibodies herein include chimeric antibodies, humanized antibodies, and human antibodies.

[0088] According to the present invention, "chimeric" monoclonal antibodies are antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, provided that they exhibit the desired biological activity.

[0089] According to the present invention, the "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin.

[0090] For the most part, humanized antibodies are human immunoglobulins in which residues from a hypervariable region of the recipient are replaced with residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity.

[0091] In some cases, residues in the framework region (FR) of human immunoglobulin are replaced with corresponding non-human residues.

[0092] Additionally, humanized antibodies may include residues that are not found in either the recipient antibody or the donor antibody.

[0093] In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for the FR substitution(s) as indicated above.

[0094] The humanized antibody may optionally comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin.

[0095] An example of a humanized antibody is trastuzumab.

[0096] According to the present invention, a "complete antibody" is an antibody that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3.

[0097] The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.

[0098] As described in more detail below, the amino acid sequence and structure of an antibody heavy chain variable domain may be considered, but is not limited to, four framework regions or "FRs," which are referred to in the art and hereinafter as "framework region 1" or "FRV"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4," respectively, which framework regions are interrupted by three complementarity determining regions or "CDRs," which are referred to in the art as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3," respectively.

[0099] As used herein, the terms "complementarity determining region" or "CDR" in the context of antibodies refer to variable regions of the H (heavy) or L (light) chains (also abbreviated as VH and VL, respectively) and contain the amino acid sequences capable of specifically binding to antigenic targets.

[0100] These CDR regions represent the fundamental specificity of the antibody for a particular antigenic determinant structure.

[0101] Such regions are also called "hypervariable regions."

[0102] "CDRs represent non-contiguous stretches of amino acids in the variable regions, but regardless of species, the positional locations of these critical amino acid sequences in the variable regions of the heavy and light chains were found to have similar locations in the amino acid sequences of the variable chains.

[0103] The variable heavy and light chains of all canonical antibodies each have 3 CDR regions, each non-contiguous with the others (called L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy (H) chains.

[0104] The amino acid residues of a variable domain of an antibody heavy chain variable domain (including a VHH or VH) are numbered according to the general numbering of heavy chain variable domains given by Kabat et al. (Sequence of Proteins of Immunological Interest, US Public Health Services, NI H Bethesda, Md., Publication No. 91), as applied to the camelid VHH domains in the article by Riechmann and Muyldermans.

[0105] According to this numbering, FR1 of a heavy chain variable domain comprises the amino acid residues at positions 1 to 30, CDR1 of a heavy chain variable domain comprises the amino acid residues at positions 31 to 35, FR2 of a heavy chain variable domain comprises the amino acid residues at positions 36-49, CDR2 of a heavy chain variable domain comprises the amino acid residues at positions 50-65, FR3 of a heavy chain variable domain comprises the amino acid residues at positions 66-94, CDR3 of a heavy chain variable The heavy chain variable domain comprises the amino acid residues at positions 95-102, and FR4 of a heavy chain variable domain comprises the amino acid residues at positions 103-1-13.

[0106] In this regard, it should be noted that—as is well known in the art for VHH domains—the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.Generally speaking, however, it can be said that, according to Kabat numbering and regardless of the number of amino acid residues in the CDRs, position 1 according to Kabat numbering corresponds to the beginning of FR1 and vice versa, position 36 according to Kabat numbering corresponds to the beginning of FR2 and vice versa, position 66 according to Kabat numbering corresponds to the beginning of FR3 and vice versa, and position 103 according to Kabat numbering corresponds to the beginning of FR4 and vice versa.

[0107] Preferably, the complete antibody has one or more effector functions.

[0108] Rituximab and trastuzumab are examples of complete antibodies.

[0109] According to the present invention, an "antibody fragment" comprises a portion of a complete antibody, comprising the antigen-binding and / or variable region of the complete antibody.

[0110] Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-domain antibodies.

[0111] F(ab')2, Fab, Fab', and Fv are antigen-binding fragments that can be generated from the variable region of IgG and IgM. F(ab')2 fragments contain two antigen-binding regions joined at the hinge by disulfides.

[0112] F(ab')2 fragments generally lack most, but not all, of the Fc region.

[0113] Fab' fragments can be formed by the reduction of F(ab')2 fragments.

[0114] Fab' fragments contain a free sulfhydryl group that can be alkylated or used in conjugation with an enzyme, toxin, or other protein of interest.

[0115] Fab' fragments are derived from F(ab')2; therefore, they may contain a small portion of Fc. Fab is a monovalent fragment produced from IgG and IgM, consisting of the VH, CH1 and VL, CL regions, linked by an intramolecular disulfide bond.

[0116] Fv fragments refer to the smallest fragments produced from IgG and IgM that contain a complete antigen-binding site.

[0117] Fv fragments have the same binding properties and similar three-dimensional binding characteristics as Fab.

[0118] The VH and VL chains of Fv fragments are held together by non-covalent interactions.

[0119] scFv fragments (single-chain variable fragments) consist of only the variable domains of the VH and VL heavy and light (L) chains of antibodies, linked together by a flexible peptide linker. A "single-domain antibody," also known as a "nanobody," is a monomeric fragment of an antibody that binds to an antigen.

[0120] Camelid antibodies (called VHH) can be generated from conventional IgG by obtaining or modifying monomeric and stable VH or VL domains.

[0121] Generally, it should be noted that the term "heavy chain variable domain," as used herein in its broadest sense, is not limited to a specific biological source or a specific preparation method.

[0122] For example, as will be discussed in more detail below, heavy chain antibody-derived heavy chain variable domains (i.e., VHH) as described herein may be obtained by (1) isolating the VHH domain from a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) “camelizing” a naturally occurring VH domain from any animal species, particularly a mammalian species, such as a human, or expressing a nucleic acid encoding such a camelized VH domain; (4) “camelizing” an “antibody domain” or “Dab” as described by Ward et al., or by expressing a nucleic acid encoding such a camellated VH domain (5) using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (6) by preparing a nucleic acid encoding a VHH using nucleic acid synthesis techniques, followed by expression of the resulting nucleic acid; and / or (7) by any combination of the foregoing.

[0123] As used herein, the term "monovalent" when referring to an antibody fragment, such as a VHH or functional fragments thereof, means an antibody fragment in monomeric form.

[0124] A monovalent antibody fragment contains only one binding site.

[0125] In this context, the binding site of an antibody fragment, such as a VHH or functional fragments thereof, encompasses one or more "complementarity determining regions" or "CDRs" of an antibody fragment that are directed against or specifically bind to a particular site, determining region, portion, domain, or stretch of amino acid residues of a target of interest. As used herein, the term "unlabeled" when referring to an antibody fragment, such as a VHH or functional fragments thereof, means an antibody fragment that does not contain any foreign polypeptide sequence (e.g., contains only a VHH sequence, or a fragment thereof).

[0126] Examples of foreign polypeptide sequences include carboxy-terminal polypeptide tags, e.g., a His tag, a cysteine-containing tag (e.g., a GGC tag), and / or a Myc tag.

[0127] The term "bispecific" when referring to an amino acid sequence, in particular an antibody fragment, such as a VHH, as described herein implies that either a) two or more of the binding sites of an amino acid sequence as described herein are directed against or bind specifically to the same target of interest but not to the same (i.e., different) site, determinant, part, domain or stretch of amino acid residues of that target, the amino acid sequence as described herein is said to be "bispecific" (in the case of two binding sites on the amino acid sequence) or multispecific (in the case of more than two binding sites on the amino acid sequence) or b) two or more binding sites of an amino acid sequence as described herein are directed against or bind specifically to different target molecules of interest.

[0128] The term "multispecific" is used when more than two binding sites are present on the amino acid sequence as described herein.

[0129] Accordingly, a "bispecific" amino acid sequence or antibody fragment, such as a "bispecific" VHH or a "multi-specific" amino acid sequence or antibody fragment, such as a "multispecific" VHH as used herein, will have the meaning of an amino acid sequence, in particular an antibody fragment, such as a VHH, as described herein, comprising respectively two or at least two binding sites, these two or more binding sites having a different binding specificity.

[0130] Thus, an amino acid sequence, particularly an antibody fragment, such as a VHH, as described herein is considered "bispecific" or "multispecific" if respectively two or more different binding regions exist in the same monomeric amino acid sequence.

[0131] The total number of amino acid residues in a heavy chain variable domain of an antibody (including a VHH or VH) may be between 110 and 130, is preferably between 112 and 115, and is most preferably 113. It should be noted, however, that parts, fragments or analogs of a heavy chain variable domain of an antibody are not particularly limited in length and / or size, as long as such parts, fragments or analogs retain (at least part of) the functional activity, and / or retain (at least part of) the binding specificity of the original heavy chain variable domain of an antibody from which such parts, fragments or analogs are derived.

[0132] Parts, fragments or analogues retaining (at least part of) the functional activity and / or retaining (at least part of) the binding specificity of the original heavy chain variable domain of an antibody from which such parts, fragments or analogues are derived are also referred to herein as "functional fragments" of a heavy chain variable domain.

[0133] The antibodies or antibody fragments are prepared by conventional methods known to those skilled in the art, by immunizing a mammal, a llama or a camel for the production of VHH.

[0134] For example, the human antigen protein Her2 is used to immunize a llama, to obtain a high-quality nanobody gene library. The antigens in such a configuration were used to screen the nanobody gene library (VHH) using phage display technology (phage display of a heavy chain antibody gene library), thus obtaining nanobody genes (VHH) with Her2 specificity. Then, the genes are transferred into E. coli, thus obtaining strains that can be efficiently expressed in E. coli with high specificity.

[0135] The antibody may be operatively linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecules to increase the half-life or stability or otherwise enhance the antibody.

[0136] For example, the antibody may be linked to one of a variety of non-protein polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0137] According to an advantageous embodiment of the invention, the first structure of the supramolecular tetrameric peptide complex corresponds to formula (I) where n and n' are 1. The antibodies or antibody fragments A and A' are identical or different. According to another advantageous embodiment of the invention, the first structure of the supramolecular tetrameric peptide complex corresponds to formula (I) where n is 1 and r is 0.

[0138] According to an advantageous embodiment of the invention, the antibody or antibody fragment is chosen from a monoclonal antibody, a VHH, or a scFV, preferably a VHH.

[0139] According to an advantageous embodiment of the invention, the antibody or antibody fragment is directed against at least one antigen chosen from: the antigens of the cluster of differentiation (CD), the identification number of which varies between CD1a and CD363 and is preferably chosen from CD3, CD4, CD13, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD30, CD31, CD33, CD34, CD37, CD38, CD39, CD40, CD44, CD47, CD52, CD56, CD66e, CD70, CD72, CD73, CD74, CD79, CD79b, CD80, CD86, CD117, CD138, CD194, CD205, CD227 or CD248, even more preferably CD3, CD16, CD28 and CD38, or

[0140] 4-1 BB, 5AC, 5T4 (trophoblastic glycoprotein, TPBG, 5T4, Wnt-activated inhibitory factor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, Alpha intergrin, Alpha v beta6, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, AOC3 (VAP-1), B7-H3, BAFF (B-cell activating factor), BCMA, B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, CCL11 (CC motif chemokine 11), CCR4 (CC chemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA (Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), dumping factor A, cMet, CRIPTO, FCSF1 R (Colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CSP4,CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184), chemokine CXC receptor type 4, cyclic ADP ribose hydrolase, cyclin B1, CYP1 B1, DLL3 (delta-like-ligand 3), DLL4 (delta-like-ligand 4), DPP4 (dipeptidyl-peptidase 4), DR5 (TRAIL-R2), ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD105), Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 fusion gene) ETS), ETV6-AML, FAP (Fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrin II, beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen, Frizzled receptor, Fucosyl GM1, Ganglioside GD2, G-28, GD3, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor a-chain,growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C, guanylyl cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase-C receptor, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), lgG4, HGF / SF (hepatocyte growth factor / scattering factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), Idiotype, IGF1 R (IGF-1, insulin-like growth factor receptor 1) ), IGHE, IFN-y, IL-1 , IL-2 receptor (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL- 9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL -23, IL31 RA, ILGF2 (insulin-like growth factor 2), integrins (a4, allbp3, aVp3, a4p7, ct5[31 , a6p4, a5[35, av[35),interferon gamma-induced protein, ITGA2, ITGB2, , Legumain, LFA-1 (lymphocyte function-associated antigen 1, RH, LINGO-1 , LMP2, LTA, MAD-CT-1 , MAD -CT-2, MAGE-1 , MAGE-2, GE A1, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (macrophage migration inhibitory factor or glycosylation inhibitory factor (GIF)), MS4A1 (membrane-spanning 4-domains subfamily A member 1), MSLN (mesothelin), MUC1 (cell surface-associated mucin 1 (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1), MelanA / MART1, ML-IAP, MPG, MS4A1 (membrane-spanning 4-domain subfamily A), MYCN, myelin-associated glycoprotein, myostatin, NA17, NARP-1, NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, Neural apoptosis-regulated proteinase 1, NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1, NY-ESO-1, OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1, P21, non-mutant p53, P97, Page4, PAP, anti-(N-glycolylneuraminic acid) paratope, PAX3, PAX5, PCSK9, PDCD1 (PD-1, CD279), PDGF-Ro (platelet-derived growth factor receptor alpha),PDGFR-p, PDL-1, PLAC1, PMEL 17, Proteinase3 (PR1), PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), RANKL, RhoC, mutant Ras, RGS5, ROBO4, RON, ROR1, SART3, sclerostin, SLAMF7, SSX2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein 72), survivin, T cell receptor, T cell transmembrane protein, TEM1 (tumor endothelial marker 1), TENB2, TGF-a, TGF-p (transforming growth factor beta), TGF-[31,TGF-[32 (transforming growth factor beta 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), TNF, TNF-a, TNFRSF8, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblastic glycoprotein), TRAIL-R1 (Tumor necrosis aporosis Inducing ligand Receptor 1), tumor-associated calcium signal transducer 2,tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2 or vimentin, WT1, XAGE 1, TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblastic glycoprotein), TRAIL-R1 (Tumor necrosis aporosis Inducing ligand Receptor 1), TRAILR2 (Death receptor 5 (DR5)), TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1, TNFRSF10B (tumor necrosis factor receptor superfamily member 10B), TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblastic glycoprotein), TRAIL-R1 (Tumor necrosis aporosis Inducing ligand Receptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor-specific glycosylation of MUC1,TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, WT1, , NKG2D, NKp46, and GPRC5D.,

[0141] According to an advantageous embodiment of the invention, the antibody or antibody fragment is chosen from a) A VHH directed against the HER2 antigen, or CD38 (preferably of sequences respectively SEQ ID NO: 10, or SEQ ID NO: 11) b) An anti-HER2 monoclonal antibody such as trastuzumab According to a particular embodiment of the invention, the VHH directed against the HER2 antigen comprises the following 3 CDRs:

[0142] CDR1: GITFMR YA (SEQ ID NO: 12)

[0143] CDR2: INSGGTT (SEQ ID NO: 13)

[0144] CDR3: NAR WV KPQFIDNNY (SEQ ID NO: 14)

[0145] According to a particular embodiment of the invention, the VHH directed against the CD38 antigen comprises the following 3 CDRs:

[0146] CDR1: GIILRIYD (SEQ ID NO: 15)

[0147] CDR2: ITSRGST (SEQ ID NO: 16)

[0148] CDR3: NADHT FA GVY (SEQ ID NO: 17)

[0149] According to an advantageous embodiment of the invention, the first structure of the supramolecular tetrameric peptide complex is represented by the following formulas:

[0150] (12) VHH-L1- (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO:

[0151] 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ

[0152] ID NO: 9)-L2

[0153] (13) VHH-L1- (SEQ ID NO: 4 or SEQ ID NO: 5)-L2

[0154] (14) VHH-L1- (SEQ ID NO: 4 or SEQ ID NO: 5)-L2 or mimetic peptides, analogs, or homologs thereof, preferably with VHH directed against the HER2 antigen, or CD38 (preferably of sequences respectively SEQ ID NO: 10, or SEQ ID NO: 11)

[0155] According to an advantageous embodiment of the invention, the first peptide P1 is directly linked to one or two or more identical or different antibodies or antibody fragment(s).

[0156] According to another advantageous embodiment of the invention, the first peptide P1 is linked to one or two or more identical or different antibodies or antibody fragment(s) via one or two spacers L1 or L2.

[0157] According to an advantageous embodiment of the invention, the spacers L1 and L2 are chosen from the group consisting of a carbon chain comprising from 1 to 60 carbon atoms, said chain being linear or branched, saturated or unsaturated and optionally comprising one or more heteroatoms preferably chosen from nitrogen, oxygen and sulfur, an alkyl chain comprising between 2 and 60 carbon atoms, said chain being linear or branched, and an alkenyl chain comprising between 2 and 60 carbon atoms, said chain being linear or branched, said alkyl and alkenyl chains optionally interrupted by one or more aryl groups, and / or one or more heteroatoms or groups chosen from the group consisting of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -NR'-C(O)-NR'-, -NR'-C(O)-O-, -OC(O)NR', ​​-C(S)NR'-, -NR'-C(S)-, -NR'-C(S)-NR',said alkyl and alkenyl chains optionally substituted by one or more of the atoms or groups selected from the group consisting of -OR', -COOR', -SR', -NR'2, each R' being independently H or a C1-C6 alkyl.,

[0158] According to another advantageous embodiment of the invention, the spacer L1 is chosen from the group consisting of an amino acid (denoted AA) or a peptide chain of 2 to 100 amino acids (denoted (AA)2-100), preferably of 2 to 60 amino acids (denoted (AA)2-6o), of 10 to 50 amino acids (denoted (AA)io-so), even more preferably of 40 to 50 amino acids (denoted (AA)o-so).

[0159] According to another advantageous embodiment of the invention, the spacer L2 is chosen from the group consisting of an amino acid (denoted AA) or a peptide chain of 2 to 60 amino acids (denoted (AA)2-BO), preferably of 2 to 30 amino acids (denoted (AA)2-3o), of 2 to 25 amino acids (denoted (AA)2-2s), even more preferably of 15 to 25 amino acids (denoted (AA)IS-2S).

[0160] For example, the expression "a peptide chain of 2 to 60 amino acids, preferably 40 to 50 amino acids" means 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids and preferably 40, 41, 42, 43, 44, 45, 46, 47, 49, 49, or 50 amino acids.

[0161] According to an advantageous embodiment of the invention, the first peptide P1 is linked to an antibody or antibody fragment via a spacer L1 comprising a peptide chain of 2 to 100 amino acids (denoted (AA)2-100), preferably of 2 to 60 amino acids (denoted (AA)2-60), of 10 to 50 amino acids (denoted (AA)10-50), even more preferably of 40 to 50 amino acids (denoted (AA)40-50) and the spacer L2 is chosen from the group consisting of an amino acid (denoted AA) or a peptide chain of 2 to 60 amino acids (denoted (AA)2-60), preferably of 2 to 30 amino acids (denoted (AA)2-30), of 2 to 25 amino acids (denoted (AA)2-25), even more preferably of 15 to 25 amino acids (denoted (AA)15-25).

[0162] According to an advantageous embodiment of the invention, the first peptide P1 is linked to a first antibody or antibody fragment via a spacer L1 covalently by an amide bond engaging the N-terminal end of the peptide P1 and the C-terminal end of the peptide chain of the spacer L1.

[0163] According to another embodiment of the invention, the first peptide P1 is linked to a second antibody or antibody fragment via a spacer L2 covalently by an amide bond engaging the C-terminal end of the peptide P1 and the N-terminal end of a peptide chain of the spacer L2.

[0164] According to another embodiment of the invention, the spacer L1 or L2 comprises a polyethylene glycol (PEG) domain.

[0165] The PEG domain comprises at least one ethylene unit.

[0166] In some embodiments, the PEG domain is linked to the N-terminus of the peptide comprising at least one P1 or P2 oligomerization domain.

[0167] In some embodiments, the PEG domain is linked to the C-terminus of the peptide comprising at least one P1 or P2 oligomerization domain.

[0168] The PEG domain may be of the formula -(O-CH2-CH2)n-OH, -(O-CH2-CH2)nO-Ci-4alkyl, -(CH2-CH2-O)n-CH2-CH2-OH, or -(CH2-CH2-O)n-CH2-CH2-O-Ci-4alkyl, where n is an integer from 1 to 60, preferably from 1 to 10.

[0169] The PEG domain may be linked to the peptide comprising at least one P1 or P2 oligomerization domain by a spacer which in some embodiments comprises -NH2-, -COO- or any other suitable group known to those skilled in the art. Linkers and PEGylation methods are described, for example, in Turecek et al. (Journal of Pharmaceutical Sciences 2016, 105, 460-475).

[0170] According to an advantageous embodiment of the invention, the first structure of the supramolecular tetrameric peptide complex is represented by the following formulas:

[0171] (l5) VHH-(AA)2-6o - (SEQ ID NO : 1 or SEQ ID NO : 2 or SEQ ID NO : 3 or SEO ID

[0172] NO : 4 or SEQ ID NO : 5 or SEQ ID NO : 6 or SEQ ID NO : 7 or SEQ ID NO : 8 or SEQ ID NO : 9)-L2

[0173] (le) VHH-(AA)2-6O - (SEQ ID NO : 4 or SEQ ID NO : 5)-L2 (l7) VHH-(AA)4O-5O - (SEQ ID NO : 1 or SEQ ID NO : 2 or SEQ ID NO : 3 or SEQ ID NO : 4 or SEQ ID NO : 5 or SEQ ID NO : 6 or SEQ ID NO : 8 or SEQ ID NO : 8 SEQ ID NO : 9)-L2

[0174] (Is) VHH-(AA) 4O -5O - (SEQ ID NO : 4 or SEQ ID NO : 5)-L2 with L2 is a spacer as defined above

[0175] (l9) VHH-(AA)2-60 - (SEQ ID NO : 1 or SEQ ID NO : 2 or SEQ ID NO : 3 or SEQ ID

[0176] NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-(AA) 2-3 o

[0177] (ho) VHH-(AA)2-6O - (SEQ ID NO: 4 or SEQ ID NO: 5)-(AA)I5-25

[0178] (In) VHH-(AA)4O-5O - (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-(AA) 2-3 o

[0179] (I12) VHH-(AA)4O-5O - (SEQ ID NO: 4 or SEQ ID NO: 5)- (AA)I5-25 or mimetic peptides, analogues, or homologues thereof, preferably with VHH directed against the HER2 antigen, or CD38 (preferably of sequences respectively SEQ ID NO: 10, or SEQ ID NO: 11).

[0180] 3. Chelating groups

[0181] According to another embodiment of the invention, the chelating group (B) is chosen from the following compounds: bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (OPTA), N'45-[acetyl(hydroxy)amino]pentyl]-N45-[[445-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide (deferoxamine / DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A) 1,4 ,7,10-tetracyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10 tetraacetic acid (DOTAGA), 1,4,7,10-tetraazacyclododecane N,N',N",N'" 1,4,7,10-tetra(methylene)phosphonic acid (DOTMP), 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) and its derivatives, N,N'-dipyridoxylethylenediamineN,N'-diacetate5,5'-bis(phosphat) (DPDP), diethylenetriamine acid N,N',N" penta(methylene) phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'- tetraacetic acid (EDTA), ethylene glycol-0,0-bis(2-aminoethyl)-N,N,N',N'- tetraacetic acid (EGTA), N,Nbis(hydroxybenzyl)-ethylenediamine-N,N'- diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate (HP-D0A3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), 1,4,7-triazacyclononan-1- succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"-tetrakis(carbamoylmethyl) (TETAM), 1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane (NOTAM), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11- tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetrakis(methylene phosphonate) (DOTP), 1,4,7-tetrakis(methylene phosphonate)-1,4,7-triazacyclononane (NOTP), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), tris(hydroxypyridinone) (THP), terpyridinbis(methyleneaminotetraacetic acid) (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10- tetraazacyclotridecane-N,N',N", Nm-tetraacetic acid (TRITA), 34[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl1]-1,4,7-triazonan-1-yl]methyl-hydroxy-phosphoryl]propanoic acid, triethylenetetraaminehexaacetic acid (TTHA) and other bispidine-type chelators, preferably DOTAGA.,

[0182] According to another embodiment of the invention, the metal (M) or the metal cation (M 2+ or M 3+ or M 4+) having an atomic number Z greater than or equal to 40, for example from 40 to 213, complexed with the chelating group, having radiosensitizing properties and / or contrast enhancement properties for MRI imaging, is chosen from metals or lanthanides, and more preferably Au, Ag, Pt, Pd, Sn, Ta, Zr, Tb, Tm, Ce, Dy, Er, Eu, La, Nd, Pr, Lu, Yb, Bi, Hf, Ho, Pm, Sm, In and Gd, and mixtures thereof.

[0183] According to an advantageous embodiment of the invention, the metal (M) or the metal cation (M 2+ or M 3+ or M 4+ ) is chosen from gadolinium (Gd) or the Gd cation 3+ , hafnium (Hf) or the Hf cation 4+ , and bismuth (Bi) or the cation Bi +3 .

[0184] According to another advantageous embodiment of the invention, the metal (M) or the metal cation (M 2+ or M 3+ or M 4+) is attached to the chelator group by at least two coordination bonds. According to an advantageous embodiment of the invention, the second structure of the supramolecular tetrameric peptide complex is represented by the following formulas:

[0185] (ll2) (SEQ ID NO : 1 or SEQ ID NO : 2 or SEQ ID NO : 3 or SEQ ID NO : 4 or SEQ ID NO : 5 or SEQ ID NO : 6 or SEQ ID NO : 7 or SEQ ID NO : 8 or SEQ ID NO : 9)-L3-[DOTAGA@(M)] m preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-L3-DOTAGA@(M),

[0186] (Ils) (SEQ ID NO : 1 or SEQ ID NO : 2 or SEQ ID NO : 3 or SEQ ID NO : 4 or SEQ ID NO : 5 or SEQ ID NO : 6 or SEQ ID NO : 7 or SEQ ID NO : 8 or SEQ ID NO : 9)-L3-[DOTAGA@(Bi, Hf or Gd)] m of preference (SEQ ID NO : 4 or SEQ ID NO : 5)-L3-DOTAGA@(Bi, Hf or Gd) or peptide mimetics, analogs, or homologues thereof.

[0187] According to another embodiment of the invention, the second peptide P2 is linked to one or two or more chelating group(s) via a spacer L3.

[0188] According to the present invention, the spacer L3 comprises a group formed by a “click” chemistry reaction well known to those skilled in the art, in particular the spacer L3 comprises a group chosen from a [1,2,3]-triazole, isoxazole, isoxazoline, oxadiazoles, a pyrazole, a dihydropyrazine, an amide, a maleimide, a hydrazone and an oxime.

[0189] These groups are represented in the formulas of the present invention by the term "click".

[0190] According to another advantageous embodiment of the invention, the spacer L3 is selected from the group consisting of a C1-C16 alkyl group, preferably C1-C8, heteroalkyl C2-C8, alkylcycloalkyl, heterocycloalkyl; aryl C3-C8, Ar-alkyl, heterocyclic, carbocyclic, cycloalkyl, heteroalkylcycloalkyl, alkylcarbonyl, heteroaryl, ester, ether, amine, imine, hydrazine, hydrazone, semicarbazide, carbazide, alkoxyamine, alkoxylamine, urethane, acyloxylamine, glycoside, peptide containing 1-8 amino acids, a polyethyleneoxy group of formula (OCH2CH2) P or (OCH2CH(CH3)) P, linear or branched where p is an integer from 1 to 50, preferably from 1 to 10 (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) or a combination of said groups and further comprises a group selected from a [1,2,3]-triazole ring, isoxazole, isoxazoline, oxadiazoles, a pyrazole, a dihydropyrazine, an amide, a hydrazone and an oxime. Click chemistry, catalyzed or not, is widely used for protein-small molecule or protein-protein couplings.

[0191] Strategies for introducing an azide (N3) or azadibenzocyclooctyne (DBCO) or bicyclo [6.1.0] nonyne (BON) function onto proteins or peptides can be cited. Among the large number of bioconjugations described in the literature, chemical reactions described as being site-specific, allowing the derivatization of peptides at their N or C-terminal ends.

[0192] Click Chemistry was introduced by Sharpless in 2001 to develop a set of unambiguous and scalable reactions that can be carried out under mild conditions, without tedious purification, without by-product formation, have a broad substrate spectrum, are physiologically stable and / or compatible with biological media, and lead to high yields with atom economy. The main click reactions consist of the formation of energetically very favorable carbon-heteroatom bonds, including a nucleophilic ring-opening reaction or a cycloaddition reaction. A widely represented reaction type in click chemistry is the Cu(l)-catalyzed alkyne-azide cycloaddition mentioned above. When click reactions are compatible with chemical functions and biological media, whether in vitro or in vivo, these reactions are said to be bioorthogonal.

[0193] These Azide / Alkyne cycloadditions are known by the abbreviation 'CuAAC' (Copper ion catalyzed AAC) and are now available in many other reactions catalyzed by various metals (Metal-catalyzed Azide-Alkyne Additions: MAAC).

[0194] Another approach to improve the reactivity of Alkynes and avoid the use of catalyst (toxic copper in bioconjugation), was introduced by Jewett, JC; Bertozzi, CR (Chem. Soc. Rev. 2010, 39, 1272-1279) with "constrained" Alkynes.

[0195] Different types of strained alkynes have been proposed, including nonynes (BCN), and even more so cyclooctynes ​​(DBCO, DIBO). Requiring no catalyst (cytotoxic Cu(l)(l I)), these new “Copper-free” reactions are also very fast even at room temperature. They are called 'Strain-promoted Alkyne-azide cycloadditions' (SPAAC).

[0196] According to the present invention, the particularly advantageous “click” reactions are summarized in the following table:

[0197] More specifically, reagents can be coupled by peptide bonding, as described in Montalbetti, CAGN, F algue B. Tetrahedron 2005, 61, 10827-10852. A coupling method using click chemistry can also be used, involving groups of the type: -N3, -CN, -COCH.

[0198] According to an advantageous embodiment of the invention, the second structure of the supramolecular tetrameric peptide complex is represented by the following formulas:

[0199] (113) (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9) -

[0200] L3-[DOTAGA@( Bi, Hf or Gd)] m preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-L3-DOTAGA@(Bi, Hf or Gd)

[0201] (114) (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9) - click-[DOTAGA@(M)] m preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-click-DOTAGA@(M), preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-click-DOTAGA@(Bi, Hf or Gd) with "click" represents a group comprising a [1,2,3]-triazole, isoxazole, isoxazoline, oxadiazoles, a pyrazole, a dihydropyrazine, an amide, a maleimide, a hydrazone or an oxime ring.

[0202] According to an advantageous embodiment of the invention, the supramolecular tetrameric peptide complex comprises:

[0203] (a) a first structure represented by the following formulas:

[0204] (h) A-L1- (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-L2 preferably

[0205] A-L1- (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2

[0206] (12) VHH-L1- (SEQ ID NO : 1 ou SEQ ID NO : 2 ou SEQ ID NO : 3 ou SEQ ID

[0207] NO : 4 ou SEQ ID NO : 5 ou SEQ ID NO : 6 ou SEQ ID NO : 7 ou SEQ ID

[0208] NO : 8 ou SEQ ID NO : 9)-L2

[0209] (13) VHH-L1- (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2

[0210] (14) VHH-L1- (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2

[0211] (15) VHH-(AA)2-6o - (SEQ ID NO : 1 ou SEQ ID NO : 2 ou SEQ ID NO : 3 ou SEQ ID NO : 4 ou SEQ ID NO : 5 ou SEQ ID NO : 6 ou SEQ ID NO : 7 ou SEQ ID NO : 8 ou SEQ ID NO : 9)-L2

[0212] (le) VHH-(AA)2-6O - (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2

[0213] (17) VHH-(AA)4O-5O - (SEQ ID NO : 1 ou SEQ ID NO : 2 ou SEQ ID NO : 3 ou SEQ ID NO : 4 ou SEQ ID NO : 5 ou SEQ ID NO : 6 ou SEQ ID NO : 7 ou SEQ ID NO : 8 ou SEQ ID NO : 9)-L2

[0214] (18) VHH-(AA)4O-5O - (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2

[0215] (19) VHH-(AA)2-6o - (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-(AA) 2.30

[0216] (ho) VHH-(AA)2.6O - (SEQ ID NO: 4 or SEQ ID NO: 5)-(AA)IS-25 (In) VHH-(AA)4O-5O - (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-(AA)2-3o

[0217] (I12) VHH-(AA) 4O -5O - (SEQ ID NO: 4 or SEQ ID NO: 5)- (AA)I5-25 or mimetic peptides, analogues, or homologues thereof, preferably with VHH directed against the HER2 antigen, or CD38 (preferably of sequences respectively SEQ ID NO: 10, or SEQ ID NO: 11) and

[0218] (b) a second structure represented by the following formulas:

[0219] (111) (SEQ ID NO: 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9)-L3-[B@(M)]m de préférence (SEQ ID NO: 4 or 5)-L3-B@(M)

[0220] (112) (SEQ ID NO: 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9)-L3-[DOTAGA@(M)] m de préférence (SEQ ID NO: 4 or 5)-L3-DOTAGA@(M),

[0221] (113) (SEQ ID NO : 1 ou 2 ou 3 ou 4 ou 5 ou 6 ou 7 ou 8 ou 9)-L3-[DOTAGA@( Bi, Hf or Gd)] m de préférence (SEQ ID NO: 4 or 5)-L3-DOTAGA@(Bi, Hf or Gd)

[0222] (114) (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-click-[DOTAGA@(M)] m or mimetic peptides, analogs, or homologues thereof, preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-click-DOTAGA@(M), preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-click-DOTAGA@(Bi, Hf or Gd) with "click" represents a group comprising a [1,2,3]-triazole, isoxazole, isoxazoline, oxadiazoles, a pyrazole, a dihydropyrazine, an amide, a maleimide, a hydrazone or an oxime.wherein the sequences SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9, preferably the sequences SEQ ID NO: 4 and SEQ ID NO: 5 or mimetic peptides, analogues, or homologues thereof associate to form a primary homodimer and / or a primary heterodimer, which associate to form one or more type(s) of “dimers of dimers”.

[0223] Preparation

[0224] The peptides comprising at least one oligomerization, preferably tetramerization, domain described herein (including truncations, analogs, etc.) may be prepared using well-known synthetic chemical techniques, for example, using solid-phase synthesis methods but preferably recombinant DNA methods, for example, as described in the example below. Examples of methods for recombinant expression of peptides and proteins are described in Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989).

[0225] The term "recombinant," when used in reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all. The term "host cell" means a cell that is capable of undergoing transformation, transfection, transduction, conjugation, etc., with a nucleic acid construct or expression vector.Host cells may be derived from plants, bacteria, yeast, fungi, insects, animals, etc. In some embodiments, the host cell comprises Escherichia coli.

[0226] Purification

[0227] The peptides and / or peptide complexes of the invention are purified by reverse-phase HPLC and mass spectrometry (MS).

[0228] The separation of peptides and / or peptide complexes can be followed by both UV detection and mass spectrometry.

[0229] According to a second aspect, the invention relates to a composition comprising at least one supramolecular peptide complex as detailed herein, in particular a plurality of complexes representing an effective amount of supramolecular peptide complexes as detailed herein, and a pharmaceutically acceptable excipient.

[0230] The effective amounts are determined by those skilled in the art during pharmacokinetic and pharmacodynamic studies.

[0231] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other undesirable reaction when administered to an animal or human. As used herein, the term "pharmaceutically acceptable carrier" includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and the like. In some embodiments, a carrier comprises a solution at neutral pH. In some embodiments, a carrier comprises a salt. In some embodiments, a carrier comprises a buffered solution. In some embodiments, a carrier comprises phosphate-buffered saline.

[0232] Excipients

[0233] The pharmaceutical formulation of the invention may further comprise one or more pharmaceutically acceptable excipients selected from solvents, stabilizers, surfactants, buffering agents, antimicrobial preservatives, protectants, antioxidants and chelating agents.

[0234] According to the present invention, a "solvent" is any pharmaceutically acceptable ingredient (i.e., safe and non-toxic for administration to a human or other mammal) and useful for the preparation of a liquid formulation, such as an aqueous formulation.

[0235] Examples of solvents include water such as sterile water for injection (WFI) or bacteriostatic water for injection (BWFI), a pH-buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution, and combinations thereof.

[0236] Preferably, the solvent is sterile water for injection or bacteriostatic water for injection (BWFI).

[0237] According to the present invention, stabilizers are compounds that increase the stability of proteins, particularly against unfolding and aggregation. Preferably, the stabilizer is approved by the authorities as a suitable additive or excipient in pharmaceutical formulations.

[0238] The stabilizer can be a saccharide.

[0239] The term "saccharide" includes monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.

[0240] Examples of saccharides herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc.

[0241] Preferably, the formulation comprises a non-reducing disaccharide as a stabilizing agent, such as a saccharide selected from the group of trehalose and sucrose.

[0242] The concentration of the stabilizer in the pharmaceutical formulation of the invention is preferably between 1 and 500 mM, 15 and 250 mM, or 150 and 250 mM, or is about 210 mM.

[0243] Surfactants are generally added to protein formulations to reduce the exposure of hydrophobic regions and thus decrease protein-protein interactions and interface-induced aggregation, also prevented by competition for adsorption sites.

[0244] Examples of surfactants include polysorbate (e.g., polysorbate 20 and polysorbate 80); poloxamer (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octylglycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetylbetaine; lauroamidopropyl-, cocamidopropyl-jinoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; methylcocoyl-, or disodium methyloleyl-taurate; polyethylene glycol, polypropylene glycol and copolymers of ethylene and propylene glycol.

[0245] Other examples of pharmaceutically acceptable surfactants include polyoxyethylene-sorbitan fatty acid esters (Tween), polyethylenepolypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers, e.g., polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).

[0246] The most suitable polyoxyethylenesorbitan fatty acid esters are polysorbate 20 (sold under the brand name Tween 20™) and polysorbate 80 (sold under the brand name Tween 80™).

[0247] The most suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™.

[0248] The most suitable polyoxyethylene alkyl ethers are those sold under the brand name Brij™.

[0249] The most suitable alkylphenol-polyoxyethylene ethers are sold under the trade name Triton-X.

[0250] The surfactant is preferably a non-ionic surfactant, preferably a polysorbate, e.g. selected from the group consisting of polysorbate 20, polysorbate 80 and polyethylene polypropylene copolymer.

[0251] The concentration of surfactant in the pharmaceutical formulation of the invention is preferably between 0.01 and 0.1% (w / v), or 0.01 and 0.08% (w / v), or 0.025 and 0.075% (w / v).

[0252] According to the present invention, the term "buffering agent" refers to an agent which allows the pH of a solution to be kept constant by the action of its conjugated acid / base components.

[0253] Examples of buffering agents that will control pH include acetate, succinate, gluconate, histidine, citrate, glycylglycine, and other organic acid buffers.

[0254] A suitable buffer in the present invention is a histidine buffer.

[0255] A "histidine buffer" is a buffer containing the amino acid histidine.

[0256] Examples of histidine buffers include histidine chloride (e.g., L-histidine hydrochloride monohydrate), histidine acetate, histidine phosphate, histidine sulfate. A "preservative" is a compound that may be added to the present formulations to reduce contamination by and / or the action of bacteria, fungi, or other infectious agent.

[0257] The addition of a preservative can, for example, facilitate the production of a multi-use (multi-dose) formulation.

[0258] Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long chain), and benzethonium chloride.

[0259] Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and w-cresol.

[0260] A "protective agent," as generally used herein, is a substance that, when combined with a protein, significantly reduces the chemical and / or physical instability of the protein upon freeze-drying and / or subsequent refrigerated storage.

[0261] Examples of protectants include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, polyethylene glycol), or polypropylene glycol); and combinations thereof.

[0262] Additional examples of protective agents include gelatin, dextrins, modified starch, and carboxymethylcellulose.

[0263] Preferred sugar alcohols are compounds obtained by reduction of mono- and disaccharides, such as lactose, trehalose, maltose, lactulose and maltulose.

[0264] Other examples of sugar alcohols are glucitol, maltitol, lactitol, and isomaltulose.

[0265] The protectant may be added to the pre-lyophilized formulation in a "lyoprotectant" amount. This means that after lyophilization of the protein in the presence of a lyoprotectant amount of the protectant, the protein essentially retains its physical and chemical stability and integrity.

[0266] An “antioxidant,” as generally used herein, is a pharmaceutically acceptable excipient generally used to limit oxidation reactions and maintain the stability and safety of proteins.

[0267] Examples of antioxidants are ascorbic acid, sodium metabisulfite, histamine, methionine, ascorbic acid, glutathione, vitamin E, polyethyleneimine.

[0268] The antioxidant is preferably methionine, especially L-methionine.

[0269] The concentration of antioxidant in the pharmaceutical formulation of the invention is preferably between 5 and 25 mM, more preferably between 5 and 15 mM.

[0270] According to a third aspect, the invention relates to a composition comprising at least one supramolecular peptide complex as detailed herein, in particular a plurality of supramolecular peptide complexes as detailed herein as a radiosensitizing agent and to a composition comprising at least one supramolecular peptide complex as detailed herein, in particular a plurality of complexes representing an effective amount of supramolecular peptide complexes as detailed herein, for use in the treatment of a tumor after activation by external radiotherapy, by ionizing radiation, preferably X-rays or after activation by vectorized internal therapy. The invention further relates to a composition comprising a sufficient amount of supramolecular peptide complexes as detailed herein for use as a contrast agent.

[0271] The use of radiosensitizers has been proposed for many years. A radiosensitizing compound is a compound that works in combination with radiation to induce a more effective response and increase therapeutic efficacy. Compounds are typically targeted that include heavy elements with high atomic numbers and that interact directly with radiation, increasing its likelihood of interaction, causing damage to the target cells.

[0272] Part of the irradiated energy is then absorbed and deposited locally around the radiosensitizers and can produce secondary electrons, Auger electrons, Compton electrons, ionizations, photons, free radicals for example, or even simply a thermal rise.

[0273] For cancer therapies, one goal is to locally increase the dose to the tumor, thus radiosensitizers must be preferentially accumulated in tumors compared to healthy tissues.

[0274] The radiosensitizing effect of the supramolecular peptide complexes of the invention is linked to the presence of atoms with a high Z, for example between 40 and 213. Indeed, the incident radiation will interact with the supramolecular peptide complex of the invention and cause physical and then chemical and biological reactions which will increase the damage at the cellular level. Schematically, the human body is mainly made up of water and our tissues are considered as such during dose deposition calculations by radiotherapy software. The concentration of a sufficient quantity of metal atoms of the supramolecular peptide complexes of the invention in the tumor then makes it possible to increase the dose deposition and the cellular damage within the tumor.It is important to distinguish the increase in the physical dose "dose enhancement" which corresponds to the energy deposited in the volume considered in connection with the presence of the supramolecular peptide complexes of the invention and the radiosensitization which corresponds to the increase in biological effects observed in the presence of the supramolecular peptide complexes of the invention.

[0275] According to the present invention, applications in external and internal radiotherapy are distinguished.

[0276] 1. External radiotherapy

[0277] External beam radiation therapy uses ionizing radiation sources external to the patient to treat a tumor target.

[0278] Physical effects

[0279] After administration to the patient, the supramolecular peptide complexes of the invention are activated by ionizing radiation. The physical interactions will depend on the nature of the incident beam (photons, electrons, carbon ions, protons, alpha) as well as the energy used. In radiotherapy departments, the photons used have an energy spectrum of the order of MV. At this energy, the mass absorption coefficient of the tissues is similar to that of the heavy atoms (high Z) present in the supramolecular peptide complexes of the invention. In certain clinical applications, the photonic radiation may be of the order of 250 kVp. The macroscopic increase in the physical dose deposited in connection with these supramolecular peptide complexes of the invention comprising a heavy metal (atomic number Z greater than or equal to 40, for example from 40 to 213) is therefore considered to be low.The activation of the supramolecular peptide complexes of the invention by ionization is actually attributed to the secondary electrons emitted in the beam track. In the case of photons, the two main mechanisms of interaction with matter are the photoelectric effect and the Compton effect. These two effects lead to the production of secondary electrons also interacting with matter and allowing the activation of the supramolecular peptide complexes of the invention. In a second step, the supramolecular peptide complexes of the invention thus activated relax via specific electronic mechanisms. The rearrangement within the heavy atoms (high Z) of the supramolecular peptide complexes of the invention leads to the emission of fluorescence photons and Auger electrons. These Auger electrons then generate a high-density focal ionization over a distance of nearly 10 nm.When these atoms, Auger emitters, are concentrated within a supramolecular peptide complex of the invention, the emitted electrons can excite neighboring heavy atoms. This then induces cascade excitation processes "Auger electron cascade" leading to a high dose deposition locally around the supramolecular peptide complexes of the invention, at a microscopic or even nanoscopic scale.

[0280] Physicochemical effects

[0281] The electrons produced by the electronic relaxation of the supramolecular peptide complexes of the invention can directly ionize biomolecules and induce direct biological damage or create free radicals such as HO. from the water present in large quantities in the irradiated tissues. In the presence of supramolecular peptide complexes of the invention, the electrons emitted by the latter will react with the surrounding water molecules and produce clusters of radicals. It is the action of these radicals locally concentrated in the peripheral volume of the supramolecular peptide complexes of the invention (from 3 to 20 nm) which explains the induction of complex damage in the biomolecules of the environment leading to cell death. The secondary electrons can also interact with the molecular oxygen O2 present in the tissues.Since oxygen is a powerful oxidant, it has a very high affinity for electrons and can thus capture the electron emitted during the ionization of a molecule, preventing its immediate recombination. These phenomena lead to the formation of reactive oxygen species (ROS), which can also lead to cell death.

[0282] Biological effects

[0283] Once the physical phase of interaction between the radiation and the supramolecular peptide complexes of the invention has passed, the creation of free radicals and reactive oxygen species (ROS) has been demonstrated, which will react with the cell membranes, DNA, or proteins, thus leading to toxicity at the cytoplasmic and nuclear level. The damage to the mitochondria, the energy centers of the cell which possess DNA and whose mobilization of cytochrome C can lead to cell death by apoptosis also seems to be one of the explanatory elements of cellular radiosensitization.

[0284] Gadolinium (Z=64), a rare earth metal, is already well known in medical applications in the form of chelate for its use as an MRI contrast agent. The nanoparticles currently under clinical evaluation are AGulX: for "Activated Guided Irradiation by X-ray" produced by the company NH Theraguix SA (Grenoble, France). These are small nanoparticles < 5 nm injected intravenously before irradiation. AGulX is a drug with "theranostic" vocation (neologism from the contraction of therapeutic and diagnostic) with therapeutic properties as a radiosensitizing agent but also as an MRI contrast agent.

[0285] In some embodiments, the supramolecular tetrameric peptide complexes comprise, as a high-Z element, a rare earth metal, or a mixture of rare earth metals.

[0286] For example, said supramolecular tetrameric peptide complexes may comprise, as a high-Z element, gadolinium, bismuth, or a mixture thereof.

[0287] In some embodiments, said method of treatment comprises a first step comprising administering an effective amount of said high-Z element containing supramolecular tetrameric peptide complexes as radiosensitizing agents to said subject in need thereof, prior to the first exposure to radiotherapy.

[0288] Advantageously, said subject may be exposed to at least one or more additional sessions of magnetic resonance imaging-guided radiotherapy, without additional administration of a magnetic resonance imaging contrast agent.

[0289] Typically, said subject is exposed to 2 or more sessions of magnetic resonance imaging-guided radiotherapy after a single administration of an effective amount of said high-Z-containing supramolecular tetrameric peptide complexes, for example, 2 to 7 sessions.

[0290] In a more particular embodiment, said subject is exposed to 2 or more sessions of radiotherapy guided by magnetic resonance imaging of 5 to 7 days, generally with a minimum delay of 2 or 3 days between each session.

[0291] In some embodiments, the subject is exposed to a dose of ionizing radiation per magnetic resonance imaging-guided radiation therapy session of about 3 Gy to about 20 Gy, and the total dose is preferably administered in a maximum of 10 fractions, e.g., in 1 to 10 fractions.

[0292] The tumor targeted by the method of the invention may be a solid tumor, preferably chosen from

[0293] • primary tumors of the cervix, rectum, lung, head and neck, prostate, colorectal, liver and pancreas and

[0294] • bone metastases.

[0295] According to one embodiment of the invention, the targeted tumor is selected from colon and rectal cancer, stomach or pancreatic cancer, breast cancer, ovarian cancer, testicular cancer, adenocarcinoma, small cell cancers, squamous cell cancers, medullary thyroid cancer, cellular epithelioma of the thyroid, liver cancer, multiple myeloma, invasive bladder cancer and ENT cancer.

[0296] In some embodiments, said supramolecular tetrameric peptide complexes are administered as an injectable solution at a concentration of 50 to 150 mg / mL, and preferably 80 to 120 mg / mL, for example 100 mg / mL, preferably by intravenous injection.

[0297] For example, a therapeutically effective amount administered for magnetic resonance imaging-guided radiotherapy is from 50 mg / kg to 150 mg / kg, typically from 80 to 120 mg / kg, e.g., 100 mg / kg. As used herein, the term "radiosensitizer" would be readily understood by those skilled in the art and generally refers to the process of increasing the sensitivity of cancer cells to radiotherapy (e.g., photon radiation, electron radiation, proton radiation, heavy ion radiation, etc.).

[0298] The supramolecular tetrameric peptide complex of the present invention contains at least one, preferably two or more high-Z metal element(s) or cation(s) and has radiosensitizing properties and / or contrast enhancing properties for MRI imaging is for use in methods of treating a tumor in a subject in need thereof, the method comprising

[0299] (i) administering a supramolecular tetrameric peptide complex containing a high Z element, greater than 40, for example comprised from 40 to 213 exhibiting contrast enhancement for magnetic resonance imaging (MRI) and / or radiosensitizing properties, to a subject in need thereof, and,

[0300] (ii) exposing said subject to ionizing radiation.

[0301] The invention further relates to supramolecular tetrameric peptide complexes containing a high Z element, greater than 40 for example comprised from 40 to 213 or a pharmaceutical composition comprising said peptide complexes for use in the manufacture of a medicament for the treatment of a tumor in a subject in need thereof, the treatment comprising

[0302] (i) administering a supramolecular tetrameric peptide complex containing a high Z element, greater than 40, for example comprised from 40 to 213 exhibiting contrast enhancement for magnetic resonance imaging (MRI) and / or radiosensitizing properties, to a subject in need thereof, and,

[0303] (ii) expose said subject to ionizing radiation.

[0304] In advantageous embodiments, said supramolecular tetrameric peptide complexes containing a high Z element, greater than 40, for example comprised from 40 to 213, exhibit both contrast-enhancing properties for magnetic resonance imaging (MRI) and radiosensitizing properties.

[0305] It is understood that the amount of supramolecular tetrameric peptide complex administered is an amount sufficient for the treatment of a tumor. As used herein, the term "treat" or "treatment" refers to one or more of (1) disease inhibition; e.g., inhibiting a disease, condition, or disorder in an individual who is suffering from or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., stopping the further development of the pathology and / or symptoms); and (2) disease amelioration; e.g., improving a disease, condition, or disorder in an individual who is suffering from or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms), e.g., decreasing the severity of the disease or reducing or alleviating one or more symptoms of the disease.

[0306] External beam radiation therapy involves exposing cancer cells in a tumor to ionizing radiation emitted in a beam by a device called a "linear particle accelerator" located near the patient; they pass through the skin and other organs or tissues to reach the tumor.

[0307] In one particular mode, the linear accelerator is equipped with a CT scanner (cone-beam computed tomography - CBCT) or a magnetic resonance imager (MRI) that allows an image of the patient to be obtained at each treatment, and makes it possible to determine more precisely what types of changes occur in the tumor, if any, from one visit to the next.

[0308] According to another particular mode, a modulation of the dose of ionizing radiation delivered is ensured by a multi-leaf collimator which moves back and forth in order to target the tumor with the best possible dose.

[0309] An MRI is performed before the actual radiotherapy step to verify tumor internalization of the peptide complex of the present invention and / or the contrast agent.

[0310] In particular, in reference to the treatment of a tumor, the term "treatment" may refer to the inhibition of tumor growth, or to the reduction of tumor size.

[0311] The terms “patient” and “subject” used herein interchangeably refer to any mammal.

[0312] Preferably, the subject is a human being, including for example a subject carrying a tumor.

[0313] In particular embodiments, the radiotherapy is guided by MRI. The use of the supramolecular tetrameric peptide complexes as described allows for better visualization and monitoring of the lesions, making it possible to maximize the volume of the tumor treated by radiotherapy while minimizing the deleterious effect on healthy tissues.

[0314] In a particular mode, the subject is exposed to radiotherapy guided by MRI (MR-Linac).

[0315] In specific embodiments, said tumor is a solid tumor.

[0316] External beam radiotherapy according to the present application is particularly well suited for the treatment of tumors in sites affected by inter- and intrafraction movements such as the thorax, abdomen and pelvis.

[0317] Thus, in an advantageous embodiment, said tumor is located in one or more of the following sites:

[0318] • Abdomen, pancreas / duodenum, kidney, liver

[0319] • Pelvis and lower limbs, especially lower gastrointestinal, prostate, bladder,

[0320] • Head, neck, brain and central nervous system (glioblastoma)

[0321] • Thorax, including lung and mediastinum, esophagus, bones, bone marrow, breast.

[0322] In a particular embodiment, said tumor is selected from the group consisting of

[0323] (i) primary tumor of the cervix, rectum, lung, breast, head and neck, prostate, bladder, colorectal, liver and pancreas or

[0324] (ii) bone or liver metastases, bone marrow cancer (myeloma).

[0325] The method of treating cancer of the present invention comprises a step of administering said supramolecular tetrameric peptide complexes containing a high Z, greater than 40, for example comprised from 40 to 213 as described above, to a patient.

[0326] The amount administered should be sufficient for the use of the supramolecular tetrameric peptide complexes as a contrast agent for MRI and / or radiosensitizing agent during radiotherapy.

[0327] Preferably, the supramolecular tetrameric peptide complexes are administered in an amount sufficient for combined use as an MRI contrast agent and a radiosensitizing agent during MRI image-guided radiotherapy, for example, when the metal ion is Gd. 3+ .

[0328] Supramolecular tetrameric peptide complexes can be administered to the subject by different possible routes such as local (intra-tumoral (IT), intra-arterial (IA)), subcutaneous, intravenous (IV), intradermal, respiratory (inhalation), intra-peritoneal, intramuscular, intrathecal, intraocular or oral.

[0329] In specific embodiments, the supramolecular tetrameric peptide complexes are administered intravenously.

[0330] Typically, when using fractionated radiotherapy, supramolecular tetrameric peptide complexes can be injected once a week over several radiotherapy sessions.

[0331] As used herein, the term "radiotherapy" is used for the treatment of diseases of an oncological nature with irradiation corresponding to ionizing radiation.

[0332] Typically, said ionizing radiations are photons, for example; X-rays.

[0333] Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface or deeper in the body.

[0334] The higher the energy of the X-ray beam, the deeper the X-rays can penetrate into the target tissue.

[0335] Linear accelerators produce X-rays of increasingly greater energy.

[0336] The use of machines to focus radiation (such as X-rays) on a cancerous site is called external beam radiation therapy.

[0337] Ionizing radiation is typically 2 MV to 25 MV, particularly between 4 MV and 18 MV, typically 4 MV or 6 MV.

[0338] Typically, prior to radiation therapy, a pre-treatment computed tomography (CT) scan and MRI are acquired.

[0339] The target and organs at risk can be defined by a radiation oncologist based on pre-treatment data. At the beginning of each treatment session, the patient is positioned on the treatment table, a new MRI is performed, and compared to the original scan used to create the radiation treatment plan.

[0340] If anything on the ultrasound has changed, the radiation treatment plan can be adjusted to account for tumor and organ movement.

[0341] Once the highly specialized team is satisfied with the radiotherapy plan and targeting, the patient will receive their treatment.

[0342] The radiation beams are precisely shaped to maximize the dose received by the target while minimizing the dose received by surrounding healthy tissue.

[0343] If the tumor or a critical organ exceeds a limit set by the physician, the radiation beam stops automatically; when the target returns to within the predefined limit, treatment resumes automatically.

[0344] This ensures that the right amount of radiation is delivered to the right location.

[0345] In the method of the present invention, the high Z-containing supramolecular tetrameric peptide complexes administered to the subject prior to radiotherapy are used either as a contrast agent for magnetic resonance imaging, or as a radiosensitizing agent for radiotherapy, or preferably, both as a contrast agent for MRI imaging and as a radiosensitizing agent.

[0346] The skilled person in the field of MRI-guided radiotherapy knows how to determine an appropriate dosage and application program, depending on the nature of the disease and the patient's constitution.

[0347] In particular, the person knows how to assess dose-limiting toxicity (DLT) and determine the maximum tolerated dose (MTD) accordingly.

[0348] The amount of radiation used in photon radiotherapy is measured in gray (Gy) and varies depending on the type and stage of cancer being treated.

[0349] For curative cases, the typical total dose for a solid tumor ranges from 20 to 120 Gy, usually 25 to 100 Gy.

[0350] Many other factors are considered by radiation oncologists when choosing a dose, including whether the patient is receiving chemotherapy, the patient's comorbidities, whether radiation therapy is given before or after surgery, and how successful the surgery was. The total dose is usually fractionated (spread out over time).

[0351] The amount and schedules (planning and administration of ionizing radiation, dose fraction, fraction administration schedule, total dose alone or in combination with other anticancer agents, etc.) are defined for any disease / anatomical site / stage of disease, patient setting / age.

[0352] A typical conventional fractionation schedule for adults for the methods of the present invention may be 1.8 to 3.0 Gy per day, five days per week, for example for 2 to 8 consecutive weeks.

[0353] In particular embodiments, said radiotherapy consists of exposing the subject to a total dose of ionizing radiation of between 25 and 80 Gy, for example 30 Gy.

[0354] Considering the combined effect of the supramolecular tetrameric peptide complexes and ionizing radiation according to the present method obtained with a high dose of ionizing radiation, in a specific embodiment, the dose of ionizing radiation exposed to the patient's tumor is advantageously hypofractionated.

[0355] For example, a dose per fraction of at least 3 Gy, and for example between about 3 Gy and about 20 Gy, or between 5 and 7 Gy, is exposed to the patient's tumor and the total radiation dose is delivered in a few fractions (usually, but not necessarily not more than 10 fractions, for example between 1 and 10 fractions).

[0356] In a specific embodiment in which the subject has pancreatic cancer, the radiation therapy applied according to the methods described herein comprises exposing the subject to 6 sessions of MRI image-guided radiation therapy with an MR-Linac system, with a fraction of 8 Gy per session.

[0357] 2. Internal vectorized radiotherapy

[0358] According to an advantageous embodiment, the peptide complexes described in the present application can be activated by a radioactive source, for example a radionuclide (radioactive isotope) linked to a biological molecule capable of targeting a particular organ or tissue of a human body.

[0359] Internal radiotherapy treatments use nuclear medicine based on the injection of molecules containing a radioactive isotope (radionuclide). Radionuclides are atoms whose nuclei are unstable due to their relative composition of protons and neutrons. The main radionuclides used for therapy are emitters of α, γ, and Auger electrons. The therapeutic use of these radiations will depend on the energy and the distance they travel through matter.

[0360] Internal radiotherapy is based on the use of radionuclides emitting a, p, and y radiation to destroy cancer cells (Table 1). This type of radiotherapy is particularly used to treat small tumor masses. Different types of radioactive sources and internal radiotherapy techniques can be applied depending on the nature and location of the tumor. [Table 1 ] Internal radiotherapy involves administering, orally or intravenously, a radionuclide (radioactive isotope) coupled to a carrier molecule in order to specifically target and irradiate tumor cells. The ligands used can be antibodies or antibody fragments. The use of sufficiently selective carriers makes this technique particularly useful for treating small tumors and metastases spread throughout the body.

[0361] Thus, according to a particular embodiment of the invention, the peptide complexes described in the present application are activated by a radionuclide (radioactive isotope) linked to a biological molecule targeting the same antigen as the peptide complex or another antigen present on the cells of the tumor targeted by the peptide complex of the invention.

[0362] According to an advantageous embodiment of the invention, a pharmaceutical composition in particular comprising said peptide complexes is intended to be used in the manufacture of a medicament intended for the treatment of a tumor in a subject in need thereof, the treatment comprising

[0363] (i) administering a supramolecular tetrameric peptide complex containing a high Z element, and targeting a given antigen present on a tumor cell, and,

[0364] (ii) administering a radionuclide linked to an antibody or antibody fragment targeting the same said antigen as said supramolecular peptide complex or another antigen present on said same targeted tumor cell.

[0365] According to a particular embodiment of the invention, the supramolecular tetrameric peptide complex is administered before the radionuclide (radioactive isotope) bound to an antibody or antibody fragment. Preferably, the administration of the peptide complex is carried out from 12 hours to 24 hours before the administration of the radionuclide (radioactive isotope), preferably 24 hours.

[0366] According to another particular embodiment of the invention, the supramolecular tetrameric peptide complex and the radionuclide (radioactive isotope) bound to an antibody or antibody fragment are administered simultaneously. According to a particular embodiment of the invention, monoclonal antibodies specifically targeting antigens or receptors overexpressed on the surface of tumor cells can be used in immunotherapy in order to vectorize radioisotopes. This approach makes it possible to specifically target the tumor while reducing side effects on healthy tissues.

[0367] According to a particular embodiment of the invention, the antigens or receptors overexpressed on the surface of the tumor cells correspond to the antigens cited above.

[0368] According to another particular embodiment of the invention, the tumor markers can be expressed in different cancers or be specific to a precise tissue origin (table).

[0369] Table 2: Summary of the main tumor markers specific for epithelial tumors used clinically. (ACE: Carcino-Embryo Antigen, CA: Cancer Antigen, PSA - PAP Prostate Specific Antigen - Prostatic Acid Phosphatase, AFP [3HCG pHCG: Alphafetoprotein Beta- Chorionic Gonadotropin Hormone, BCMA: B-lymphocyte maturation antigen, TCT: Thyroglobulin Thyro-Calcitonin, NSE: Neuron Specific Enolase, SCC: Squamous Cell Carcinoma, TPA: Tissue Polypeptide Antigen)

[0370] 3. Contrast agents

[0371] In another embodiment of the invention, the peptide complex is used as a contrast agent.

[0372] Advantageously, supramolecular tetrameric peptide complexes are used as contrast agents for magnetic resonance imaging.

[0373] By contrast agent, we mean here any product or composition used in medical imaging with the aim of artificially increasing the contrast allowing the visualization of a particular anatomical structure (for example certain tissues or organs) or pathological anatomical elements (for example tumors) in relation to neighboring or non-pathological structures.

[0374] Imaging agent means any product or composition used in medical imaging for the purpose of creating a signal enabling a particular anatomical structure (for example certain tissues or organs) or pathological structure (for example tumors) to be visualized in relation to neighboring or non-pathological structures.

[0375] The operating principle of the contrast or imaging agent depends on the imaging technique used.

[0376] Imaging can be performed using magnetic resonance imaging (MRI), computed tomography (CT) imaging, positron emission tomography (PET) imaging, or any combination of these.

[0377] As used herein, the term "contrast agent" refers to a contrast agent that enhances contrast in MRI imaging.

[0378] In an advantageous embodiment of the invention, an imaging session according to the invention comprises:

[0379] (i) administering the supramolecular tetrameric peptide complexes containing a high Z element, preferably gadolinium, to a subject, and,

[0380] (ii) performing magnetic resonance imaging using a device for performing MRI imaging, known to those skilled in the art. Description of the figures

[0381] Figure 1A concerns the in vitro validation of the metal-based heterodimer library for active tumor targeting. SDS gel confirming the purity of E3@VHHs.

[0382] Figure 1 B represents the binding affinity of E3@VHHs.

[0383] Figure 1G represents a thermal shift test confirming self-assembly between K3 and E3@VHHs.

[0384] Figure 1D is for the in vitro validation of the binding affinity of various K3-E3@HER2 functionalized metals on HCC-1954 cells (HER2+ cell line).

[0385] Figure 1 shows the confirmation of the versatility of the binding affinity-based approach after replacement of anti-HER2 VHH with anti-CD38 VHH on KMS-18 cells (CD38+ cell line).

[0386] Figure 2A shows the in vitro radiosensitization properties of the metal@K3-E3@VHH complex dictated by the atomic number of the metal. A. ICP-MS measurements after 30 minutes of incubation of various metal@K3-E3@HER2 complexes on HCC-1954 cells.

[0387] Figure 2B shows the Quantification of yH2AX foci at early and late time points after radiotherapy after treatment with / without metal@K3-E3@HER2 on CHC-1954 cells.

[0388] Figure 2C is for a clonogenicity assay performed with metal@K3-E3@HER2 on HCC-1954 cells 30 minutes after incubation on HCC-1954 cells.

[0389] Figure 2D shows the quantification of reactive oxygen species (H2O2) without / with 6Gy irradiation with and without metal@K3-E3@HER2 metal incubated for 30 minutes before irradiation on HCC-1954 cells.

[0390] Figure 2E shows the Quantification of yH2AX foci at early and late time points after radiotherapy after treatment with / without metal@K3-E3@HER2 on SKOV3 cells.

[0391] Figure 2F shows a clonogenicity assay performed with metal@K3-E3@HER2 on SKOV3 cells 30 minutes after incubation.

[0392] Figure 2G is for the quantification of reactive oxygen species (H2O2) without / with 6Gy irradiation with and without metal@K3-E3@HER2 metal incubated for 30 minutes before irradiation on SKOV3 cells. Figure 3A is for the preclinical validation of the radiosensitization properties of metal@K3-E3@HER2 complexes during external beam radiotherapy. A. Biodistribution study performed by fluorescence imaging in the tumor and major organs. The inserts represent the relationship between the fluorescent signal and the ICP-MS quantification. The specificity of active tumor targeting is validated by a direct comparison with a non-specific complex (Bi@K3-E3@eGFP).

[0393] Figure 3B shows a pharmacokinetic profile of the three complexes confirming rapid elimination from the blood. The inserts represent the half-life (T1 / 2) and the area under the curve between 0 and 48 hours (AUC0-48h).

[0394] Figure 3C shows a treatment plan based on biodistribution and pharmacokinetic profile.

[0395] Figure 3D shows tumor growth involution with / without treatment with a single administration of Gd@K3-E3@HER2 or Bi@K3-E3@HER2 complexes. Hematoxylin and eosin staining of major organs at day 50 after study inclusion. Ex vivo yH2AX staining of different groups.

[0396] Figure 4A concerns the Gd@K3-E3@CD38 complex as a theranostic complex and for anti-BCMA radionuclide therapy. A. Treatment plan carried out for this study.

[0397] Figure 4B shows tumor dissemination validated by bioluminescence imaging (BLI), tumor-targeted efficacy validated by magnetic resonance imaging (MRI), and anti-BCMA@177Lu monitored by micropositron emission tomography (pPET).

[0398] Figure 4C shows longitudinal quantification of tumor uptake of anti-BCMA antibody-linked radionuclide therapy and Gd@K3-E3@CD38 complex.

[0399] Figure 4D shows a waterfall plot of tumor volume evolution at day 20 after Gd@K3-E3@CD38 administration.

[0400] Figure 4E shows associated Kaplan-Meier curves comparing treatment outcomes for different study groups.

[0401] Figure 4F is a biodistribution study of anti-BCMA antibody-linked radionuclide therapy and Gd@K3-E3@CD38 complex in heart, kidney, and liver. Figure 5A is a UV (272 nm), PDA (total scan), MS (TIC m / z = 100 - 2000) chromatogram of DBCO-Peg4-DOTAGA, DOTAGA-anhydride (7.87 mg, 17.2 pmol, 1.8 eq) was added to the DBCO-Peg4-NH2 solution (350 ml, 27.4 mM, 9.6 pmol, 1.0 eq). DIPEA (10 µl) was added before the MR was diluted with DMSO (50 µl) and sonicated.

[0402] Figure 5B concerns a mass spectrum (integrated over the main peak in TIC) of DBCO-Peg4-DOTAGA

[0403] Figure 5C is a UV (272 nm), PDA (full scan), MS (TIC m / z = 100 - 2000) chromatogram of DBCO-Peg4-DOTAGA-Gd. To an aq. solution of DBCO-Peg4-DOTAGA (150 µl, 1.0 mM, 90 nM, 1.0 eq) was added aq-NH4Cl (1.6 µl, 1.0 M, 11 eq) and aq-GdCl3 (1.6 µl, 100 mM, 1.1 eq). Complexation was monitored by MS. After reaction overnight at 20°C, complete complexation was observed. No decomplexation in LC-MS was observed nor Gd exchange in the presence of excess Cu(ll).

[0404] Figure 5D concerns a mass spectrum (integrated over the main peak in TIC) of DBCO-Peg4-DOTAGA-Gd.

[0405] Figure 5E is an HRMS of the doubly charged DBCO-Peg4-DOTAGA-Gd complex and simulation of the isotopic pattern.

[0406] Figure 5F concerns the synthesis of K3-DOTAGA-Gd. To a solution of K3-Azide in PBS (4.3 mg / ml, 50 μl, 35 nmol, 1.0 eq) was added an aq solution. of DBCO-Peg4-DOTAGA-Gd (72 μl, 1.0 mM, 72 nmol, 2.0 QE). Mass spectrum (direct infusion) of K3-DOTAGA-Gd. Very pronounced z = 6 - 9 signals

[0407] Figure 5G is SEC-HRMS of E3@HER2. Deconvolution. E3-dimers (B).

[0408] Figure 5H is SEC-HRMS of K3-Dotaga-E3@HER2. Deconvolution. E3-dimers (A), E3-dimers +1 K3 (B), and K3 / E3 heterotetramers (C).

[0409] Experimental part

[0410] Expression and purification of recombinant peptide structures

[0411] The peptide structures as described in the application, in particular the first peptide structure, correspond to fusion proteins comprising an antibody or an antibody fragment covalently linked to a peptide oligomerization domain.

[0412] The peptide structure of formula A-L1-P1-L2 is represented in the experimental part by E3@VHHs (E3 corresponds to P1, and VHH corresponds to A; for convenience the spacers L1 and L2 are not considered in the abbreviated form of the first peptide structure).

[0413] The peptide structure of formula P2-L3-B@(M) is represented in the experimental part by Metal@K3 (K3 corresponds to P2, and Metal corresponds to B@(M); for convenience the spacer L3 and the chelating group B are not considered in the abbreviated form of the second peptide structure).

[0414] E3@VHHs peptide structures (i.e., E3@HER2, E3@CD38, and E3@eGFP) were expressed in E. coli BL21 (DE3) pLysS at 20 °C by autoinduction in ZYM-5052 medium. Recombinant peptide structures were purified on cobalt-saturated HiTrap™ chelating resin (1 mL) (GE Healthcare Saclay, France) and then loaded onto a Superdex 200 Increase 10 / 300 GL column (GE Healthcare, Biosciences AB, Sweden) operating at a flow rate of 0.4 mL / min. Fractions were separated on SDS-PAGE gradient gels and analyzed by Coomasie blue.

[0415] The DNA sequences encoding the recombinant E3@VHHs peptide constructs were synthesized by Integrated DNA Technologies (IDT) and inserted into the pETOM plasmid containing a c-myc sequence, resulting in the pETOM-E3@VHHs plasmids, respectively. The sequences of DNA constructs were verified by DNA sequencing.

[0416] <h2 style=";text-align:left;direction:ltr">The sequences E3@VHH uses with its help:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0417] <h2 style=";text-align:left;direction:ltr"> E3@GFP :<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0418] <h2 style=";text-align:left;direction:ltr"> MGCSQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREVWA GMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEY WGQGTQVTVSSSGRGSKLASTPLGDTTHTSGNNTSSSPQPKKKPLDGEYFTLQIR GRERFEMFRELNEALELEDAQAGKEPGGSGGARYRTSHHHHHHH (SEQ ID NO: 18)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0419] <h2 style=";text-align:left;direction:ltr"> E3@HER2:<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0420] <h2 style=";text-align:left;direction:ltr"> MECVQLVESGGGLVQAGGSLRLSCATSGITFMRYALGWYRQSPGKQREMVASINS GGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNARWVKPQFIDNNY WGQGTQVTVSSSGRGSKLASTPLGDTTHTSGNNTSSSPQPKKKPLDGEYFTLQIR GRERFEMFRELNEALELEDAQAGKEPGGSGGARYRTSHHHHHH (SEQ ID NO: 19)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0421] <h2 style=";text-align:left;direction:ltr"> E3@CD38 :<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0422] <h2 style=";text-align:left;direction:ltr"> MACQVQLQESGGGLVQPGGSLRLSCAASGIILRIYDMGWYRQAPGKQRELVAAITS RGSTNYADSVKGRFTISRDNAENTVSLQMNSLKPGDTAVYYCNADHTFAGVYWGQ GTQVTVSSSGRGSKLASTPLGDTTHTSGNNTSSSPQPKKKPLDGEYFTLQIRGRER FEMFRELNEALELEDAQAGKEPGGSGGARYRTSHHHHHH (SEQ ID NO: 20)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0423] Separation and analysis of proteins by non-denaturing polyacrylamide gel electrophoresis

[0424] The complexes were resolved on a non-denaturing polyacrylamide gradient gel (5-20%) at pH 8.8 and analyzed by the Typhoon FLA 9500 biomolecular imager (GE Healthcare) at 470 and 555 nm.

[0425] Preparation of the second peptide structure Metal@K3 (Chemical conjugate)

[0426] The following K3 peptide: Asn-Asn-Thr-Ser-Ser-Ser-Pro-GIn-Pro-Lys-Lys-Lys-Pro-Leu- Asp-Gly-Glu-Tyr-Phe-Thr-Leu-GIn-lle-Arg-Gly-Arg-Glu-Glu-Phe-Arg-Lys-Lys-Phe- Arg-Lys-Asn-Lys-Ala-Leu-Glu-Leu-Lys-Asp-Ala-GIn-Ala-Gly-Lys-Glu-Pro-Gly, synthesized by Polypeptide Group, France is conjugated with Azido-PEG4-C2-carboxylic acid (p53tet-E343K / E346K-azido-PEG4-Nter).

[0427] NNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRKLNKALELKDAQAGKEPG (SEQ ID NO: 21)

[0428] Step 1: DBCO + DOTA-GA anhydride + Gd

[0429] 78 μl at 100 mM (7.8 pmol) of DBCO-PEG4-amine (Broadpharm) is added in 282 μl of DMSO. 15 mg (32.5 pmol) of DOTA-GA anhydride (Chematech) is added. 7 ml at 5 mM (35 pmol) of gadolinium (GdCl3.6H2O, Alfa Aesar) is then added (stirring for 4 h). 51

[0430] Step 2: K3 grafting

[0431] DBCO reacts with K3 azide to produce D0TAGA-K3 (stirring for 2 hours at room temperature).

[0432] 1.3 pmol of K3 (K3-azido-PEG4-Nter, PolyPeptide) is added into 1.851 ml of PBS. The solution from step 1 is added dropwise (with stirring overnight)

[0433] Step 3: 3kDa purification with PBS (x64) to obtain the final K3@DOTAGA(Gd3+).

[0434] Obtaining supramolecular tetrameric peptide complexes

[0435] The heterotetramers represented in the experimental part by Metal@K3- E3@VHHs were formed by mixing the recombinant peptide structures E3@VHHs and Metal@K3 with a molar ratio of 1:1, for 1 h at room temperature. Purification was carried out by centrifugation.

[0436] Cell lines. Human breast cancer cells (MDA-MB-231, HCC-1954) were obtained from the American Type Culture Collection (ATCC, USA). Myeloma cancer cells (MM.1 S, KMS12-BM) were generously provided by the Ghobrial laboratory (Dana-Farber Cancer Institute, Boston, MA). Fetal bovine serum (FBS) was purchased from VWR.® MDA-MB-231, KMS18, KMS12-BM, and MM.1 S cell lines were cultured in RPMI 1640 with L-glutamine (#L0500-500, VWR, France). HCC-1954 and SKOV3 cells were cultured in RPM1 1640 with L-glutamine and HEPES (#10-041-CV / 702523, Dutsher). All media were supplemented with 10% FBE and 10,000 units / ml of penicillin and streptomycin (PS, #P06-07100, Dutsher). All cell lines were maintained in a humidified incubator supplied with 5% CO2, maintained at 37°C and regularly tested for mycoplasma contamination.

[0437] In vitro binding assays. The in vitro specificity of the different metal@K3-E3@VHH complexes was assessed by flow cytometry. For flow cytometric analysis, 1 to 2x10 5 Cells per condition were treated with concentrations ranging from 100 nM to 1 pM in FACS buffer (PBS, 2% FBS, 2 mM EDTA) for 5 minutes at room temperature. Cells were then washed three times with PBS, with centrifugation at 300 * g for 5 minutes between each wash. Flow cytometry data acquisition was performed using the MACSQuant® Analyzer 10 flow cytometer (Miltenyi), and subsequent analysis was performed using FlowJoTM software (V. 10.8.2). All results were normalized by subtracting the passive effect obtained with E3@eGFP.

[0438] DNA damage assays. All metal@K3-E3@VHHs treatment groups were incubated at (0.4 mg / mL metals) in HCC1954 or SKBR3 cells overnight before irradiation with 2 Gy. At 30 min or 24 h after treatment, the treated cells were then fixed with 4% paraformaldehyde in PBS for 30 min. The fixed cells were blocked with 1% bovine serum albumin, 10% fetal calf serum, and 0.3% tritonX-100 in PBS before being stained overnight at 4 °C with an anti-yH2AX antibody (Millipore). Following previously reported protocols, secondary anti-mouse AlexaFluor-594 (Abeam)-conjugated IgG was used for staining, and micrographs were obtained using an upright Carl Zeiss microscope with an HXP 120C light source and a 63x / 1.4 plan-apochromat oil objective. Semi-quantitative analyses were conducted to compare the number of foci per cell expressing yH2Ax.Signal intensities of individual foci were quantified using CelIProfiler cell imaging software (version 3.1.8).

[0439] Clonogenic assays. The different metal@K3-E3@VHH treatment groups evaluated (0.4 mg / mL metals) were incubated with HCC1954 and SKBR3 cells overnight before cell irradiation with a dose of 2, 4, 6, 8, or 10 Gy, which was delivered with a 220 kVp X-ray beam (Small Animal Radiation Research Platform (SARRP), Xstrahl). Following previously reported protocols, treated cells were then incubated for 6 hours, then counted, reseeded into 10 cm dishes (at 450 cells per plate), and allowed to grow for an additional 10 days before being stained with a dye solution composed of 1% crystal violet in 10% ethanol; clones were then manually counted.

[0440] Reactive oxygen species assays. A total of 10,000 cells / well of HCC1954 or SKBR3 were seeded into 96-well plates and cultured for 24 hours. The cells were then incubated with different concentrations of metal@K3-E3@VHH complexes overnight and washed with PBS to remove metal@K3-E3@VHH complexes that were not internalized by the cells. Next, the cells were incubated with 10 μM dihydrorhodamine 123 (DHR123) for 3 hours. Before irradiation, the cells were washed with PBS to remove excess DHR. Irradiations were performed with a single irradiation fraction of 4 Gy using 220 kVp irradiation (SARRP). The fluorescence signal was measured 3 h after irradiation using a plate reader (SpectraMax ID5, Molecular Devices) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Mouse studies All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals as defined by the Institutional Care and Use Committee of the University of Strasbourg (APAFIS: # 38306-2022082410083076 V5). Adult female BALB / c and BALB / c nude mice (Charles River, France; 6–8 weeks old) were acclimated upon arrival, maintained at a constant temperature of 22°C, and exposed to a 12–12-hour light-dark cycle for at least 7 days before the start of any experimentation. They were maintained in a pathogen-free environment with continuous access to food and water throughout the experimental procedures.

[0441] Tumor mouse models Subcutaneous breast cancer model. A total of 1,5.10 6HCC-1954-Luc+ cells were mixed at a 1:1 (v / v) ratio with growth factor-reduced Matrigel (Perkin Elmer) and injected subcutaneously into the flanks of mice (balb / c nude mice, female, 6-8 weeks old, Charles River Lab) to generate tumors. Tumor volume was measured using a caliper and calculated as follows: [(width) 2 x length] / 2. Mice were randomly assigned to study groups once the tumor reached 150 mm 3 of volume. A multiple myeloma (MM) xenograft mouse model was established by intravenously injecting 1.5 million MM.1 SLUC+ cells into the tail veins of 6- to 8-week-old female SCID / beige mice (from Charles River Laboratory). Tumor growth was assessed weekly using bioluminescence imaging (BLI) with the IVIS Spectrum system from Perkin Elmer, and data were collected using Living Image 4.5. Upon detection of a tumor signal in the spine, mice were randomly assigned to treatment groups. Weekly animal health checks were performed based on the endpoints. Treatment was discontinued after 12 days, and study endpoints included hindlimb paralysis or loss of greater than 20% of initial body weight.

[0442] Studies of and biodistribution Pharmacokinetic and biodistribution studies were conducted in healthy mice and tumor-bearing mice after intravenous injections of 0.03 pmole / kg (based on the amount of dye) of metal@K3-E3@HER2, metal@K3-E3@CD38, and metal@K3-E3@eGFP complexes. Mice were euthanized at various time points: 30 minutes, 1 hour, 3 hours, 6 hours, 24 hours, 48 ​​hours, and 72 hours, with three mice per group at each time point. Blood samples were collected by cardiac puncture after euthanasia and stored in EDTA-coated tubes. Simultaneously, all harvested organs were imaged ex vivo using an IVIS system (PerkinElmer) for biodistribution analysis and then analyzed by ICP-MS for the evaluation of the correlation between the two quantification modalities.To quantify tissue fluorescence and generate biodistribution profiles, collected organ homogenates were transferred to a 96-well plate and subjected to fluorescence imaging following the same experimental procedure.

[0443] Blood samples were subjected to ICP-MS processing to analyze the pharmacokinetics (PK) of the blood compartment, which was then fitted into a two-component model using standard procedures. The biodistribution study with HCC-1954 tumor-bearing mice (n = 3 / group) was performed on a 7 Tesla BioSpec 70 / 20 preclinical MRI scanner (Bruker BioSpin, USA). For in vivo studies, an equivalent dose of 0.5 mM Gd@K3-E3@VHH (HER2 for the HCC-1954 mouse model or CD38 for the MM.1 S mouse model) was injected intravenously. A T1 RARE-VTR sequence using a repetition time of 9000 ms, an echo time of 19.6 ms, and a flip angle of 180° was used. The acquisition matrix size and the reconstructed matrix were 400 x 200 pixels, the field of view was 200 x 200 μm 2and the slice thickness was 3 mm. Animals were pre-scanned and then imaged at 2, 15, 30, and 45 min, and 1, 3, and 6 h after Gd@K3-E3@VHH injection. A region of interest was drawn through the tumor and other vital organs, and the T1 contrast was measured and correlated to its respective calibrations (ParaVision (v. 5.1)). For T1 phantom relaxivity measurements, different dilutions of Gd@K3-E3@VHH (0.01 mM to 0.5 mM) were prepared in deionized water and imaged using the exact setup above. Signal amplitudes were calculated for the average, and T1 relaxivity measurements were calculated for absolute quantification of Gd@K3-E3@VHH.

[0444] Bioluminescence imaging. 1 million MM.1S cells were administered intravenously. To monitor tumor dissemination, mice were injected (ip) with luciferin (100 μl) and anesthetized with 5% isoflurane. The animals were then randomly assigned to each study group, and tumor dissemination was monitored by bioluminescence signal using the MS system (Perkin Elmer).

[0445] External beam radiotherapy. Using the HER2+ mouse mode, mice received a single dose of each group of metal@K3-E3@VHH complex by intravenous tail injection 30 minutes before irradiation with 5 fractions of 2Gy, which were delivered with a 220 kVp X-ray beam collimated to the tumor size and using the SARRP platform. Tumor size was then monitored daily using a caliper, and animals were removed from the study when their tumor reached >3 cm in the longest axis, when they showed a body weight loss of >15%, or when they were moribund.

[0446] Toxicity studies. Daily monitoring of animal body weight began on the day of injection. After a period of 21 days, the mice were euthanized, and blood samples were collected by submandibular puncture for the evaluation of basic metabolic profiles, complete blood count, and differential white blood cell count. Subsequently, major organs were removed, subjected to H&E staining, and submitted for histological analyses by a board-certified veterinary pathologist.

[0447] Statistical analysis. Data are represented as mean ± standard deviation. All results were analyzed using GraphPad Prism 9.5.1. Statistical analysis was performed using an unpaired nonparametric t-test for comparison of multiple groups at a single time point (Mann-Whitney test) or one-way ANOVA for comparison of groups. Significance was determined at the following thresholds: *P<0.05, **P<0.01, and ***P<0.001.

[0448] Results and discussion

[0449] Synthesis and characterization of the targeting specificity of the metal@K3- library

[0450] E3@VHH. Study com in vitro radiosensitization in the context of a external. To ensure fair biological comparisons between distinct metal@K3-E3@VHH complexes, a comprehensive longitudinal uptake study was first conducted using HER2+ breast cancer cells (HCC-1954), incubated with 1 mM concentrations of the peptides (Figure

[0451] 2A). In each formulation, HER2+ receptors were targeted using the E3@HER2 peptide. Quantification of metal concentrations by inductively coupled plasma mass spectrometry (ICP-MS) revealed an initial increase in binding within the first 15 minutes of incubation, followed by a gradual decrease in signal intensity over time, attributed to an exocytosis phenomenon. As a result, each formulation showed comparable behavior throughout the study duration, confirming the feasibility of evaluating the radiosensitization properties of each formulation in subsequent radiotherapy studies.

[0452] For the comparative analysis of radiosensitization effects on cells pre-exposed to distinct metal@K3-E3@HER2 complexes, a quantitative study of DNA double-strand break (DSB) repair was conducted, evaluating the abundance of gH2AX foci in two HER2+ cell lines (HCC-1954 and SKBR3). Consistent with the results associated with various radiosensitizers (radioenhancers), a significant increase in acute and residual gH2AX foci was demonstrated in all groups subjected to irradiation combined with metal treatments compared to untreated but irradiated cells (P value <0.05) (Figure 2B).During the acute phase following exposure to a dose of 4 Gy XRT, no discernible disparity was noted between the different metallic peptides, while significant differences were observed in residual gH2AX foci directly correlated with the atomic number of the metallic element used. Subsequently, clonogenic assays were performed to elucidate the link between residual double-stranded DNA damage and overall cell viability (Figure 2C). Consistently, an inverse relationship was noted, in which peptides charged with higher Z numbers were correlated with decreased cell viability, reaffirming the influence of elemental physics governing the characteristics of these radiosensitizing agents.Finally, these results were supported by the quantification of the resulting reactive oxygen species (ROS), through the evaluation of hydrogen peroxide (H2O2) species, where significant differences were observed between all metallic formulations and directly related to the atomic numbers Z (Figure 2D).

[0453] The enhancement of fractionated external beam radiation is dictated by the atomic number of the metal@K3-E3@HER2 complex in the HER2+ breast cancer model.

[0454] Following these in vitro results, the therapeutic efficacy of these formulations was validated in the context of subcutaneous HER2+ breast cancer xenograft tumor models. A comparison of Gd@K3-E3@HER2 and Bi@K3-E3@HER2 peptides was performed as both exhibited interesting imaging characteristics; the former was detectable by magnetic resonance imaging (MRI) and the latter was detectable by computed tomography (CT) in addition to being visible by fluorescence imaging (FLI) upon functionalization with a Cy5.5 dye.Based on the fluorescence signal and ex vivo quantification by ICP-MS, which show a linear correlation, the study of biodistribution in the main organs, as well as in tumors, reveals a pattern similar to that observed with ultra-small nanoparticles used as radiosensitizing agents (radioenhancers), showing high uptake in the kidneys 24 hours after intravenous administration, independent of VHH or metal functionalized to E3-K3 heterodimers.

[0455] Significantly, in the HER2+ breast cancer model, Gd@K3-E3@HER2 and Bi@K3-E3@HER2 complexes demonstrated significantly enhanced tumor uptake compared to non-targeted peptides (Gd@K3-E3@GFP), with values ​​of 10.8 ± 0.4, 11.1 ± 0.6, and 2.3 ± 0.6 %ID / g, respectively, 24 hours after intravenous administration (Figure 3A). For all three compounds, the pharmacokinetic (PK) profiles remained similar, with no statistically significant differences in half-life or area under the curve between 0 and 48 hours (corresponding to the last ICP-MS quantification data point) (Figure 3B). Furthermore, a therapeutic study was conducted using a fractionated radiotherapy plan of 5x2Gy 24 hours after a single administration of radioenhancers (Figure 3C). After tumor growth (Figure 3D), all non-irradiated groups behaved similarly.When irradiated, the untreated group exhibited tumor outgrowth similar to that of the group treated with the nonspecific radioenhancer Gd@K3-E3@GFP, with a slight delay during the first 30 days after randomization into the study. Importantly, both Gd@K3-E3@HER2 and Bi@K3-E3@HER2 radioenhancers significantly shrank tumors compared to irradiated mice (P-value < 0.001). More interestingly, consistent with the in vitro results, Bi@K3-E3@HER2 outperformed Gd@K3-E3@HER2 (P-value < 0.01). Ex vivo, at day 60 after randomization, hematoxylin and eosin (H&E) staining was performed on all major organs as well as tumors to assess potential side effects resulting from the presence of radiosensitizing agents in local organs.No anatomopathological differences were observed between all groups, except in mouse tumors irradiated with Bi@K3-E3@HER2, where a significant amount of necrosis was evident, confirming an increase in local dose deposition (Figure 3E). Furthermore, ex vivo fluorescent imaging of yH2AX confirmed the increased amount of DNA damage induced by both radiosensitizing agents (Figure 3F). These results support the potential of both peptide-based tumor-targeting radiosensitizing agents. Importantly, these results suggest that within the same scaffold, metal selection plays a crucial role in enhancing radiosensitizing properties.

[0456] Proof-of-concept study of internal radioligand (radionuclide) radiotherapy enhanced with Gd@K3-E3@CD38 in a mouse model of multiple myeloma.

[0457] In the present study, the use of Gd@K3-E3@CD38 was validated in a preliminary proof-of-concept study for BCMA@177Lu-enhanced radioligand (radionuclide) internal radiotherapy in the setting of multiple myeloma (Figure 4A). Accordingly, bioluminescence imaging was used to validate tumor dissemination in mice and determine the optimal randomization time, magnetic resonance imaging was used to monitor tumor uptake of Gd@K3-E3@CD38, and positron emission tomography (PET) was performed to monitor radioligand (radionuclide) internal radiotherapy. Based on pharmacokinetic (PK) profiles and MRI, BCMA@177Lu antibody was administered intravenously 24h after radioenhancers.This time point was chosen based on the highest tumor uptake of Gd@K3 to E3@CD38 24 hours after administration with tumor retention reaching a plateau for approximately 96 hours (Figure 4B). Thanks to the long tumor retention offered by the targeted peptide, it was observed through waterfall diagrams performed at day 25 (Figure 4C) a significant shrinkage of the tumor volume when the mice were treated with the radioenhancers. This tumor shrinkage was directly related to a significant survival (6 out of 6 mice alive at day 60 and 4 complete responses) (P-value < 0.001) (Figure 4D). Regarding toxicity in the main organs, although the Gd@K3-E3@CD38 peptide complex was still present in the heart, at the time of administration of the radioligand (radionuclide linked to an antibody), renal and hepatic elimination was observed.Therefore, there was no nonspecific toxicity, as observed in hematoxylin and eosin (H&E) staining at day 25 (Figure 4F).

[0458] Conclusion

[0459] The results presented here not only advance the understanding of the physical aspects of radiosensitization mechanisms, but also have the potential to inform the development of future radiosensitizing agents, thus contributing to the continued evolution of cancer treatment strategies. The feasibility of developing a first-in-class peptide-based radiosensitizing agent with controlled metal loading was demonstrated, and we were thus able to compare for the first time different metals and their radiation-enhancing properties in biological models. Finally, this study confirmed that the targeted peptide platform described here enhances external and internal radiation therapy.The improved tumor retention induced by the tumor-targeting properties of the peptide platform allows for a single administration to improve fractionated radiotherapy plans, justifying direct interest in clinical settings. In addition, the proof-of-concept study was validated by demonstrating the possibility of using this platform to improve the efficacy of radioligand-based therapies (radionuclides linked to an antibody) in multiple myeloma tumor models. Such an approach can lead either to a decrease in radiotracer concentrations or to a local increase in therapeutic efficacy depending on the clinical need in order to improve the therapeutic window.

Claims

Claims 1. Supramolecular tetrameric peptide complex comprising: (a) a first structure represented by the following formula (I): (I) (A)n-(L1)x-P1-(L2)x'-(A')rï preferably A-L1-P1-L2 or A-L1- 1 , in which A and A are an antibody or antibody fragment P1 is a first peptide comprising at least one oligomerization domain, preferably tetramerization domain, L1 and L2 are spacers that independently represent a carbon chain or a peptide, with n being an integer of 1 or 2, and n' being an integer of 0 or 1 or 2 x and x' independently represent a number of 0 or 1, (b) a second structure represented by the following formula (II): (II) P2-L3-[B@(M)] m in which M represents a metal cation B is a chelating group binding at least one metal cation of a heavy metal (M) having an oxidation state (M 2+ , M 3+ , M 4+ ) having an atomic number Z greater than or equal to 40, in which the metal cation (M 2+ , M 3+ , M 4+ ) is selected from Au, Ag, Pt, Pd, Sn, Ta, Zr, Tb, Tm, Ce, Dy, Er, Eu, La, Nd, Pr, Lu, Yb, Bi, Hf, Ho, Pm, Sm, In and Gd, and mixtures thereof, preferably the metal cation is chosen from the Gd cation 3+ , the Hf cation 4+ , and the cation Bi +3 . P2 is a second peptide comprising at least one oligomerization domain, preferably tetramerization domain, L3 is a spacer, which preferably comprises a group selected from a [1,2,3]-triazole, isoxazole, isoxazoline, oxadiazoles, a pyrazole, a dihydropyrazine, an amide, a maleimide, a hydrazone and an oxime, with m being an integer of 1, 2, 3 or 4, preferably 1 or 2, @ represents a non-covalent bond between the chelating group and the metal cation of a heavy metal (M) having an oxidation state (M 2+ , M 3+ , M 4+ ) wherein the first and second peptides P1 and P2 comprise a tetramerization domain selected from the group consisting of peptide sequences comprising: - the tetramerization domain of human p53 protein (SEQ ID NO: 1) GEYFTLQIRGRERFEMFRELNEALELKDAQA - the tetramerization domain of the mutant protein p53E343K (SEQ ID NO: 2) GEYFTLQIRGRERFEMFRKLNEALELKDAQA - the tetramerization domain of the mutant protein p53E346K (SEQ ID NO: 3) GEYFTLQIRGRERFEMFRELNKALELKDAQA - the tetramerization domain of the mutant protein p53E343K / E346K (SEQ ID NO: 4) GEYFTLQIRGRERFEMFRKLNKALELKDAQA - the tetramerization domain of the mutant protein p53K351 E (SEQ ID NO: 5) GEYFTLQIRGRERFEMFRELNEALELEDAQA - the tetramerization domain of p63 (SEQ ID NO: 6); DELLYLPVRGRETYEMLLKIKESLELMQYLP - the tetramerization domain of p73 (SEQ ID NO: 7); EDTYYLQVRGRENFEILMKLKESLELMELVP - the tetramerization domain of the Xenopus laevis p53 protein (SEQ ID NO: 8); EEIFTLRIKGRSRYEMIKKLNDALELQESLD - the tetramerization domain of the rainbow trout p53 protein (SEQ ID NO: 9) DEIYTLQIRGKEKYEMLKKFNDSLELSELVP or peptide mimetics, analogs, or homologs of SEQ ID NO: 1 to 9, which associate and form primary homodimers and / or primary heterodimers, which associate and form "dimers of dimers", and wherein the peptide complex is a radiosensitizing agent or a contrast agent.

2. A supramolecular tetrameric peptide complex according to claim 1, wherein the first and second peptides P1 and P2 which are different and represent either the p53E343K / E346K mutant (SEQ ID NO: 4), or the p53E351 K mutant (SEQ ID NO: 5), or mimetic peptides, analogs, or homologues thereof, associate and form primary homodimers and / or primary heterodimers, which associate and form "dimers of dimers" of formula (III): and (III) @@ represents non-covalent bonds between the α-helical tetramerization domains of peptides P1 and P2.

3. Supramolecular tetrameric peptide complex according to claim 1, wherein the antibody or antibody fragment is selected from a monoclonal antibody, a VHH, or a scFV, preferably a VHH.

4. Supramolecular tetrameric peptide complex according to claim 3, wherein the antibody or antibody fragment is directed against at least one antigen chosen from: the antigens of the cluster of differentiation (CD), the identification number of which varies between CD1a and CD363 and is preferably chosen from CD3, CD4, CD13, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD30, CD31, CD33, CD34, CD37, CD38, CD39, CD40, CD44, CD47, CD52, CD56, CD66e, CD70, CD72, CD73, CD74, CD79, CD79b, CD80, CD86, CD117, CD138, CD194, CD205, CD227 or CD248, even more preferably CD3, CD16, CD28 and CD38, or 4-1 BB, 5AC, 5T4 (trophoblastic glycoprotein, TPBG, 5T4, Wnt-activated inhibitory factor 1 or WAIF1), adenocarcinoma antigen, AGS-5, AGS-22M6, activin receptor-like kinase 1, AFP, AKAP-4, ALK, Alpha intergrin, Alpha v beta6, androgen receptor, angiopoietin 2, angiopoietin 3, annexin A1, AOC3 (VAP-1), B7-H3, BAFF (B-cell activating factor), BCMA, B-lymphoma cell, bcr-abl, Bombesin, BORIS, C5, C242 antigen, CA125 (carbohydrate antigen 125, MUC16), CA-IX (or CAIX, carbonic anhydrase 9), CALLA, CanAg, CCL11 (CC motif chemokine 11), CCR4 (CC chemokine receptor type 4, CD194), CCR5, CD3E (epsilon), CEA (Carcinoembryonic antigen), CEACAM3, CEACAM5 (carcinoembryonic antigen), CFD (Factor D), Ch4D5, Cholecystokinin 2 (CCK2R), CLDN18 (Claudin-18), dumping factor A, cMet, CRIPTO, FCSF1 R (Colony stimulating factor 1 receptor, CD115), CSF2 (colony stimulating factor 2, granulocyte-macrophage colony stimulating factor (GM-CSF)), CSP4,CTLA4 (cytotoxic T lymphocyte-associated protein 4), CTAA16.88 tumor antigen, CXCR4 (CD184), chemokine CXC receptor type 4, cyclic ADP ribose hydrolase, cyclin B1, CYP1 B1, DLL3 (delta-like-ligand 3), DLL4 (delta-like-ligand 4), DPP4 (dipeptidyl-peptidase 4), DR5 (TRAIL-R2), ED-B, EGFL7 (EGF-like domain-containing protein 7), EGFR, EGFRII, EGFRvIII, Endoglin (CD105), Endothelin B receptor, Endotoxin, EpCAM (epithelial cell adhesion molecule), EphA2, Episialin, ERBB2 (Epidermal Growth Factor Receptor 2), ERBB3, ERG (TMPRSS2 fusion gene) ETS), ETV6-AML, FAP (Fibroblast activation protein alpha), FCGR1, alpha-fetoprotein, fibrin II, beta chain, fibronectin extra domain-B, FOLR (folate receptor), folate receptor alpha, folate hydrolase, Fos-related antigen, Frizzled receptor, Fucosyl GM1. Ganglioside GD2, G-28, GD3, GloboH, Glypican 3, N-glycolylneuraminic acid, GM3, GMCSF receptor a-chain,growth differentiation factor 8, GP100, GPNMB (transmembrane glycoprotein NMB), GUCY2C (guanylate cyclase 2C, guanylyl cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase-C receptor, HER1 (human epidermal growth factor receptor 1), HER2, HER2 / neu, HER3 (ERBB-3), lgG4, HGF / SF (hepatocyte growth factor / scattering factor), HHGFR, HIV-1, histone complex, HLA-DR (human leukocyte antigen), HLA-DR10, HLA-DRB, HMWMAA, Hsp90, hTERT, ICAM-1 (intercellular adhesion molecule 1), Idiotype, IGF1 R (IGF-1, insulin-like growth factor receptor 1), IGHE, IFN-y, IL-1, IL-2 receptor (interleukin 2 receptor), IL-4, IL-5, IL-6, IL-6R (interleukin 6 receptor), IL-9, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-20, IL-22, IL -23, IL31 RA, ILGF2 (insulin-like growth factor 2), integrins (a4, allb|33, aV[33, a4[37, a5pi , a6p4, a7p7, alip3, a5p5, avp5), interferon gamma-induced protein, ITGA2, ITGB2, KIR2D, LCK,Legumain, LFA-1 (lymphocyte function-associated antigen 1, CD11a), LHRH, LINGO-1, LMP2, LTA, MAD-CT-1, MAD-CT-2, MAGE-1, MAGE-2, MAGE-3, MAGE A1, MAGE A3, MAGE 4, MARTI, MCP-1, MIF (macrophage migration inhibitory factor or glycosylation inhibitory factor (GIF)), MS4A1 (membrane-spanning 4-domains subfamily A member 1), MSLN (mesothelin), MUC1 (cell surface-associated mucin 1 (MUC1) or polymorphic epithelial mucin (PEM)), MUC1-KLH, MUC16 (CA125), MCP1 (monocyte chemotactic protein 1), MelanA / MART1 , ML-IAP, MPG, MS4A1 (membrane-subfamily A spanning 4 domains), MYCN, myelin-associated glycoprotein, myostatin, NA17, NARP-1 , NCA-90 (granulocyte antigen), Nectin-4 (ASG-22ME), NGF, Neural apoptosis-regulated proteinase 1 , NOGO-A, Notch receptor, nucleolin, Neu oncogene product, NY-BR-1 , NY-ESO-1 , OX-40, OxLDL (oxidized low-density lipoprotein), OY-TES1 , P21 , non-mutant p53, P97, Page4 , PAP,anti-(N-glycolylneuraminic acid) paratope, PAX3, PAX5, PCSK9, PDCD1 (PD-1, CD279), PDGF-Ra (platelet-derived growth factor receptor alpha), PDGFR-p, PDL-1, PLAC1, PMEL 17, Proteinase3 (PR1), PSMA, PSA, PSCA, Rabies virus glycoprotein, RHD (Rh polypeptide 1 (RhPI), CD240), RANKL, RhoC, mutant Ras, RGS5, ROBO4, RON, ROR1, SART3, sclerostin, SLAMF7, SSX2, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), STEAP2, STn, TAG-72 (tumor associated glycoprotein 72), survivin, T cell receptor, protein T cell transmembrane, TEM1 (tumor endothelial marker 1), TENB2, TGF-a, TGF-p (transforming growth factor beta), TGF- [31,TGF-|32 (transforming growth factor beta 2), Tie (CD202b), Tie2, TIM-1 (CDX-014), TNF, TNF-a, TNFRSF8, TNFRSF10B (transforming growth factor receptor superfamily member 10B, necrosis factor receptor superfamily member 13B), TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblastic glycoprotein), TRAIL-R1 (Tumor necrosis aporosis Inducing ligand Receptor 1), tumor-associated calcium signal transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, Tyrosinase, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2 or vimentin, WT1, XAGE 1, TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblastic glycoprotein), TRAIL-R1 (Tumor necrosis aporosis Inducing ligand Receptor 1 ), TRAILR2 (Death receptor 5 (DR5 )), TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309 ), VEGFR-1 , VEGFR2, or vimentin, WT1 , XAGE 1 , TNFRSF10B (tumor necrosis factor receptor superfamily member 10B),TNFRSF-13B (tumor necrosis factor receptor superfamily member 13B), TPBG (trophoblastic glycoprotein), TRAIL-R1 (Tumor necrosis aporosis Inducing ligand Receptor 1), TRAILR2 (Death receptor 5 (DR5)), tumor-associated calcium signal transducer 2, tumor-specific glycosylation of MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, or vimentin, WT1, XAGE 1, MUC1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, WT1, XAGE 1, TWEAK receptor, TYRP1 (glycoprotein 75), TRP-2, VCAM-1 (CD106), VEGF, VEGF-A, VEGF-2 (CD309), VEGFR-1, VEGFR2, WT1, XAGE 1, NKG2D, NKp46, and GPRC5D, preferably the antibody or antibody fragment is selected from a) A VHH directed against the HER2 antigen, or CD38 (preferably of sequences respectively SEQ ID NO: 10,or SEQ ID NO: 11) b) An anti-HER2 monoclonal antibody such as trastuzumab., 5. Supramolecular tetrameric peptide complex according to claim 1, wherein -the spacer L1 is chosen from the group consisting of an amino acid (denoted AA) or a peptide chain of 2 to 100 amino acids (denoted (AA)2-100), preferably of 2 to 60 amino acids (denoted (AA)2-60), from 10 to 50 amino acids (denoted (AA)10-50), even more preferably from 40 to 50 amino acids (denoted (AA)40-50), and -the spacer L2 is chosen from the group consisting of an amino acid (denoted AA) or a peptide chain of 2 to 60 amino acids (denoted (AA)2-60), preferably of 2 to 30 amino acids (denoted (AA)2-30), of 2 to 25 amino acids (denoted (AA)2-25), even more preferably of 15 to 25 amino acids (denoted (AA)15-25).

6. Supramolecular tetrameric peptide complex according to claim 1, wherein the chelating group (B) is selected from the following compounds: bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'45-[acetyl(hydroxy)amino]pentyl]-N45-[[445-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide (deferoxamine / DFO), 4,11-bis(carboxymethyl)-1,4,8,11- tetraazabicyclo[6.6.2]hexadecane (DO2A) 1 ,4 ,7,10-tetracyclododecane- N,N',N",N'"-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10 tetraacetic acid (DOTAGA), 1 ,4,7,10 tetraazacyclododecane N,N',N",N'" 1 ,4,7,10-tetra(methylene) phosphonic acid (DOTMP), 1 ,4,7-triazacyclononane-1 ,4-diacetic acid (NODA) and its derivatives, N,N'dipyridoxylethylenediamineN,N'diacetate5,5 '-bis(phosphat) (DPDP),diethylenetriamine N,N',N" penta(methylene) phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethyleneglycol-0,0-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,Nbis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), 1,4,7-triazacyclononan-1 acid -succinic-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazacyclotetradecane-N,N',N",N"-tetrakis(carbamoylmethyl) (TETAM), 1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane (NOTAM), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,7,10-tetraazacyclododecane 1,4,7,10-tetrakis(methylene phosphonate) (DOTP), 1,4,7-tetrakis(methylene phosphonate)-1,4,7-triazacyclononane (NOTP), I' acid, 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), tris (hydroxypyridinone) (THP), terpyridinbis (methyleneam tetraacetic acid (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-tetraazacyclotridecane-N,N',N",Nm-tetraacetic acid (TRITA), 34[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl1]-1,4,7-triazonan-1-yl]methyl-hydroxy-phosphoryl]propanoic acid, triethylenetetraaminehexaacetic acid (TTHA) and other bispidine chelators, preferably DOTAGA.

7. A supramolecular tetrameric peptide complex according to any preceding claim, which comprises (a) une première structure représentée par les formules suivantes : (11 ) A-L1- (SEQ ID NO : 1 ou SEQ ID NO : 2 ou SEQ ID NO : 3 ou SEQ ID NO : 4 ou SEQ ID NO : 5 ou SEQ ID NO : 6 ou SEQ ID NO : 7 ou SEQ ID NO : 8 ou SEQ ID NO : 9)-L2 de préférence A-L1- (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2 (12) VHH-L1- (SEQ ID NO : 1 ou SEQ ID NO : 2 ou SEQ ID NO : 3 ou SEQ ID NO : 4 ou SEQ ID NO : 5 ou SEQ ID NO : 6 ou SEQ ID NO : 7 ou SEQ ID NO : 8 ou SEQ ID NO : 9)-L2 (13) VHH-L1- (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2 (15) VHH-(AA)2-60 - (SEQ ID NO : 1 ou SEQ ID NO : 2 ou SEQ ID NO : 3 ou SEQ ID NO : 4 ou SEQ ID NO : 5 ou SEQ ID NO : 6 ou SEQ ID NO : 7 ou SEQ ID NO : 8 ou SEQ ID NO : 9)-L2 (16) VHH-(AA)2-60 - (SEQ ID NO : 4 ou SEQ ID NO : 5)-L2 (17) VHH-(AA)40-50 - (SEQ ID NO : 1 ou SEQ ID NO : 2 ou SEQ ID NO : 3 ou SEQ ID NO : 4 ou SEQ ID NO : 5 ou SEQ ID NO : 6 ou SEQ ID NO : 7 ou SEQ ID NO : 8 ou SEQ ID NO : 9)-L2 (18) VHH-(AA)40-50 - (SEQ ID NO: 4 or SEQ ID NO: 5)-L2 (I9) VHH-(AA)2-60 - (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-(AA)2-30 (110) VHH-(AA)2-60 - (SEQ ID NO: 4 or SEQ ID NO: 5)-(AA)15-25 (111) VHH-(AA)40-50 - (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)-(AA)2-30 (112) VHH-(AA)40-50 - (SEQ ID NO: 4 or SEQ ID NO: 5)- (AA)15-25 or mimetic peptides, analogues, or homologues thereof, preferably with VHH directed against the HER2 antigen, or CD38 (preferably of sequences respectively SEQ ID NO: 10, or SEQ ID NO: 11) and (b) a second structure represented by the following formulas: (111) (SEQ ID NO : 1 or SEQ ID NO : 2 or SEQ ID NO : 3 or SEQ ID NO : 4 or SEQ ID NO : 5 or SEQ ID NO : 6 or SEQ ID NO : 7 or SEQ ID NO : 8 or SEQ ID NO : 9)-L3-[B@(M)] (112) (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)- L3-[DOTAGA@(M)] m preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-L3-DOTAGA@(M), (113) (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)- L3-[DOTAGA@(Bi, Hf or Gd)] m preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-L3-DOTAGA@(Bi, Hf or Gd) (114) (SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9)- click-[DOTAGA@(M)] m preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-click-DOTAGA@(M), preferably (SEQ ID NO: 4 or SEQ ID NO: 5)-click-DOTAGA@(Bi, Hf or Gd) or mimetic peptides, analogs, or homologues thereof, with "click" represents a group comprising a [1,2,3]-triazole, isoxazole, isoxazoline, oxadiazoles, a pyrazole, a dihydropyrazine, an amide, a maleimide, a hydrazone or an oxime. wherein the sequences SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9, preferably the sequences SEQ ID NO: 4 and SEQ ID NO: 5 or mimetic peptides, analogues, or homologues thereof, associate and form primary homodimers and / or primary heterodimers, which associate and form "dimers of dimers", preferably of formula (III).

8. Pharmaceutical composition comprising at least one supramolecular peptide complex, in particular a plurality of complexes representing an effective amount of supramolecular peptide complexes according to one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. A pharmaceutical composition according to claim 8, for use in the treatment of a tumor in a subject in need thereof or for use in MRI imaging, the use comprising (i) administering a supramolecular tetrameric peptide complex containing a high Z element exhibiting contrast enhancement for MRI imaging and / or radiosensitizing properties, to a subject in need thereof, and, in the case of treatment (ii) expose said subject to ionizing radiation.

10. Pharmaceutical composition according to claim 8, for use in the treatment of a tumor in a subject in need thereof, the treatment comprising (i) administering a supramolecular tetrameric peptide complex containing a high Z element, and targeting a given antigen present on a tumor cell, and, (ii) administering a radionuclide linked to an antibody or antibody fragment targeting the same said antigen as said supramolecular peptide complex or another antigen present on the same targeted tumor cell.

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