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WO2024028282A1 - Site-specific modification by a bbb-shuttle of antibody-based entities for crossing the blood-brain barrier - Google Patents

Site-specific modification by a bbb-shuttle of antibody-based entities for crossing the blood-brain barrier Download PDF

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Publication number
WO2024028282A1
WO2024028282A1 PCT/EP2023/071199 EP2023071199W WO2024028282A1 WO 2024028282 A1 WO2024028282 A1 WO 2024028282A1 EP 2023071199 W EP2023071199 W EP 2023071199W WO 2024028282 A1 WO2024028282 A1 WO 2024028282A1
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WIPO (PCT)
Prior art keywords
antibody
group
bond
shuttle
disulfide
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PCT/EP2023/071199
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French (fr)
Inventor
Macarena SÁNCHEZ NAVARRO
Meritxell TEIXIDÓ TURÀ
Ernest GIRALT LLEDÓ
Original Assignee
Gate2Brain, S.L.
Universitat De Barcelona
Fundació Institut De Recerca Biomèdica (Irb Barcelona)
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Publication of WO2024028282A1 publication Critical patent/WO2024028282A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention relates to the field of conjugates with antibodies, and its use to the treatment and diagnosis of diseases that need that antibody cross the blood-brain barrier.
  • Monoclonal antibodies have revolutionized the treatment of several diseases, especially in oncology.
  • Antibody-based treatments target leukemia and solid tumors in many organs.
  • brain tumors remain practically intractable with biotherapeutics and most small molecules.
  • BBB blood-brain barrier
  • BTB blood-tumor barrier
  • BM Brain metastasis
  • BM Brain metastasis
  • HER-2 human epidermal growth factor receptor
  • mAbs to treat BM or primary brain tumors remains elusive, mainly due to the lack of antibody penetration across the BBB and BTB.
  • WO2015/001015A1 describes specific apamin-derived peptides comprising the KAPETAL fragment, which are useful as shuttles as they have the ability to cross the BBB and are capable of facilitating the transport into the brain of drugs or other diagnostically useful substances which by themselves cannot cross the BBB.
  • Example 22 (Cetuximab-V3-Ap3-NH2) and Example 23: (Cetuximab-V2-Ap5a-NH2), the AP5 peptide corresponds to MiniAp4 peptide, a cyclic peptidomimetic derived from bee venom which displays high resistance to proteolysis and negligible toxicity and immunogenicity, being V2and V 3 as follows:
  • V3 linker bonds 1 and 2 are connected to the side chain of a lysine of the antibody.
  • all the previous methods based on modifications of lysine such as the ones disclosed in the previous documents do not allow to control where the peptides are located when they are incorporated in a monoclonal antibody.
  • the present inventors have developed a conjugate of a mAb antibody and selected peptide shuttles linked to the antibody through a bromomaleimide linker at specific sites of the antibody, and which effectively crosses the blood-brain barrier.
  • the linker used in the present invention allows a peptide incorporation strategy in which the incorporation of the shuttle peptides into the monoclonal antibody is controlled and allows knowing where they are located.
  • This strategy is based on incorporating the peptides through the -SH groups of the reduced antibody.
  • this strategy may be applied to any monoclonal antibody providing homogeneous conjugates by site-specific modification of the antibody, a highly desirable feature for a pharmacological product, enhancing therapeutic index and facilitating product manufacturing and profiling.
  • Trastuzumab (Herceptin®) is an FDA-approved antibody against HER2, which is widely used in clinics to treat breast cancer.
  • the conjugation has been achieved by reducing interchain disulfide bridges and rebridging them using dibromomaleimide, which enables high control over the number of peptides anchored to every antibody molecule.
  • the conjugate shows enhanced transport across the blood-brain barrier in an in vitro cell model with respect to trastuzumab alone or with respect to a conjugate of trastuzumab with the Angiopep-2 peptide disclosed by Anthony Regina et al., in “ANG4043, a novel brain-penetrant Peptide-mAb conjugate, is efficacious against HER2-positive Intracranial Tumors in Mice”, mct.aacrjournals.org.
  • a first aspect of the present invention relates to an antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof, which comprises from 1-6 peptides of formula P inserted in the disulfide bonds of the antibody in the form of a -P-(W)s-Y and joined to the sulfides through a linker -[(l_i)-(l_2)- (L3)m-; wherein:
  • Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof;
  • the disulfide bonds are any disulfide bond initially present in the antibody and that is capable of structurally retaining sulfide bonds in the structure upon reduction conditions, the disulfide bond being selected from the group consisting of a naturally present interchain disulfide bond of the antibody, a naturally present intrachain disulfide bond of the antibody, and a disulfide bond introduced in the antibody by genetic engineering;
  • Li is a linker selected from the group consisting of Li a and L ; q is an integer from 1 to 6;
  • L2 is a biradical composed from 2 to 8 biradicals selected from the group consisting of LA, LB, LC and has the formula -LA-(LB) U -LC-
  • D is a substance attached to the linker L3 selected from a biologically active substance, a substance for use in a diagnostic method; and a radioligand for radiotherapy;
  • P is a biradical of a single peptide, equal or different, which is selected from the group consisting of: (a) a peptide which comprises the amino acid sequence X1KAPETALX2 with an intrapeptide bond between the Xi and X2 which is an amide bond; wherein Xi is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4- diaminobutanoic acid); and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid); i.e.
  • SEQ ID N0:1 X1KAPETALX2
  • a peptide having 12-20 amino acids residues in length having at least an intrapeptide bond which is a disulfide or diselenide bond, and comprises an amino acid sequence which is: X3KAPETALX4AAA; having at least an intrapeptide disulfide or diselenide bond between X3 and X4, wherein X3 and X4 are equal and are selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines); i.e.
  • peptide which comprises the amino acid sequence XI KAPETALX 2 wherein Xi is selected from the group consisting of Dap and Dab; and X 2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid) (SEQ ID NO:7) being a linear peptide;
  • S indicates sulfide
  • a second aspect of the present invention relates to a process for preparing an antibody shuttle conjugate as defined above, comprising: a) reducing the disulfide bridges of the antibody; b) rebridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), and c) optionally, carrying out a hydrolysis; wherein q; L2, L3, D, P, m, W, s and Y are as defined in the antibody shuttle conjugate of formula (I);
  • a third aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle conjugate of the present invention, together with appropriate amounts of pharmaceutically acceptable carriers or excipients.
  • a four aspect of the present invention relates to the antibody shuttle conjugate of the present invention as defined above, for use as a medicament.
  • a fifth aspect of the present invention relates to the antibody shuttle conjugate of the present invention as defined above for use in the treatment of Central Nervous System (CNS) disorders in a mammal, including a human.
  • CNS Central Nervous System
  • a sixth aspect of the present invention relates to the antibody shuttle conjugate of the present invention as defined above for use as a diagnostic agent.
  • FIG 1. H NMR of DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetic acid).
  • FIG. 13 C NMR of DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetic acid).
  • Example 1 FIG 3. LIPLC traces and MS spectra of the Comparative Example 2 and Example 3. LIPLC chromatograms are recorded at 220 nm in a 2-min linear gradient from 0 to 100% of MeCN (0.036% TFA) in H 2 O (0.045% TFA).
  • FIG 4. The site-specific Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Trastuzumab-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) were generated and characterized, a) ASC synthetic scheme; b) Mass characterization of Trastuzumab-DBM- TTDS-SEQ ID NO: 15 (also named Tz-Ang2, Comparative Example 3) (top) and Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also named Tz-MiniAp4, Example 4) (down) by LCT-Premier. Deconvoluted spectra are shown.
  • FIG. Mass characterization of Anti- Trastuzumab (Anti- Idiotype) Alexa Fluor® 647- conjugated Antibody (AF647 modified Trastuzumab (also named Tz) (a), Trastuzumab- DBM-TTDS-SEQ ID NO: 15 (also named Tz-Ang2, Comparative Example 3) (b) and Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also named Tz-MiniAp4, Example 4) (c) by LCT-Premier. Antibodies are deglycosylated. Deconvoluted spectra are shown.
  • FIG. Mass characterization of Trastuzumab (also named Tz) (a), Trastuzumab-DBM- TTDS-SEQ ID NO: 15 (also named Tz-Ang2, Comparative Example 3) (b) and Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also named Tz-MiniAp4, Example 4) (c) after immunoprecipitation of acceptor well from HBBBCMTA by LCT-Premier. The raw data (top) and the deconvoluted spectra (down) are shown.
  • FIG 7. Stability studies in mouse serum of the BBB-shuttles peptides present in Example 4 and Comparative Example 3.
  • BT474 and SKBR3 cells were aliquoted and incubated with Tz, Tz-Ang2 or Tz-MiniAp4 at 4°C for 2 hours.
  • Anti-human dylight 650 was used to detect I g 1 .
  • FIG 11. Cell cycle arrest analysis of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) treated cells.
  • SKBR3, BT-474 or MDA-MB-231 cells were serum starved and stimulated with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) (100 nM) for 5 days.
  • amino acids cited herein are L-amino acids.
  • the 1-letter code and the 3-letter code have been used indistinctly.
  • diaminopropionic acid (Dap) diaminopropionic acid (Dap), Diaminobutiric (Dab), Selenocystein (Sec) and Penicillamine (Pen).
  • Dap diaminopropionic acid
  • Dab Diaminobutiric
  • Dbu Dbu
  • an antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof which comprises from 1-6 peptides of formula P inserted in the disulfide bonds of the antibody in the form of a -P-(W)s-Y and joined to the sulfides through a linker -[(Li )-(l_2)- (l_3)m- ; wherein, Z, Li , L2, L3, D, m, P, W, S, Y and n have the meaning mentioned above.
  • the antibody shuttle conjugate of the present invention is that where the disulfide bonds initially present in the antibody are capable of structurally retaining the sulfide bonds in the structure upon reduction conditions and of forming between them new bridges by reaction of the sulfide groups of the reduced disulfide bonds of the antibody with the linker Li, to incorporate the biradical below between the sulfide groups of the antibody.
  • the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof may be in the form of a formulation comprising the antibody or their salts and excipients.
  • appropriate excipients for Bevacizumab (Avastin) are trehalose dihydrate, sodium phosphate, polysorbate 20, and water for injectable preparations
  • appropriate excipients for Pertuzumab (Perjeta) are Glacial acetic acid, L-histidine, Sucrose, Polysorbate 20, and water for injectable preparations
  • appropriate excipients for are Cetuximab (Erbitux): Sodium Chloride, Glycine, Polysorbate 80, Citric Acid Monohydrate, Sodium Hydroxide, and water for Injection Preparations
  • appropriate excipients for trastuzumab (Herceptin) are for instance: L-histidine hydrochloride monohydrate, L- histidine, a,a-trehalose, or polysorbate 20 dihydrate.
  • the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof is that wherein the linker L2 is a biradical selected from the group consisting of:
  • the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof is that wherein n is an integer from 1 to 4.
  • the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof is that wherein n is 4, i.e. , it has four peptides incorporated within the antibody structure.
  • all the peptides are equal.
  • the disulfide bond of the antibody is an interchain bond. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the disulfide bond of the antibody is an intrachain bond. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the disulfide bond of the antibody is a disulfide bond formed by genetic engineering.
  • the antibody shuttle conjugate is that wherein P is a biradical of a peptide selected from the group consisting of:
  • TIHCO ⁇ (b) a peptide having 9-20 ammo acids residues in length having at least an intrapeptide bond which is a disulfide bond, and comprises an amino acid sequence which is: CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that is SEQ ID NO:9: CKAPETALCAAA
  • a peptide having 9-11 amino acids residues in length having at least an intrapeptide bond which is a disulfide bond and consists of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that are
  • SEQ ID NO: 10 CKAPETALC
  • SEQ ID NO: 12 CKAPETALCAA
  • the lines between two amino acids of the sequences above or below represent the intrapeptide bond between the side chains of the two amino acids.
  • the lines between two amino acids of the sequences above or below represent the intrapeptide bond between the side chains of the two amino acids.
  • the antibody shuttle conjugate is that wherein, P is a biradical of a peptide selected from the group consisting of: (a) the peptide having the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond (SEQ ID NO:7); (b) the peptide having the amino acid sequence CKAPETALC having at least an intrapeptide disulfide bond between cysteines in position 1 and 9 (SEQ ID NO: 10; and (c) the peptide having the amino acid sequence DapKAPETALD (SEQ ID NO:14).
  • P is a biradical of a peptide selected from the group consisting of: (a) the peptide having the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond (SEQ ID NO:7); (b) the peptide having the amino acid sequence CKAPETALC having at least an intrapeptide disulfide bond between cysteines in position 1 and 9 (
  • the antibody shuttle conjugate is that wherein P is a biradical of the peptide DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond (SEQ ID NO:7).
  • the antibody shuttle conjugate is that wherein P is a biradical of a peptide having one intrapeptide bond. In another particular embodiment, the antibody shuttle conjugate is that wherein P is a biradical of a peptide having two intrapeptide bonds.
  • Antibodies generally consist of two heavy chains (HCs) and two light chains (LCs) folded into constant and variable domains, although some antibodies, such as camelid antibodies, contain only 2 heavy chains. They can occur individually as monomers (e.g. IgG), or in multimers of two (e.g. IgA) to five units (e.g. IgM). They have disulfide intrachain bonds.
  • the antibodies for the purposes of the present invention may be chimeric, humanized or fully human antibodies. In the sense of the present invention the antibodies can be natural antibodies or recombinant antibodies.
  • the antibody is a therapeutical antibody.
  • the antibody is for diagnosis.
  • the antibody is an antibody-drug conjugate (ADC).
  • the antibody can also be an antibody fragment, provided it contains at least one disulfide bond and provided that it maintains the function of the antibody from which it is derived In particular, its therapeutic or diagnostic activity.
  • the antibody may be an antibody fragment such as Fab, scFV, (Fab)2, diabody, triabody, tetrabody, minibody, or nanobody.
  • disulfide bonds can be incorporated for instance into the antibodies or fragments thereof by incorporating cys by mutagenizing a nucleic acid sequence of an antibody and replacing one or more amino acid residues by cysteines to encode the cysteine engineered antibody (see CA2957354A1).
  • Antibody fragments can offer several advantages compared to full-length antibodies.
  • the antibody shuttle conjugate is that wherein the antibody comprises a heavy chain constant domain of the type of IgA, IgD, I g E, IgG or IgM.
  • Antibodies of the IgA type can be divided into two isotopes: I gA1 or lgA2.
  • Antibodies of the IgG class can be divided into four isotypes: lgG1 , lgG2, lgG3 and lgG4. classes of antibodies are encompassed within the scope of the present invention.
  • the antibody shuttle conjugate is that wherein the antibody comprises a light chain constant domain, e.g., of the type of kappa or lambda.
  • the antibody shuttle conjugate is that wherein the antibody or antibody fragment according to the present invention comprises:(a) an immunoglobulin constant region; (b) an IgG 1 constant region; or (c) a human IgG 1 constant region.
  • I gG 1 is preferred as it is the most widely used isotype for anticancer mAbs, as well as the most effective IgG isotype in mediating ADCC (antibody-dependent cellular cytotoxicity).
  • the antibody is a monoclonal chimeric, humanized, or fully human antibody. Antibody humanization allows reducing immunogenicity by decreasing the mouse content of monoclonal antibodies.
  • Common techniques developed for the production of fully human monoclonal antibodies can be used such as phage display, where a library of human antibodies is expressed on the surface of phage and subsequently selected and amplified in E. coli, and transgenic mice expressing a human antibody repertoire.
  • the antibody shuttle conjugate is that wherein the antibody is: (a) is multispecific.
  • Multispecific antibodies is a class of engineered antibody and antibody-like proteins that combine multiple specific antigen binding elements in a single construct.
  • the antibody shuttle conjugate is that wherein the antibody is: heterodimeric bispecific antibodies which are traditional IgG molecule with one arm targeting one antigen and the other targeting a second antigen.
  • the antibody shuttle conjugate is that wherein the antibody is a bispecific antibody fusion which is a non-standard IgG molecule. IgG is elongated at its N-terminus on the corresponding heavy and light chains by an additional variable domain of a second antibody.
  • the antibody shuttle conjugate is that wherein the antibody is a trispecific antibody which is also a non-standard IgG molecule.
  • the same technologies used to generate bispecific antibodies can also be combined to generate trispecific antibodies with varying valences.
  • the antibody shuttle conjugate is that wherein the antibody is fusion of a scFv to an IgG via the attachment of the scFv to the N or C-terminus of the heavy or light chain.
  • the antibody shuttle conjugate of the invention is that wherein the antibody is selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab, Pertuzumab, Aducanumab, Bapineuzumab, Nimotuzumab, and Necitumumab.
  • the antibody shuttle conjugate of the invention is that where the antibody is selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab and Pertuzumab.
  • the antibody shuttle conjugate of the invention is that where the antibody is Trastuzumab.
  • Trastuzumab (Herceptin®), an FDA-approved antibody against HER2, which is widely used in clinics to treat breast cancer.
  • the antibody shuttle conjugate of the invention is that which is Trastuzumab-DBM-TTDS-Dap-Lys-Ala- Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • the antibody shuttle conjugate of the invention is that where the antibody is Cetuximab.
  • Cetuximab Erbitux®
  • an FDA-approved antibody against EGFR which is widely used in clinics to treat colorectal cancer and head and neck cancer.
  • the antibody shuttle conjugate of the invention is that which is Cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Cx-DBM-TTDS- SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp sidechain carboxylic acid.
  • DBM corresponds to linker Li a .
  • TTDS corresponds to linker l_2a.
  • DBM Lia has the formula:
  • the antibody shuttle conjugate of the invention is that where the antibody is Bevacizumab.
  • Bevacizumab (Avastin ®), an FDA-approved antibody against VEGF-A, which is widely used in clinics to treat colon cancer, lung cancer, glioblastoma and renal-cell carcinoma.
  • the antibody shuttle conjugate of the invention is that which is Bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu- Thr-Ala-Leu-Asp-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • the antibody shuttle conjugate of the invention is that where the antibody is Pertuzumab.
  • Pertuzumab Perjeta ®
  • an FDA-approved antibody against HER2 which is widely used in clinics to treat breast cancer.
  • the antibody shuttle conjugate of the invention is that which is Pertuzumab-DBM-TTDS- Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • the antibody shuttle conjugate of the invention is that where the antibody is Aducanumab, a lgG1 antibody against an epitope of the p-amyloid protein.
  • the antibody shuttle conjugate of the invention is that where the antibody is Bapineuzumab, an anti-Ap-amyloid lgG1 antibody. Both antibodies are useful for the treatment of Alzheimer disease.
  • the antibody shuttle conjugate of the invention is that where the antibody is Nimotuzumab, a lgG1 anti-EGFR antibody. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Necitumumab, IgG 1 anti-EGFR antibody. Both antibodies are useful for the treatment of brain metastases.
  • the formulation comprising the antibody shuttle conjugate of the invention is that where D is present, yielding to an antibody-drug shuttle conjugate, an antibodyradioligand shuttle conjugate, or an antibody-diagnostic agent shuttle conjugate.
  • the biologically active substance is a pharmaceutical active ingredient.
  • the antibody-drug shuttle conjugate is that where the antibody is cetuximab and D is SN38.
  • the antibody-drug shuttle conjugate according to the invention is that where the antibody is cetuximab, the drug is SN38, the peptide is MiniAp4, and the linker is TTDS-DBM.
  • D is biotin-Ytrium 90 or Biotin- Iodine 131 or 123. They can be joined through streptavidin to the antibody shuttle conjugate of the present invention, where the streptavidin is attached to a lateral chain of the amino acids of the biradical Lswhen present in the antibody shuttle conjugate of the present invention.
  • D is a contrast agent for a magnetic resonance imaging method (MRI), such as Gadolinium, in particular, Gd-DTPA (Magnevist®).
  • MRI magnetic resonance imaging method
  • Gadolinium may be complexed with DOTA which is attached to a lateral chain of the amino acids of the biradical Lswhen present in the antibody shuttle conjugate of the present invention or to an amino acid derivative by a feasible bond.
  • the antibody shuttle conjugates of the present invention may be prepared by a process comprising: a) reducing the disulfide bridges of the antibody; b) rebridging the disulfide bridges, i.e. forming new bridges, by reacting the -SH groups of the antibody with a dibromomaleimide-peptide (DBM-peptide) of formula (II), DBM-P-(W) S -(Y), in which the dibromomaleimide is attached to the peptide via the N-terminus of the peptide, wherein DBM, P, W, Y and s is as defined for the antibody shuttle conjugate of formula (I).
  • DBM-peptide dibromomaleimide-peptide
  • the binding of the DBM-peptide to the antibody is possible thanks to the reactivity of the cysteines forming the disulfide bridges. This method allows a high control of the number of peptides anchored to each antibody molecule.
  • the antibody is trastuzumab.
  • the antibody shuttle conjugate of the present invention may be defined by its preparation process. Accordingly, an antibody shuttle conjugate obtainable by the process defined above is also considered part of the present invention.
  • the antibody shuttle conjugate as defined above may be obtainable by reacting the hydrosulfide groups of the reduced disulfide bonds of the corresponding antibody or of a fragment thereof with a dibromomaleimide-peptide of formula (II) as defined above to rebridge the disulfide bond with the biradical below incorporated between the disulfide bridges of the antibody.
  • the antibody shuttle conjugate as defined above may be obtainable by a process comprising: a) reducing disulfide bridges of the antibody; b) rebridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), c) optionally, carrying out a hydrolysis; wherein q; L2, L3, D, P, m, W, s, and Y are as defined in the antibody shuttle conjugate of formula (I).
  • the disulfide bonds are interchain bonds. All the embodiments defined above for the conjugate of formula (I) as product per se are also embodiments of the process for its preparation.
  • the shuttle peptide used in the conjugates of the present invention can be generated wholly or partly by chemical synthesis.
  • the amino acids required for the preparation of compounds of formula (I) are commercially available.
  • the compounds of formula (I) can be prepared easily, for example by synthesis in liquid-phase or, preferably, by solid-phase peptide synthesis, for which there are a number of procedures published (see M. Amblard, et al., "Methods and protocols of modern solid-phase peptide synthesis. Molecular Biotechnology 2006, Vol. 33, p. 239-254).
  • the compounds of formula (I) can also be prepared by any combination of liquid-phase synthesis and/or solid-phase synthesis.
  • linker dibromomaleimide For example, by synthesizing the body of the peptide P through solid-phase synthesis and subsequently removing protecting groups in solution.
  • the binding to the linker dibromomaleimide can be performed in solid-phase or in solution.
  • the construction of the linker can also be prepared by any combination of liquid-phase synthesis and/or solidphase synthesis.
  • the linker used is 3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)acetic acid or similar derivatives thereof.
  • the compound of formula (II) can be prepared by a process comprising: reacting a peptide derivatized with the linkers with 3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)acetic acid by solid phase peptide synthesis or in solution to yield the DBM-peptide.
  • the compound of formula (II) with a linker L can be obtained by the corresponding compound of formula (II) with a linker Li a by hydrolysis.
  • the hydrolysis can be carried out in mild pH basic conditions,
  • the DBM peptide of formula (II) has the following formula:
  • DBM peptide of formula (II) has the following formula:
  • the antibody shuttle conjugate of the invention is that where the linker Li is L .
  • This conjugate may be prepared by hydrolysis in mild basic pH conditions from the corresponding antibody shuttle conjugate having as Li linker the linker Lla.
  • the antibody shuttle conjugates of the present invention may be in the form of a pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable salts used herein encompasses any salt formed from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids or bases. There is no limitation regarding the salts, except that if used for therapeutic purposes, they must be pharmaceutically acceptable. As some of the compounds of formula (I) are basic compounds, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids.
  • Such acids include, for instance, chlorohydric, acetic, benzenesulfonic, benzoic, camphor sulfonic, citric, ethansulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, methane sulfonic, phosphoric, succinic, sulfuric, tartaric, p-toluensulfonic acid, and the like.
  • antibodies in the form of a salt according to the present invention are Bevacizumab (Avastin) in the form of a salt with trehalose dihydrate, sodium phosphate, polysorbate 20 salts; Pertuzumab (Perjeta) in the form of a salt with glacial acetic acid, L- histidine, Sucrose, or Polysorbate 20; cetuximab (Erbitux) in the form of a salt formed with sodium Chloride, glycine, polysorbate 80, citric acid monohydrate, or sodium hydroxide, trastuzumab (Herceptin) in the form of a salt with L-histidine hydrochloride monohydrate, L-histidine, a,a-trehalose, or polysorbate 20 dihydrate
  • compositions of formula (I) can be carried out by methods known in the art. For instance, they can be prepared from the conjugate, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate pharmaceutically acceptable base or acid in water or in an organic solvent or in a mixture of them.
  • a pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle conjugate as defined above, together with appropriate amounts of pharmaceutically acceptable carriers or excipients is also part of the invention.
  • pharmaceutical composition refers to a mixture of a compound described herein with other chemical components, such as diluents or carriers.
  • the pharmaceutical composition facilitates administration of the compound to an organism.
  • pharmaceutically acceptable excipients or carriers refers to pharmaceutically acceptable material, composition or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must be also suitable for use in contact with tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a benefit/risk ratio.
  • therapeutically effective amount refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • the particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.
  • pharmaceutically acceptable excipient refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • Suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
  • compositions of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • compositions of the present invention may be administered in parenteral form suitable for injection such as intravenous bolus injections, intravenous infusion, implantation into the body, oral, intratecal, or intranasal.
  • An antibody shuttle conjugate as defined above for use as a medicament is also part of the invention. This includes the antibody shuttle conjugates as well as the antibody-drug shuttle conjugates according to the present invention.
  • the term “medicament” as used herein is synonymous of a pharmaceutical or veterinary drug (also referred to as medicine, medication, or simply drug) use to cure, treat or prevent disease in animals, including humans, as widely accepted. Drugs are classified in various ways. One key distinction is between traditional small-molecule drugs, usually derived from chemical synthesis, and biopharmaceuticals, which include recombinant proteins, vaccines, blood products used therapeutically (such as I VIG), gene therapy, monoclonal antibodies and cell therapy (for instance, stem-cell therapies).
  • An antibody shuttle conjugate as defined above for use in the treatment of CNS disorders in a mammal, including a human is also part of the invention.
  • This aspect can also be formulated as the use antibody shuttle conjugate as defined above for the preparation of a medicament for the treatment of CNS disorders in a mammal, including a human.
  • the invention also relates to a method of treatment of a mammal, including a human, suffering from or being susceptible of suffering from a CNS disorder, said method comprising the administration to said patient of a therapeutically effective amount of antibody shuttle conjugate as defined above, together with pharmaceutically acceptable excipients or carriers.
  • the antibody shuttle conjugate is for use in a CNS disorder which is cancer.
  • the antibody shuttle conjugate of the present invention is for use in the treatment of a primary brain tumor.
  • the antibody shuttle conjugate of the present invention is for use in the treatment of brain metastasis (BM) is a major complication in several types of cancers, particularly in lung, melanoma, and breast cancers.
  • BM brain metastasis
  • the antibody shuttle conjugates of the present invention can be used in the same manner as other known chemotherapeutic agents, i.e. , in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. They may be used alone or in combination with other suitable bioactive compounds.
  • the antibody shuttle conjugates of the present invention are for use in the treatment of cancer in a mammal, including a human in combination therapy with a chemotherapeutic agent.
  • the antibody-drug shuttle conjugates in combination therapy with a further chemotherapeutic agent can be used for instance for specific treatment regimens.
  • the antibody shuttle conjugate of the present invention is for use in combination with radioimmunotherapy.
  • Both the antibody shuttle conjugates as the antibody-radioligand shuttle conjugates according to the invention can be used.
  • the combination of radiation therapy and immunotherapy can be used to treat non-Hodgkin lymphoma and other types of cancer including brain tumors.
  • the antibodyradioligand shuttle conjugates according to the invention can bind to cancer cells and deliver a high dose of radiation directly to the tumor.
  • an antibody shuttle conjugate as defined above for use as a diagnostic agent is also part of the invention.
  • the antibody shuttle conjugate according to the invention is for use in a method of diagnostic of CNS disorders.
  • Cell culture-treated plates and flasks were purchased from Corning Costar. Culture medium was acquired from Lonza. XTT cell proliferation kit was purchased from Biological Industries (Cromwell, CT). Pierce® iodination beads were obtained from Pierce. Desalting columns (MiniTrap and MidiTrap G-25) were obtained from GE-Healthcare.
  • Microwave assisted solid-phase peptide synthesis was carried out on a Liberty Blue Automated Microwave Peptide Synthesizer using H-Rink amide Protide resin (loading: 0.56 mmols/g). Linear peptide was synthesized on a 0.5 mmol scale using a 5 excess of Fmoc-amino acid (0.2M) relative to the resin.
  • the test used for the identification and control of the synthesis was the following: A) Kaiser colorimetric assay for the detection of solid-phase bound primary amines (E. Kaiser et al., Anal. Biochem. 1970, vol. 34, pp. 595-598); B) p-nitro phenyl ester test for secondary amines bound to solid-phase (A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791).
  • Protocols used durinq the manual is of the com The compounds were synthesized at a 100 pmol scale using the following methods and protocols: The resin for the manual synthesis was selected depending on the group Y: If Y is a OH, the terminus will be COOH, 2-chlorotrytil chloride resin will be chosen among others available. If Y is a NH2, the terminus will be CONH2, Rink amide MBHA resin will be chosen among others available.
  • Resin initial The resin was conditioned by washing with MeOH (5 x 30 s),
  • DMF (5 x 30 s), DCM (5 x 30 s), 1% TFA in DCM (1 x 30 s and 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30s), 5 % DIEA in DCM (1 x 30 s, 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s).
  • Fmoc removal Removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was done with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each. Two additional treatments with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) (2 x 5 min) were performed to ensure the removal of the Fmoc group from secondary amines (proline).
  • the solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s).
  • the coupling reaction was carried out twice under the same conditions. The extent of coupling was checked by the Kaiser colorimetric assay.
  • the Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using 30 s treatments and two treatments of 10 minutes. If the amino acid to be deprotected was a proline, an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group.
  • the Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using 30 s treatments and two treatments of 10 minutes. If the amino acid to be deprotected was a proline, an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group.
  • method 4 scale 100 pmols: The protected amino acid (3 eq 300 pmols)), DIG
  • amino acid to be deprotected was a proline
  • an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group.
  • Protocols used during the microwave assisted automated is: The compounds were synthesized at a 500 pmol scale using the following methods and protocols: The resin for the microwave assisted automated synthesis was selected depending on the group Y: If Y is a OH, the terminus will be COOH, CI-TCP(CI) ProTide resin will be chosen among others available. If Y is a NH2, the terminus will be CONH2, Rink amide ProTide resin will be chosen among others available.
  • Resin initial conditioning The resin was conditioned by washing with MeOH (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30 s), 1% TFA in DCM (1 x 30 s and 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30s), 5 % DIEA in DCM (1 x 30 s, 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s).
  • Methods for the of the P disulfide or diselenide bond
  • the cyclization was performed in solution after the cleavage from the resin or on resin after the selective deprotection of the Cys, Sec or Pen residues.
  • the peptide was dissolved at a concentration of 100 pM in aqueous ammonium bicarbonate buffer 10 mM and pH 8.0.
  • the solution was intensely stirred for 24 h at room temperature. After that, the product was acidified with TFA to pH 2-3, frozen and lyophilized.
  • method 2 amide bond
  • the cyclization was performed on resin
  • the Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes.
  • the /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq, 1000 pmol, 56 mg) and DIEA (30 eq, 3000 pmol, 240 pL).
  • the OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq, 10 pM, 12 mg), phenyl silane (10 eq, 1000 pmol, 123 mg) in DCM (3 x 15 min).
  • the resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 x 5 min).
  • the /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq, 1000 pmol, 56 mg) and DIEA (30 eq, 3000 pmol, 240 pL).
  • the OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq, 10 pM, 12 mg), phenyl silane (10 eq, 1000 pmol, 123 mg) in DCM (3 x 15 min).
  • the resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3 x 5 min).
  • the /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq., 1000 pmols, 56 mg) and DIEA (30 eq., 3000 pmols, 240 pL).
  • the OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 pM, 12 mg), phenyl silane (10 eq., 1000 pmols, 123 mg) in DCM (3 x 15 min).
  • the resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3 x 5 min).
  • the disulfide bond can be accomplished by reaction of two thiols.
  • the thiols are dissolved at a concentration of 100 pM in aqueous ammonium bicarbonate buffer 10 mM and pH 8.0 and the solution is intensely stirred for 24 h at room temperature. After that, the solution is acidified with TFA to pH 2-3, frozen and lyophilized.
  • thioether bond is accomplished by reaction of an /V-terminal bromoacetyl group with a cysteine thiol as described in P.L.
  • ethers Ether formation can be accomplished by reaction of a hydroxyl group with a halo alkyl compound, preferably under basic conditions as described in Greene’s Protective Groups in Organic Synthesis, Fifth Edition. Peter G. M. Wuts. 2014 John Wiley & Sons, Inc. pp. 26-29.
  • esters can be accomplished by reaction of a hydroxyl group and a carboxylic acid using typical esterification conditions, such as Fischer esterification in the presence of acid catalysis, or alternatively with the reaction of the hydroxyl group with the corresponding acid chloride, as described in Greene’s Protective Groups in Organic Synthesis, Fifth Edition. Peter G. M. Wuts. 2014 John Wiley & Sons, Inc. pp. 271-279
  • thioesters The thioester bond is accomplished by reaction of a thiol with a carboxylic acid as described in M. Kazemi et al., Journal of Sulfur Chemistry, 2015, vol. 36:6, pp. 613-623. of Fmoc-TTDS-OH
  • the coupling of the Fmoc-TTDS-OH (2 equivalents) was achieved by either 2 cycles of 30 min of 4 equivalents of oxyma and 4 of N,N’- Diisopropylcarbodiimide (DIC) in DMF or 4 equivalents of DIC and 4 of HOBt in DCM during 2 h.
  • DIC Diisopropylcarbodiimide
  • NanoMate Advanced BioSciences, Ithaca, NY, USA aspirated the samples from a 384-well plate (protein Lobind) with disposable, conductive pipette tips, and infused the samples through the nanoESI Chip (which consists of 400 nozzles in a 20 x 20 array) towards the mass spectrometer.
  • Spray voltage was 1.70 kV and delivery pressure were 0.50 psi; the ionization was NanoESI, positive ionization.
  • NMR experiments were carried out on a Bruker Avance III 600 MHz spectrometer equipped with a TCI cryoprobe. Samples were prepared by dissolving compounds in 90% H2O/10% D2O at 3-4 mM and pH was adjusted to 2-3. Chemical shifts were referenced to internal sodium-3-(trimethylsilyl)propane sulfonate (DSS). Suppression of the water signal was achieved by excitation sculpting. Residue specific assignments were obtained from 2D total correlated spectroscopy (TOCSY) and correlation spectroscopy (COSY) experiments, while 2D nuclear Overhauser effect spectroscopy (NOESY) permitted sequence specific assignments. 13C resonances were assigned from 2D 1 H13C HSQC spectra.
  • TOCSY total correlated spectroscopy
  • COSY correlation spectroscopy
  • NOESY nuclear Overhauser effect spectroscopy
  • Amino Acid Analysis was performed to assess the amino acids present and the amount obtained for each peptide. To this end, ion exchange chromatographic analysis after acid hydrolysis was performed. The samples were hydrolyzed with 6 M HCI at 110 °C for 16 h. They were then evaporated to dryness at reduced pressure and dissolved in 20 mM aqueous HCI. Finally, the amino acids were modified using the AccQ Tag protocol from Waters and analyzed by ion exchange HPLC. For amino acid analysis, 100 pL of peptide (1 mg/mL) was added to 100 pL HCI (12 M) and 20 pL aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization reagent. This mixture was left overnight at 110°C. The liquid was fully evaporated and 200 pL 20 mM HCI was added before the Waters AccQ-Tag protocol was performed.
  • BBS Borate buffered saline
  • EDTA ethylenediaminetetraacetic acid
  • PBS Phosphate-buffered saline
  • TCEP Tris(2- carboxyethyl)phosphine hydrochloride
  • HHL heavy-heavy-light
  • HH heavy-heavy
  • HL heavylight
  • He heavy chain
  • Lc light chain
  • the column outlet was directly introduced into the electrospray ionization (ESI) source of a Waters LCT-Premier XE mass spectrometer (TOF).
  • Capillary voltage and cone voltage were set to 3000 V and 100 V respectively.
  • Desolvation and source temperatures were set to 350°C and 120°C, respectively.
  • Cone and desolvation gas flow were set to 50 L/h and 600 L/h, respectively.
  • the mass spectrometer acquired full MS scans (400-4000 m/z) working in positive polarity mode.
  • Output parameters were as follows: mass range 5000-70000 and resolution 1 Da/channel. A uniform Gaussian model was used, with the corresponding peak widths at half height.
  • DBM ((3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetic acid) was prepared as described in Mol. Pharm. 12, 3986-3998 (2015).
  • glycine 0.294 mg, 3.91 mmol
  • acetic acid 20 mL
  • the reaction mixture was heated to 100 °C overnight.
  • the solution was concentrated under vacuum and purified by silica gel chromatography (eluent DCM/MeOH 9:1).
  • Example 2 Preparation of NH2-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (NH2- TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • the cyclization was performed on resin following cyclization method 2:
  • the Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes.
  • the /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq., 1000 pmols, 56 mg) and DIEA (30 eq., 3000 pmols, 240 pL).
  • the OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 pM, 12 mg), phenyl silane (10 eq., 1000 pmols, 123 mg) in DCM (3 x 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 x 5 min).
  • Example 3 Preparation of DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM- TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • NH2-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH 2 (NH2-TTDS-SEQ ID NO: 1) prepared as in example 2, and using DBM prepared as in example 1, coupling method 2 was used to achieve compound DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- Asp-NH 2 (DBM-TTDS-SEQ ID NO: 1).
  • the coupling method 4 was applied using Fmoc-L-Tyr(tBu)-OH (137.7 mg).
  • the subsequent amino acids were coupled sequentially as follows using coupling method 4:
  • the cyclization was performed on resin following cyclization method 2:The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes. The /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq., 1000 pmols, 56 mg) and DIEA (30 eq., 3000 pmols, 240 pL).
  • the OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 pM, 12 mg), phenyl silane (10 eq., 1000 pmols, 123 mg) in DCM (3 x 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 x 5 min).
  • Example 4 Preparation of Trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- ASP-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in example 3 and Trastuzumab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Tz-DBM-TTDS-SEQ ID NO: 1.
  • Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
  • trastuzumab (111 pM, 4.9 mL, 544 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM.
  • BBS pH 8.5
  • a fresh solution of TCEP was added (10 mM, 332.2 pL, 3.26 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation.
  • TCEP was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions.
  • the DBM peptide (Example 3) in dry DMF (10 mM, 247 pL, 4.35 pmol, 8 eq.) was added to the reduced trastuzumab and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PD10 G25 columns with PBS. The final conjugates were characterized by LC-MS confirming the integrity of the antibody after peptide conjugation.
  • DBM-TTDS- Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe- Lys-Thr-Glu-Glu-Tyr-NH2 (DBM-TTDS-SEQ ID NO: 15) prepared as in Comparative Example 2 and Trastuzumab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Tz- DBM-TTDS-SEQ ID NO: 15.
  • Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
  • trastuzumab (111 pM, 4.9 mL, 544 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM.
  • BBS pH 8.5
  • a fresh solution of TCEP was added (10 mM, 332.2 pL, 3.26 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation.
  • TCEP was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions.
  • the DBM peptide (Comparative Example 2) in dry DMF (10 mM, 247 pL, 4.35 pmol, 8 eq.) was added to the reduced trastuzumab and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PD10 G25 columns with PBS. The final conjugates were characterized by LC-MS confirming the integrity of the antibody after peptide conjugation.
  • Example 5 Preparation of Cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp- NH2 (Cx-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino and Asp side-chain carboxylic acid.
  • DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in example 3 and Cetuximab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Cx-DBM-TTDS-SEQ ID NO: 1.
  • Cetuximab was obtained in its clinical form (SelleckChem, lyophilized), resuspended in sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
  • the conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017).
  • Cetuximab (1mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM.
  • a fresh solution of DTT was added (10 mM, 4 pL, 2.64 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. DTT was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions.
  • Example 6 Preparation of Bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- ASP-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in example 3 and Bevacizumab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Bv-DBM-TTDS-SEQ ID NO: 1.
  • Bevacizumab was obtained in its clinical form (HSJD, lyophilized), resuspended in sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
  • the conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017).
  • Bevacizumab (1mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM.
  • a fresh solution of DTT was added (10 mM, 4 pL, 2.64 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. DTT was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions.
  • Example 7 Preparation of Pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- ASP-NH2 (Pt-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
  • the conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017).
  • Pertuzumab (1mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM.
  • a fresh solution of DTT was added (10 mM, 4 pL, 2.64 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. DTT was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions.
  • Example 8 Stability of the shuttle in mouse serum.
  • One of the major advantages of the shuttles of this invention is that unlike the vast majority of peptides composed exclusively of L-amino acids (which are rapidly metabolized by a series of enzymes present in the serum of the blood, thus limiting their therapeutic effects), these are made with D-amino acids, thus are not recognized by the metabolic enzymes present in the serum, thereby significantly increasing their half-life in serum.
  • Example 4 Regarding to the stability studies in mouse serum of the BBB-shuttles peptides present in Example 4 and Comparative Example 3, these were incubated at a concentration of 150 pM in buffer HBSS at 37°C, in the presence of 90% mouse serum. At a range of times, 50 pL aliquots were collected to which methanol was added in order to precipitate the serum proteins. The samples were centrifuged, filtered and analyzed by HPLC in order to determine the degree of degradation. FIG.
  • Example 7 shows stability studies in mouse serum of the BBB-shuttles peptides present in Example 4 and Comparative Example 3
  • Example 9 Binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS- SEQ ID NO: 15 (Comparative Example 3) to breast cancer cells.
  • Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) was performed with increasing concentrations in ice-cold PBS for 30 minutes at 4°C. Cells were then washed and incubated with an anti-human- Dylight 650 secondary antibody (Abeam pic) in ice-cold PBS for 30 minutes at 4°C. Cells were washed with ice-cold PBS and analyzed by flow cytometry (10,000 gated events per condition).
  • FIG. 8 shows binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz- DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) to HER-2 overexpressing cells.
  • Example 10 Binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to breast cancer cells.
  • FIG. 9 shows binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to EGFR-positive MDA-MB-231 breast cancer cells.
  • Example 11 Binding of Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to breast cancer cells.
  • FIG. 10 shows binding of Pt, Pt-DBM- TTDS-SEQ ID NO: 1 (Example 7) to HER-2 overexpressing cells.
  • Cells were grown in 12-well plates in the monolayer up to 50% of confluence and serum starved overnight. Then cells were treated with PG (100 nM) and/or Tz (10 pg/ml), 24 h after stimulation, cells were trypsinized, washed twice with ice-cold PBS, fixed in 70% ethanol at -20°C for 15 min, resuspended in RNaseA 1 mg/ml (ELIRX Ltd. Gdansk, Tru) and stained with propidium iodide (2,5 pg/ml). Cell cycle was analyzed with BD LSR II flow cytometer (BD Biosciences).
  • Tz-DBM-TTDS-SEQ ID NO: 1 Example 4
  • Tz-DBM- TTDS-SEQ ID NO: 15 Comparative Example 3 treated cells.
  • SKBR3, BT-474 or MDA- MB-231 cells were serum starved and stimulated with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) (100 nM) for 5 days.
  • Cells were stained with propidium iodide and cell cycle was analyzed by flow cytometry. Results are shown in FiG. 11.
  • the reaction was stopped by removing the solution from the reaction vessel and adding it to a PD MiniTrap G-25 column (GE Healtcare) previously equilibrated with PBS.
  • the iodinated protein was dialyzed (Slide-A- Lyzer® minidialysis devices, 20 KDa, 0.5 mL) overnight against PBS to further remove the unincorporated 125 l.
  • the radioactivity of 10pL fractions was measured for 2 min using a Packard Cobra II Gamma Counter, and the protein concentration was determined using BCA analysis (Thermo Scientific).
  • the samples were diluted with Ringer Hepes to a final concentration of 100 nM.
  • Example 14 AlexaFluor 488-NHS labelling and quantification of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5); Bv, Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6), Pt, Pt-DBM-TTDS- SEQ ID NO: 1 (Example 7)
  • Example 15 Permeability assays in the in vitro human BBB cellular model
  • the amount of protein was quantified using a gamma counter and the apparent permeability using the following formula: where P app is obtained in cm/s, Q A (t) is the amount of compound at the time t in the acceptor well, VD is the volume in the donor well, t is time in seconds, A is the area of the membrane in cm and Qo(to) is the amount of compound in the donor compartment at the beginning of the experiment.
  • MS analysis proteins from the acceptor compartment were purified by immunoprecipitation with Protein A magnetic beads following manufacturer’s instructions.
  • 25 pL of beads were placed into a 1.5 microcentrifuge tube, diluted with PBST and gently mixed. The tube was placed into a magnetic stand to facilitate the supernatant removal.
  • 500 pL were added of PBS solution were added to the tube to wash the beads. After mixing the solution was removed after collecting the beads with the magnetic stand. This operation was repeated 3 times. 1 mL of acceptor solution was added and left mixing with the beads o/n at 4°C. Then the supernatant was discarded, and the beads were washed (3 x 500 pL PBST and 3 x 500 pL PBS).
  • FIG. 12 shows permeability results in the human in vitro BBB cellular model for Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) and FIG 13, FIG 14, FIG 15 show permeability results in the human in vitro BBB cellular model for Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5); Bv, Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6), Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7), respectively.
  • FIG 16 shows comparison of permeability of the conjugates.
  • Example 16 Biodistribution studies of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) in mice.
  • mice Biodistribution studies in mice were performed by ChemPartner animal facility according to protocols approved by ChemPartner Institutional Animal Care and Use Committee (IACUC) following Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.
  • CD-1 male mice (6-8 weeks) were injected with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3). (10 mg/kg) via tail vein injection. 8h after injection blood was collected for serum generation and brains were terminally collected.
  • FIG. 17 shows biodistribution studies in mice.
  • Example 17 Conjugation of DBM peptides to lgG1 mAb at pH 8.5
  • the conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017).
  • the selected monoclonal antibody (mAb) (1 mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM.
  • a fresh solution of DTT was added (10 mM, 4 pL,
  • DBM-TTDS-MiniAp4 [M+H+] theorical: 1509,269 Da; [M+H+] experimental: 1508,517 Da
  • DBM-TDS-Ang2 [M+H+] theorical: 2898,765 Da; [M+2H+/2] experimental: 1449,571 Da
  • AlexaFluor ® 488 (Aex:485/Aem:535nm) to conduct transport assay, where antibodies were assayed at 500 nm and quantification was carried out using a fluorometer.
  • Example 19 Permeability assays in the in vitro human BBB cellular model
  • the amount of protein was quantified using a gamma counter and the apparent permeability using the following formula: where P app is obtained in cm/s, QA(Q is the amount of compound at the time t in the acceptor well, VD is the volume in the donor well, t is time in seconds, A is the area of the membrane in cm and Qo(to) is the amount of compound in the donor compartment at the beginning of the experiment.
  • the results are shown in FIG. 18.
  • the results show that the permeability of the modified antibody according to the present invention (Cx-DBM_MiniAp4) is higher than the permeability of an antibody modified with a maleimide MiniAp4 as in Example 23 of WO2015/001015A1 (Cx-mal-MiniAp4).
  • FIG 19 shows the ratio of Papp of Cx modified with a SN38 conjugated peptide shuttle (MiniAp4 or Ang2) and Cx modified with the naked shuttle (MiniAp4 and Ang2) assayed at 1 pM in the human in vitro BBB cellular model.
  • the results show that the MiniAp4 conjugate increases the transport of SN38 more efficiently than the Ang2 conjugates.
  • a antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof which comprises from 1-6 peptides of formula P incorporated inserted in the disulfide bonds of the antibody in the form of a -P-(W)s-Y and joined to the sulfides through a linker -[(Ll)-(L 2 )-(L3)m-; wherein:
  • Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof;
  • the disulfide bonds are any disulfide bond that is capable of structurally retaining its structure upon reduction conditions and is selected from the group consisting of a naturally present interchain disulfide bond of the antibody, a naturally present intrachain disulfide bond of the antibody, and a disulfide bond introduced in the antibody by genetic engineering;
  • Li is a linker selected from the group consisting of Li a and Lit>;
  • L 2 is a biradical composed from 2 to 8 biradicals selected from the group consisting of LA, LB, LC and has the formula -LA-(LB) U -LC-
  • D is a radical of a substance selected form a biologically active substance, a substance for use in a diagnostic method; and a radioligand for radiotherapy;
  • P is a biradical of a peptide, equal or different, selected from the group consisting of:
  • peptide which comprises the amino acid sequence XI KAPETALX 2 wherein Xi is selected from the group consisting of Dap and Dab; and X 2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid) (SEQ ID NO:7) being a linear peptide;
  • CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that is SEQ ID NO:9: CKAPETALCAAA
  • an anticancer active pharmaceutical ingredient selected from the group consisting of auristatins, duocarmycins, PBD dimers, maytansinoids, calicheamicins, anthracyclines, camptothecines, alpha-amanitin, tubulysins, MMAE, T-
  • Clause 9 A process for preparing an antibody shuttle conjugate as defined in any of the clauses 1-8, comprising: a) reducing disulfide bridges of the antibody; b) rebridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), c) optionally, carrying out a hydrolysis; wherein q; L2, L3, D, P, m, W, s, and Y are as defined in the antibody shuttle conjugate of formula (I).
  • a pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle conjugate as defined in any of the clauses 1-8, together with appropriate amounts of pharmaceutically acceptable carriers or excipients.

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Abstract

Conjugates of antibodies and selected peptide shuttles that cross the blood-brain barrier, optionally, also conjugated with an active pharmaceutical ingredient, radiotherapy agent or diagnostic agent, pharmaceutical compositions comprising them, as well as the conjugates for use in medicine and as a diagnostic agent.

Description

SITE-SPECIFIC MODIFICATION BY A BBB-SHUTTLE OF ANTIBODY-BASED ENTITIES FOR CROSSING THE BLOOD-BRAIN BARRIER
This application claims the benefit of European Patent Application EP22382746.0 filed 01 August 2022.
Technical Field
The present invention relates to the field of conjugates with antibodies, and its use to the treatment and diagnosis of diseases that need that antibody cross the blood-brain barrier.
Background Art
Monoclonal antibodies (mAbs) have revolutionized the treatment of several diseases, especially in oncology. Antibody-based treatments target leukemia and solid tumors in many organs. However, brain tumors remain practically intractable with biotherapeutics and most small molecules.
One of the main challenges in treating brain tumors is overcoming the blood-brain barrier (BBB) and the blood-tumor barrier (BTB) in therapeutic amounts. Only 0.1-0.2% of peripherally injected doses of mAbs reach the brain parenchyma. The BBB consists of specialized endothelial cells, tightly connected and surrounded by astrocyte end-feet and pericytes, that ensures brain insulation. Although, at the core of the tumor, the BBB is replaced by the leaker BTB, at the tumor margins and in small brain metastases, the BBB may be intact and greatly hamper access of therapeutics.
Brain metastasis (BM) is a major complication in several types of cancers, particularly in lung, melanoma, and breast cancers. Breast cancer BM are especially prevalent, affecting 24% of women with stage IV breast cancer. Breast tumors overexpressing human epidermal growth factor receptor (HER-2) and those known as triple-negative display a higher incidence of BM. Although the application of mAbs has proven highly efficacious in these breast cancer types, application of mAbs to treat BM or primary brain tumors remains elusive, mainly due to the lack of antibody penetration across the BBB and BTB.
Several approaches have been explored in order to increase brain penetration of antibodies. Methods like direct injection and temporal disruption of the BBB may entail high risks for the patient. This is why, many efforts have been devoted to the development of ligands that hijack endogenous transport mechanisms across the brain endothelium. These ligands, dubbed BBB-shuttles, include antibody derivatives, endogenous proteins, peptides, and small molecules. Anthony Regina et al., in “ANG4043, a novel brain-penetrant Peptide-mAb conjugate, is efficacious against HER2-positive Intracranial Tumors in Mice” (published online mct.aacrjournals.org) discloses that the introduction of a Angiopep-2 (Ang2) to an anti- HER2 monoclonal antibody confers properties of increased uptake in brain endothelial cells as well as BBB permeability. Such introduction is through a linker and using a nonsite specific methodology. However, a limited effect of the conjugated shuttle has been observed, which could be due to the low protease-resistance of the peptide and the heterogeneous mixture obtained by randomly conjugating the peptide to surface lysines.
WO2015/001015A1 describes specific apamin-derived peptides comprising the KAPETAL fragment, which are useful as shuttles as they have the ability to cross the BBB and are capable of facilitating the transport into the brain of drugs or other diagnostically useful substances which by themselves cannot cross the BBB. This document discloses two constructs of peptides with antibodies as cargos, Example 22 (Cetuximab-V3-Ap3-NH2) and Example 23: (Cetuximab-V2-Ap5a-NH2), the AP5 peptide corresponds to MiniAp4 peptide, a cyclic peptidomimetic derived from bee venom which displays high resistance to proteolysis and negligible toxicity and immunogenicity, being V2and V3as follows:
Figure imgf000003_0001
In V3 linker bonds 1 and 2 are connected to the side chain of a lysine of the antibody, However, all the previous methods based on modifications of lysine such as the ones disclosed in the previous documents do not allow to control where the peptides are located when they are incorporated in a monoclonal antibody.
Finally, Macarena Sanchez Navarro et al.; in the Abstract “Paving the way towards the brain delivery of biotherapeutics: Modification of proteins with blood-brain barrier peptide shuttles” ECBS/LS-EuCheMS madrid (Spain) proposes the possibility of attachment of a controlled average number of peptides to different parts of proteins in order to increase its BBB penetration, in particular, it discloses the modification of GFP with MiniAp4 and with a branched version of THRre. However, nothing is said about where the peptides are introduced, how this attachment may be carried out, or about the results of the transport through the BBB or BTB. Therefore, from what is known in the art, it is derived that there is still the need of addressing the long-standing challenge of increasing antibody transport across the BBB for the treatment or diagnose of brain tumors and other CNS diseases.
Summary of Invention
The present inventors have developed a conjugate of a mAb antibody and selected peptide shuttles linked to the antibody through a bromomaleimide linker at specific sites of the antibody, and which effectively crosses the blood-brain barrier.
Unlike the methods disclosed in the prior art (see WO2015/001015A1), the linker used in the present invention allows a peptide incorporation strategy in which the incorporation of the shuttle peptides into the monoclonal antibody is controlled and allows knowing where they are located. This strategy is based on incorporating the peptides through the -SH groups of the reduced antibody. Advantageously, this strategy may be applied to any monoclonal antibody providing homogeneous conjugates by site-specific modification of the antibody, a highly desirable feature for a pharmacological product, enhancing therapeutic index and facilitating product manufacturing and profiling. In addition, the incorporation of the selected peptide shuttles has been achieved by taking advantage of the higher reactivity of the cysteines of the interchain disulfide bridges, overcoming the need for genetic engineering to introduce a reactive tag and expanding the applicability of this strategy to other mAbs.
As a way of example, the present inventors have prepared a homogenous antibody-BBB- shuttle conjugate of Trastuzumab with four copies of MiniAp4 (Tz-MiniAp4). Trastuzumab (Herceptin®) is an FDA-approved antibody against HER2, which is widely used in clinics to treat breast cancer. The conjugation has been achieved by reducing interchain disulfide bridges and rebridging them using dibromomaleimide, which enables high control over the number of peptides anchored to every antibody molecule. The conjugate shows enhanced transport across the blood-brain barrier in an in vitro cell model with respect to trastuzumab alone or with respect to a conjugate of trastuzumab with the Angiopep-2 peptide disclosed by Anthony Regina et al., in “ANG4043, a novel brain-penetrant Peptide-mAb conjugate, is efficacious against HER2-positive Intracranial Tumors in Mice”, mct.aacrjournals.org.
Accordingly, a first aspect of the present invention relates to an antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof,
Figure imgf000005_0001
which comprises from 1-6 peptides of formula P inserted in the disulfide bonds of the antibody in the form of a -P-(W)s-Y and joined to the sulfides through a linker -[(l_i)-(l_2)- (L3)m-; wherein:
Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof; the disulfide bonds are any disulfide bond initially present in the antibody and that is capable of structurally retaining sulfide bonds in the structure upon reduction conditions, the disulfide bond being selected from the group consisting of a naturally present interchain disulfide bond of the antibody, a naturally present intrachain disulfide bond of the antibody, and a disulfide bond introduced in the antibody by genetic engineering;
Li is a linker selected from the group consisting of Lia and L ;
Figure imgf000005_0002
q is an integer from 1 to 6;
Li is attached through the bonds a and b to the -S- of the disulfide bonds of the antibody and the bond c is attached to the linker L2 through an amide bond, an ester bond, or an thioester bond between the C=O group next to the bond c of the linker Li with an NH, O, or S group of the on the left side of the draw of LA below of linker L2;
L2 is a biradical composed from 2 to 8 biradicals selected from the group consisting of LA, LB, LC and has the formula -LA-(LB)U-LC-
LA is a biradical selected from the group consisting of: -NH-(CH2)r-C(=O)-;
-S-(CH2)r-C(=O)-; -O-(CH2)r-C(=O)-; -NH-(CH2)r-; -S-(CH2)r-; -O-(CH2)r-; -NH-(CH2)r-O- ; -NH-(CH2)r-NH- and -NH-(CH2)r-S-;
LB is a biradical independently selected from the group consisting of: -NH-(CH2)r-C(=O)-; -C(=O)-(CH2)r-C(=O)-; -S-(CH2)r-C(=O)-; -O-(CH2)r-C(=O)-; -NH-(CH2)r-; -C(=O)-(CH2)r- ;-S-(CH2)r-; -O-(CH2)r-; -NH-CH-((CH2)rNH2)-C(=O)-; -S-CH2-CH(NH2)-C(=O)-; -(CH2)r- C(=O)-; -(CH2)r-O-; -(CH2)r-NH-; -(CH2)r-S-; -C(=O)-(CH2)r-NH-; -C(=O)-(CH2)rO-;-C(=O)- (CH2)r-S-; -NH-(CH2)r-O-; -NH-(CH2)r-NH-; -NH-(CH2)r-S-; and combinations thereof;
LC is a biradical selected from the group consisting of: -NH-(CH2)r-C(=O)-; -NH-CH- ((CH2)r-NH2)-C(=O)-; -C(=O)-(CH2)r-C(=O)-; -S-(CH2)r-C(=O)-; -S-CH2-CH(NH2)-C(=O)-; - O-(CH2)r-C(=O)-; -(CH2)r-C(=O)-; u is an integer from 0 to 6; r’ is an integer from 1 to 5; when u=0, LA is attached to the biradical LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional group on the right side of the LA formulas and the functional groups of the left side of the LC formulas; when u=1 , LA is attached to the biradical LB through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LA formulas and the functional groups on the left side of the LB formulas; and LB is attached to the biradical LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LB formulas and the functional groups on the left side of the LC formulas; when u is higher than 1 , LB are equal or different and are attached among them through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester; being one LB terminal attached to LA through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LA formulas and the functional groups of the left side of the LB formulas; and being another LB terminal attached to LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional group on the right side of the LB formulas and the functional group on the left side of the LC formulas;
L3 is a biradical selected from the group consisting of: an amino acid selected from Lys, Orn, Dap, Dab; Glu, and Asp; an amino acid derivative selected from Lys, Orn, Dap, and Dab derivatized by having attached to the amino group of the lateral chain of the amino acid a biradical selected from the group consisting of -C(=O)-(CH2)r-C(=O)-; -C(=O)- (CH2)t-NH-; -C(=O)-(CH2)t-S-; -C(=O)-(CH2)t-O-, wherein the attachment to the amino group is through the C=O terminal group on the left side of the biradicals; an amino acid derivative selected from Glu and Asp, derivatized by having attached to the C=O group of the lateral chain of the amino acid a biradical selected from the group consisting of -NH- (CH2)trC(=O)-; -NH-(CH2)t-NH-; -NH-(CH2)t-S-; -NH-(CH2)t-O- wherein the attachment to the C=O group is through the NH group on the left side of the biradicals; and any of the previous amino acids or amino acids derivatives that have further attached CH2CH2NCH2CO2H) 4 (DOTA) or streptavidin by a feasible bond; t is an integer from 1 to 5; m is an integer selected from 0 or 1;
D is a substance attached to the linker L3 selected from a biologically active substance, a substance for use in a diagnostic method; and a radioligand for radiotherapy;
P is a biradical of a single peptide, equal or different, which is selected from the group consisting of: (a) a peptide which comprises the amino acid sequence X1KAPETALX2 with an intrapeptide bond between the Xi and X2 which is an amide bond; wherein Xi is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4- diaminobutanoic acid); and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid); i.e.
SEQ ID N0:1 : X1KAPETALX2 (b) a peptide having 12-20 amino acids residues in length having at least an intrapeptide bond which is a disulfide or diselenide bond, and comprises an amino acid sequence which is: X3KAPETALX4AAA; having at least an intrapeptide disulfide or diselenide bond between X3 and X4, wherein X3 and X4 are equal and are selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines); i.e.
Figure imgf000008_0001
(c) a peptide having 9-11 amino acids residues in length having at least an intrapeptide bond which is a disulfide or diselenide bond and consists of an amino acid sequence selected from the group consisting of XsKAPETALXe; XsKAPETALXeA; and XsKAPETALXeAA having at least an intrapeptide disulfide or diselenide bond between X5 and Xe; wherein X5 and Xe are equal and are selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines), i.e.
Figure imgf000008_0002
(d) a peptide which has 16 amino acid residues and comprises the amino acid sequence XyNXsKAPETALXgAAAX H with an intrapeptide disulfide or diselenide bond between the X7 and Xg, and between Xs and X10; wherein X7-X10 are independently selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines); provided that X7 and Xg are equal, and Xs-X are equal; i.e.
Figure imgf000008_0003
and (e) peptide which comprises the amino acid sequence XI KAPETALX2 wherein Xi is selected from the group consisting of Dap and Dab; and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid) (SEQ ID NO:7) being a linear peptide;
W is a biradical selected from the group consisting of -NH-(CH2)r-C(=O)-, and -NH-CH((CH2)rNH2)-C(=O)-; r is an integer independently selected from 1 to 5; s is an integer independently selected from 0 to 1 ;
Y is a radical selected from the group consisting of -NH2, -OH, -OR3, and -NHR3; when m=0, L3 and D are absent, and P is directly attached to LC of L2 through an amide bond formed between the C=O terminal group of LC and the amine group of the first amino acid of the peptide sequence P; when m=1 , D is present and is attached to the functional group of the lateral chain of the amino acid of linker L3 or to the amino acid derivative of linker L3 through its derivatization, wherein the attachment is through an amide, ester, disulfide, or thioester bond; L3 is attached to LC of L2 through an amide bond formed between the C=O terminal group on the left side of LC formula and the amine group of the linker L3; and P is directly attached to L3 through an amide bond formed between the C=O terminal group on the right side of LC formula and the amine group of the first amino acid of the peptide sequence P; and when s=0, P is directly attached to Y through an amide, carboxylic acid or ester bond, the bond being formed between the C=O of the C-terminal of the last amino acid of the sequence P, and the radical Y which is -NH2, -OH, -OR3, or -NHR3; and when s=1 , P is attached to a radical W through an amide bond formed with a C=O of the C-terminal of the last amino acid of the sequence P, the bond being formed between the functional groups on the left side of the draw W formulas and the functional groups (C=O) of the C-terminal of the last amino acid of the sequence P on the right side of the draw sequence; and W is attached to Y as follows: -C(=O)-NH-(CH2)r-C(=O)-Y, or -C(=O)-NH- CH((CH2)rNH2)-C(=O)-Y; n is an integer independently selected from 1 to 6; indicates the point of attachment; and
S indicates sulfide.
A second aspect of the present invention relates to a process for preparing an antibody shuttle conjugate as defined above, comprising: a) reducing the disulfide bridges of the antibody; b) rebridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), and c) optionally, carrying out a hydrolysis; wherein q; L2, L3, D, P, m, W, s and Y are as defined in the antibody shuttle conjugate of formula (I);
Figure imgf000010_0001
(II).
A third aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle conjugate of the present invention, together with appropriate amounts of pharmaceutically acceptable carriers or excipients.
A four aspect of the present invention relates to the antibody shuttle conjugate of the present invention as defined above, for use as a medicament.
A fifth aspect of the present invention relates to the antibody shuttle conjugate of the present invention as defined above for use in the treatment of Central Nervous System (CNS) disorders in a mammal, including a human.
A sixth aspect of the present invention relates to the antibody shuttle conjugate of the present invention as defined above for use as a diagnostic agent.
Brief Description of Drawings
FIG 1. 1H NMR of DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetic acid). Example 1.
FIG 2. 13C NMR of DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetic acid).
Example 1. FIG 3. LIPLC traces and MS spectra of the Comparative Example 2 and Example 3. LIPLC chromatograms are recorded at 220 nm in a 2-min linear gradient from 0 to 100% of MeCN (0.036% TFA) in H2O (0.045% TFA).
FIG 4. The site-specific Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Trastuzumab-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) were generated and characterized, a) ASC synthetic scheme; b) Mass characterization of Trastuzumab-DBM- TTDS-SEQ ID NO: 15 (also named Tz-Ang2, Comparative Example 3) (top) and Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also named Tz-MiniAp4, Example 4) (down) by LCT-Premier. Deconvoluted spectra are shown. Meal for Tz-Ang2= 159244; Mfound: 79537, 159396; Meal for Tz-MiniAp4= 153684; Mfound: 76918, 153678; c) Coomassie- stained SDS-PAGE for the trastuzumab conjugates with the DBM derivatized BBB-shuttle peptides, 1 : protein marker; 2: Trastuzumab (also named Tz) ; 3: Trastuzumab-DBM- TTDS-SEQ ID NO: 15 (also named Tz-Ang2, Comparative Example 3); 4: Trastuzumab- DBM-TTDS-SEQ ID NO: 1 (also named Tz-MiniAp4, Example 4).
FIG 5. Mass characterization of Anti- Trastuzumab (Anti- Idiotype) Alexa Fluor® 647- conjugated Antibody (AF647 modified Trastuzumab (also named Tz) (a), Trastuzumab- DBM-TTDS-SEQ ID NO: 15 (also named Tz-Ang2, Comparative Example 3) (b) and Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also named Tz-MiniAp4, Example 4) (c) by LCT-Premier. Antibodies are deglycosylated. Deconvoluted spectra are shown.
FIG 6. Mass characterization of Trastuzumab (also named Tz) (a), Trastuzumab-DBM- TTDS-SEQ ID NO: 15 (also named Tz-Ang2, Comparative Example 3) (b) and Trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also named Tz-MiniAp4, Example 4) (c) after immunoprecipitation of acceptor well from HBBBCMTA by LCT-Premier. The raw data (top) and the deconvoluted spectra (down) are shown. Mcai for Tz: 148212; Mf0Und: 148215; Mcai for Tz-Ang2= 159244: Mf0Und: 159408; Mcai for Tz-MiniAp4= 153684; Mf0Und: 76920, 153678.
FIG 7. Stability studies in mouse serum of the BBB-shuttles peptides present in Example 4 and Comparative Example 3.
FIG 8. Binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) to Her-2 overexpressing cells. BT474 and SKBR3 cells were aliquoted and incubated with Tz, Tz-Ang2 or Tz-MiniAp4 at 4°C for 2 hours. Anti-human dylight 650 was used to detect I g 1 . Amount of bound Antibody was analyzed by flow cytometry. Error bars represent the standard deviation (n = 3). P value was calculated using one-way anova multiple comparisons (SKBR3: Vehicle vs Tz p= 0.0037; Vehicle Vs. Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p=0.0007; Vehicle Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.0037; Tz Vs. Tz-DBM-TTDS-SEQ ID NO: x (Comparative Example 3) p=0.5465; Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p>0.9999; Tz-DBM-TTDS-SEQ ID NO: 2 (Comparative Example 3) vs Tz- DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.5373; BT474: Vehicle vs Tz p= 0.0004; Vehicle Vs. Tz-DBM-TTDS-SEQ ID NO: 2 (Comparative Example 3) p=0.0034; Vehicle Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.0004; Tz Vs. Tz-DBM-TTDS-SEQ ID NO: x (Comparative Example 3) p=0.5086; Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p=0.9630; Tz-DBM-TTDS-SEQ ID NO: 2 (Comparative Example 3) vs Tz- DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.7174; MDA MB 231 SKBR3: Vehicle vs Tz p= 0.9894; Vehicle Vs. Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p=0.7054; Vehicle Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.8647; Tz Vs. Tz- DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p=0.8595; Tz Vs. Tz-DBM-TTDS- SEQ ID NO: 1 (Example 4) p=0.9641; Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) vs Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.9882).
FIG 9. Binding of Cx and Cx- DBM-TTDS-SEQ ID NO: 1 (Example 5) to MDA-MB-231 cells. There are not significant differences in binding. Activity is preserved.
FIG 10. Binding of Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to BT474 cells. There are not significant differences in binding. Activity is preserved.
FIG 11. Cell cycle arrest analysis of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) treated cells. SKBR3, BT-474 or MDA-MB-231 cells were serum starved and stimulated with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) (100 nM) for 5 days. Cells were stained with propidium iodide and cell cycle was analyzed by flow cytometry. Error bars represent the standard deviation (n = 3).
FIG 12. Permeability in the human in vitro BBB cellular model for Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) (100 nM). Error bars represent the standard deviation (n = 3). P value was calculated using two-tailed t-test (Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p<0.0001; Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p<0.0001; Tz-DBM-TTDS- SEQ ID NO: 15(Comparative Example 3) vs Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.0002.
FIG 13. Permeability of Cx and Cx- DBM-TTDS-SEQ ID NO: 1 (Example 5) (1 pM) in the human in vitro BBB cellular model. Error bars represent the SEM (n = 3). The P value was calculated using two-tailed t-test (Cx vs. Cx- DBM-TTDS-SEQ ID NO: 1 (Example 5) p=0.069).
FIG 14. Permeability of Bv and Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6) (1 pM) in the human in vitro BBB cellular model. Error bars represent the SEM (n = 3). The P value was calculated using two-tailed t-test (Bv vs. Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6) p<0.001).
FIG 15. Permeability of Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) (1 pM) in the human in vitro BBB cellular model. Error bars represent the SEM (n = 3). The P value was calculated using two-tailed t-test (Pt vs. Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) p<0.0001).
FIG 16. Permeability of Cx and Cx- DBM-TTDS-SEQ ID NO: 1 (Example 5), Bv and Bv- DBM-TTDS-SEQ ID NO: 1 (Example 6) and Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) (1 pM) in the human in vitro BBB cellular model. Error bars represent the SD (n = 3).
FIG 17. Brain concentration of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz- DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) after i.v. bolus injection. Results are expressed in terms of nmol/g of tissue. Error bars represent the standard deviation (n = 3). P value was calculated using one-way anova test (Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p=0.7697; Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p=0.0323; Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) vs Tz- DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.0486). Brain to plasma ratio. mAbs were injected at 10 mg/kg in the tail vein of mice. After 8 hours, serum was collected and wholebody saline perfusion was performed. The brains were then removed and mAb were quantified by ELISA. Results are expressed in terms of brain/serum ratio for mAbs. Error bars represent the standard deviation (n = 3). P value was calculated using one-way anova test (Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p=0.2657; Tz Vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p=0.0319; Tz-DBM-TTDS-SEQ ID NO: 2 (Comparative Example 3) vs Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) p= 0.1710).
FIG. 18. Permeability of Cx, Cx-MiniAp4 (1 pM) in the human in vitro BBB cellular model. Error bars represent the SD (n = 3). The P value was calculated using one-way Anova (Cx vs. Cx-DBM-MiniAp4 p=0.0012; Cx-mal-MiniAp4 vs. Cx-DBM-MiniAp4 p=0.0007).
FIG.19. Ratio of Papp of Cx modified with a SN38 conjugated peptide shuttle (MiniAp4 or Ang2) and Cx modified with the naked shuttle (MiniAp4 and Ang2) assayed at 1 pM in the human in vitro BBB cellular model. Error bars represent the SD (n = 3). The P value was calculated using two-tailed t-test (Cx vs. Cx-MiniAp4 p=0.0061).
Detailed description of the invention
All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.
As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun.
The word “comprise” for the purposes of the present invention encompasses the case of “consisting of”.
Unless otherwise stated, the amino acids cited herein are L-amino acids. The 1-letter code and the 3-letter code have been used indistinctly. For the following amino acids, the following abbreviations have been used: diaminopropionic acid (Dap), Diaminobutiric (Dab), Selenocystein (Sec) and Penicillamine (Pen). In the context of the present invention penicillamine only embraces D-penicillamine. The diaminopropionic acid (Dap) can also be abbreviated as Dpr, and the Diaminobutiric (Dab) can also be abbreviated as Dbu.
As mentioned above, it is part of the invention an antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof,
Figure imgf000014_0001
which comprises from 1-6 peptides of formula P inserted in the disulfide bonds of the antibody in the form of a -P-(W)s-Y and joined to the sulfides through a linker -[(Li )-(l_2)- (l_3)m-; wherein, Z, Li , L2, L3, D, m, P, W, S, Y and n have the meaning mentioned above. In a particular embodiment, the antibody shuttle conjugate of the present invention is that where the disulfide bonds initially present in the antibody are capable of structurally retaining the sulfide bonds in the structure upon reduction conditions and of forming between them new bridges by reaction of the sulfide groups of the reduced disulfide bonds of the antibody with the linker Li, to incorporate the biradical below between the sulfide groups of the antibody.
Figure imgf000015_0001
The antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof may be in the form of a formulation comprising the antibody or their salts and excipients. For instance, appropriate excipients for Bevacizumab (Avastin) are trehalose dihydrate, sodium phosphate, polysorbate 20, and water for injectable preparations, appropriate excipients for Pertuzumab (Perjeta) are Glacial acetic acid, L-histidine, Sucrose, Polysorbate 20, and water for injectable preparations; appropriate excipients for are Cetuximab (Erbitux): Sodium Chloride, Glycine, Polysorbate 80, Citric Acid Monohydrate, Sodium Hydroxide, and water for Injection Preparations; and appropriate excipients for trastuzumab (Herceptin) are for instance: L-histidine hydrochloride monohydrate, L- histidine, a,a-trehalose, or polysorbate 20 dihydrate.
In a particular embodiment, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof, is that wherein Li is attached through the bonds a and b to the -S- of the disulfide bonds of the antibody and the bond c is attached to the linker L2 through an amide bond between the C=O group next to the bond c of the linker Li with an NH group of the linker L2.
In another particular embodiment, either alone or in combination with any of the embodiments above, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof, is that wherein the linker L2 is a biradical selected from the group consisting of:
Figure imgf000015_0002
Figure imgf000016_0001
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof, is that wherein n is an integer from 1 to 4. In another particular embodiment, in combination with any of the embodiments above, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof, is that wherein n is 4, i.e. , it has four peptides incorporated within the antibody structure. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, all the peptides are equal.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the disulfide bond of the antibody is an interchain bond. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the disulfide bond of the antibody is an intrachain bond. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the disulfide bond of the antibody is a disulfide bond formed by genetic engineering.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein P is a biradical of a peptide selected from the group consisting of:
(a) a peptide which comprises the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond, that is SEQ ID NO:8: DapKAPETALD
TIHCO ^ (b) a peptide having 9-20 ammo acids residues in length having at least an intrapeptide bond which is a disulfide bond, and comprises an amino acid sequence which is: CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that is SEQ ID NO:9: CKAPETALCAAA
^S-S^
(c) a peptide having 9-11 amino acids residues in length having at least an intrapeptide bond which is a disulfide bond and consists of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that are
SEQ ID NO: 10: CKAPETALC
^S-S^
SEQ ID NO:11 : CKAPETALCA
Figure imgf000017_0001
SEQ ID NO: 12: CKAPETALCAA; and
Figure imgf000017_0002
(d) a peptide which has 16 amino acid residues and comprises the amino acid sequence CNCKAPETALCAAACH with an intrapeptide disulfide bond between the first and third cysteine which are cysteines 1 and 11 , and between the second and the fourth cysteine which are cysteine 3 and 15, that is,
SEQ ID NO: 13: CNCKAPETALCAAACH
Figure imgf000017_0003
(e) a peptide which comprises the amino acid sequence DapKAPETALD (SEQ ID NO: 14).
The lines between two amino acids of the sequences above or below represent the intrapeptide bond between the side chains of the two amino acids. In a particular embodiment, the lines between two amino acids of the sequences above or below represent the intrapeptide bond between the side chains of the two amino acids.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein, P is a biradical of a peptide selected from the group consisting of: (a) the peptide having the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond (SEQ ID NO:7); (b) the peptide having the amino acid sequence CKAPETALC having at least an intrapeptide disulfide bond between cysteines in position 1 and 9 (SEQ ID NO: 10; and (c) the peptide having the amino acid sequence DapKAPETALD (SEQ ID NO:14).
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein P is a biradical of the peptide DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond (SEQ ID NO:7).
In another particular embodiment, either alone or in combination with any of the embodiments of the invention the antibody shuttle conjugate is that wherein P is a biradical of a peptide having one intrapeptide bond. In another particular embodiment, the antibody shuttle conjugate is that wherein P is a biradical of a peptide having two intrapeptide bonds.
Antibodies generally consist of two heavy chains (HCs) and two light chains (LCs) folded into constant and variable domains, although some antibodies, such as camelid antibodies, contain only 2 heavy chains. They can occur individually as monomers (e.g. IgG), or in multimers of two (e.g. IgA) to five units (e.g. IgM). They have disulfide intrachain bonds. The antibodies for the purposes of the present invention may be chimeric, humanized or fully human antibodies. In the sense of the present invention the antibodies can be natural antibodies or recombinant antibodies. In particular embodiments, the antibody is a therapeutical antibody. In another particular embodiment, the antibody is for diagnosis. In another particular embodiment the antibody is an antibody-drug conjugate (ADC).
The antibody can also be an antibody fragment, provided it contains at least one disulfide bond and provided that it maintains the function of the antibody from which it is derived In particular, its therapeutic or diagnostic activity. For example, the antibody may be an antibody fragment such as Fab, scFV, (Fab)2, diabody, triabody, tetrabody, minibody, or nanobody. For instance, disulfide bonds can be incorporated for instance into the antibodies or fragments thereof by incorporating cys by mutagenizing a nucleic acid sequence of an antibody and replacing one or more amino acid residues by cysteines to encode the cysteine engineered antibody (see CA2957354A1). Antibody fragments can offer several advantages compared to full-length antibodies. Their smaller size may enable better tissue penetration into solid tumors, and their shorter half-life is ideal if using an antibody as a radioactive imaging agent. Useful for applications in which the antibody doesn’t need to engage with the immune system, such as the blocking of a signaling molecule or receptor.
In a particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody comprises a heavy chain constant domain of the type of IgA, IgD, I g E, IgG or IgM. Antibodies of the IgA type can be divided into two isotopes: I gA1 or lgA2. Antibodies of the IgG class can be divided into four isotypes: lgG1 , lgG2, lgG3 and lgG4. classes of antibodies are encompassed within the scope of the present invention.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody comprises a light chain constant domain, e.g., of the type of kappa or lambda.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody or antibody fragment according to the present invention comprises:(a) an immunoglobulin constant region; (b) an IgG 1 constant region; or (c) a human IgG 1 constant region. In particular, I gG 1 is preferred as it is the most widely used isotype for anticancer mAbs, as well as the most effective IgG isotype in mediating ADCC (antibody-dependent cellular cytotoxicity). In a more particular embodiment, the antibody is a monoclonal chimeric, humanized, or fully human antibody. Antibody humanization allows reducing immunogenicity by decreasing the mouse content of monoclonal antibodies. This can be made, for example, by the expression of isolated human variable domain genes in E. coli. Common techniques developed for the production of fully human monoclonal antibodies can be used such as phage display, where a library of human antibodies is expressed on the surface of phage and subsequently selected and amplified in E. coli, and transgenic mice expressing a human antibody repertoire.
In also another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody is: (a) is multispecific. Multispecific antibodies is a class of engineered antibody and antibody-like proteins that combine multiple specific antigen binding elements in a single construct.
In also another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody is: heterodimeric bispecific antibodies which are traditional IgG molecule with one arm targeting one antigen and the other targeting a second antigen. In also another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody is a bispecific antibody fusion which is a non-standard IgG molecule. IgG is elongated at its N-terminus on the corresponding heavy and light chains by an additional variable domain of a second antibody.
In also another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody is a trispecific antibody which is also a non-standard IgG molecule. The same technologies used to generate bispecific antibodies can also be combined to generate trispecific antibodies with varying valences.
In also another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is that wherein the antibody is fusion of a scFv to an IgG via the attachment of the scFv to the N or C-terminus of the heavy or light chain.
In a particular embodiment, the antibody shuttle conjugate of the invention is that wherein the antibody is selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab, Pertuzumab, Aducanumab, Bapineuzumab, Nimotuzumab, and Necitumumab. In a more particular embodiment, the antibody shuttle conjugate of the invention is that where the antibody is selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab and Pertuzumab.
In a particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Trastuzumab. Trastuzumab (Herceptin®), an FDA-approved antibody against HER2, which is widely used in clinics to treat breast cancer. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that which is Trastuzumab-DBM-TTDS-Dap-Lys-Ala- Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Cetuximab. Cetuximab (Erbitux®), an FDA-approved antibody against EGFR, which is widely used in clinics to treat colorectal cancer and head and neck cancer. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that which is Cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Cx-DBM-TTDS- SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp sidechain carboxylic acid.
The term “DBM” corresponds to linker Lia. The term “TTDS” corresponds to linker l_2a. These terms in both cases have been used indistinctly.
DBM = Lia has the formula:
Figure imgf000021_0001
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Bevacizumab. Bevacizumab (Avastin ®), an FDA-approved antibody against VEGF-A, which is widely used in clinics to treat colon cancer, lung cancer, glioblastoma and renal-cell carcinoma. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that which is Bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu- Thr-Ala-Leu-Asp-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Pertuzumab. Pertuzumab (Perjeta ®), an FDA-approved antibody against HER2, which is widely used in clinics to treat breast cancer. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that which is Pertuzumab-DBM-TTDS- Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Aducanumab, a lgG1 antibody against an epitope of the p-amyloid protein. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Bapineuzumab, an anti-Ap-amyloid lgG1 antibody. Both antibodies are useful for the treatment of Alzheimer disease.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Nimotuzumab, a lgG1 anti-EGFR antibody. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate of the invention is that where the antibody is Necitumumab, IgG 1 anti-EGFR antibody. Both antibodies are useful for the treatment of brain metastases.
In a particular embodiment, either alone or in combination with any of the embodiments of the invention, the formulation comprising the antibody shuttle conjugate of the invention is that where D is present, yielding to an antibody-drug shuttle conjugate, an antibodyradioligand shuttle conjugate, or an antibody-diagnostic agent shuttle conjugate.
In a particular embodiment the biologically active substance is a pharmaceutical active ingredient. In another particular embodiment, the antibody shuttle conjugate of the invention is that wherein: the antibody is an anticancer therapeutical antibody, m=1 and the radical of the active biological substance D is a radical of an anticancer active pharmaceutical ingredient selected from the group consisting of auristatins, duocarmycins, PBD dimers, maytansinoids, calicheamicins, anthracyclines, camptothecines, alpha- amanitin, tubulysins, MMAE, T-DM1 , and PROTAC moieties.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody is an anticancer therapeutical antibody, m=1 , and D is a chemotherapeutic agent.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the antibody-drug shuttle conjugate is that where the antibody is cetuximab and D is SN38. In another particular embodiment, the antibody-drug shuttle conjugate according to the invention is that where the antibody is cetuximab, the drug is SN38, the peptide is MiniAp4, and the linker is TTDS-DBM.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, the formulation comprising the antibody is an anticancer therapeutical antibody, m=1, and D is a radioligand. This formulation is useful for radioimmunotherapy. In another particular embodiment, either alone or in combination with any of the embodiments of the invention, D is biotin-Ytrium 90 or Biotin- Iodine 131 or 123. They can be joined through streptavidin to the antibody shuttle conjugate of the present invention, where the streptavidin is attached to a lateral chain of the amino acids of the biradical Lswhen present in the antibody shuttle conjugate of the present invention.
In another particular embodiment, either alone or in combination with any of the embodiments of the invention, D is a contrast agent for a magnetic resonance imaging method (MRI), such as Gadolinium, in particular, Gd-DTPA (Magnevist®). Gadolinium may be complexed with DOTA which is attached to a lateral chain of the amino acids of the biradical Lswhen present in the antibody shuttle conjugate of the present invention or to an amino acid derivative by a feasible bond.
In another particular embodiment, the antibody shuttle conjugate of the invention is that wherein: the antibody is an antineurodegenerative therapeutical antibody, m=1, and the radical of the active pharmaceutical ingredient is a radical of an antineurodegenerative active pharmaceutical ingredient.
The antibody shuttle conjugates of the present invention may be prepared by a process comprising: a) reducing the disulfide bridges of the antibody; b) rebridging the disulfide bridges, i.e. forming new bridges, by reacting the -SH groups of the antibody with a dibromomaleimide-peptide (DBM-peptide) of formula (II), DBM-P-(W)S-(Y), in which the dibromomaleimide is attached to the peptide via the N-terminus of the peptide, wherein DBM, P, W, Y and s is as defined for the antibody shuttle conjugate of formula (I). The binding of the DBM-peptide to the antibody is possible thanks to the reactivity of the cysteines forming the disulfide bridges. This method allows a high control of the number of peptides anchored to each antibody molecule. In a particular embodiment, the antibody is trastuzumab. The antibody shuttle conjugate of the present invention may be defined by its preparation process. Accordingly, an antibody shuttle conjugate obtainable by the process defined above is also considered part of the present invention.
In particular, the antibody shuttle conjugate as defined above may be obtainable by reacting the hydrosulfide groups of the reduced disulfide bonds of the corresponding antibody or of a fragment thereof with a dibromomaleimide-peptide of formula (II) as defined above to rebridge the disulfide bond with the biradical below incorporated between the disulfide bridges of the antibody.
Figure imgf000024_0001
In another particular embodiment, the antibody shuttle conjugate as defined above may be obtainable by a process comprising: a) reducing disulfide bridges of the antibody; b) rebridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), c) optionally, carrying out a hydrolysis; wherein q; L2, L3, D, P, m, W, s, and Y are as defined in the antibody shuttle conjugate of formula (I).
Figure imgf000024_0002
In a particular embodiment of the process, the disulfide bonds are interchain bonds. All the embodiments defined above for the conjugate of formula (I) as product per se are also embodiments of the process for its preparation.
The shuttle peptide used in the conjugates of the present invention can be generated wholly or partly by chemical synthesis. The amino acids required for the preparation of compounds of formula (I) are commercially available. The compounds of formula (I) can be prepared easily, for example by synthesis in liquid-phase or, preferably, by solid-phase peptide synthesis, for which there are a number of procedures published (see M. Amblard, et al., "Methods and protocols of modern solid-phase peptide synthesis. Molecular Biotechnology 2006, Vol. 33, p. 239-254). The compounds of formula (I) can also be prepared by any combination of liquid-phase synthesis and/or solid-phase synthesis. For example, by synthesizing the body of the peptide P through solid-phase synthesis and subsequently removing protecting groups in solution. The binding to the linker dibromomaleimide can be performed in solid-phase or in solution. The construction of the linker can also be prepared by any combination of liquid-phase synthesis and/or solidphase synthesis. The linker used is 3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)acetic acid or similar derivatives thereof.
The compound of formula (II) can be prepared by a process comprising: reacting a peptide derivatized with the linkers with 3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1- yl)acetic acid by solid phase peptide synthesis or in solution to yield the DBM-peptide. The compound of formula (II) with a linker L can be obtained by the corresponding compound of formula (II) with a linker Lia by hydrolysis. The hydrolysis can be carried out in mild pH basic conditions,
In a particular embodiment, the DBM peptide of formula (II) has the following formula:
Figure imgf000025_0001
In another particular embodiment, the DBM peptide of formula (II) has the following formula:
Figure imgf000026_0001
In a particular embodiment, the antibody shuttle conjugate of the invention is that where the linker Li is L . This conjugate may be prepared by hydrolysis in mild basic pH conditions from the corresponding antibody shuttle conjugate having as Li linker the linker Lla.
The antibody shuttle conjugates of the present invention may be in the form of a pharmaceutically acceptable salt thereof. The term “pharmaceutically acceptable salts” used herein encompasses any salt formed from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids or bases. There is no limitation regarding the salts, except that if used for therapeutic purposes, they must be pharmaceutically acceptable. As some of the compounds of formula (I) are basic compounds, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for instance, chlorohydric, acetic, benzenesulfonic, benzoic, camphor sulfonic, citric, ethansulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic, malic, mandelic, methane sulfonic, phosphoric, succinic, sulfuric, tartaric, p-toluensulfonic acid, and the like.
Examples of antibodies in the form of a salt according to the present invention are Bevacizumab (Avastin) in the form of a salt with trehalose dihydrate, sodium phosphate, polysorbate 20 salts; Pertuzumab (Perjeta) in the form of a salt with glacial acetic acid, L- histidine, Sucrose, or Polysorbate 20; cetuximab (Erbitux) in the form of a salt formed with sodium Chloride, glycine, polysorbate 80, citric acid monohydrate, or sodium hydroxide, trastuzumab (Herceptin) in the form of a salt with L-histidine hydrochloride monohydrate, L-histidine, a,a-trehalose, or polysorbate 20 dihydrate
The preparation of pharmaceutically acceptable salts of the compounds of formula (I) can be carried out by methods known in the art. For instance, they can be prepared from the conjugate, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate pharmaceutically acceptable base or acid in water or in an organic solvent or in a mixture of them.
A pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle conjugate as defined above, together with appropriate amounts of pharmaceutically acceptable carriers or excipients is also part of the invention.
The term “pharmaceutical composition” refers to a mixture of a compound described herein with other chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration of the compound to an organism. The terms “pharmaceutically acceptable excipients or carriers” refers to pharmaceutically acceptable material, composition or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must be also suitable for use in contact with tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a benefit/risk ratio.
The expression "therapeutically effective amount" as used herein, refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.
The expression "pharmaceutically acceptable excipient, diluent or carrier" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
The relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
The compositions of the present invention may be administered in parenteral form suitable for injection such as intravenous bolus injections, intravenous infusion, implantation into the body, oral, intratecal, or intranasal.
An antibody shuttle conjugate as defined above for use as a medicament is also part of the invention. This includes the antibody shuttle conjugates as well as the antibody-drug shuttle conjugates according to the present invention.
The term “medicament” as used herein is synonymous of a pharmaceutical or veterinary drug (also referred to as medicine, medication, or simply drug) use to cure, treat or prevent disease in animals, including humans, as widely accepted. Drugs are classified in various ways. One key distinction is between traditional small-molecule drugs, usually derived from chemical synthesis, and biopharmaceuticals, which include recombinant proteins, vaccines, blood products used therapeutically (such as I VIG), gene therapy, monoclonal antibodies and cell therapy (for instance, stem-cell therapies).
An antibody shuttle conjugate as defined above for use in the treatment of CNS disorders in a mammal, including a human is also part of the invention. This aspect can also be formulated as the use antibody shuttle conjugate as defined above for the preparation of a medicament for the treatment of CNS disorders in a mammal, including a human. The invention also relates to a method of treatment of a mammal, including a human, suffering from or being susceptible of suffering from a CNS disorder, said method comprising the administration to said patient of a therapeutically effective amount of antibody shuttle conjugate as defined above, together with pharmaceutically acceptable excipients or carriers.
In a particular embodiment, the antibody shuttle conjugate is for use in a CNS disorder which is cancer.
In another particular embodiment, the antibody shuttle conjugate of the present invention is for use in the treatment of a primary brain tumor. In another particular embodiment the antibody shuttle conjugate of the present invention is for use in the treatment of brain metastasis (BM) is a major complication in several types of cancers, particularly in lung, melanoma, and breast cancers.
The antibody shuttle conjugates of the present invention, both the antibody shuttle conjugates as well as the antibody-drug shuttle conjugates according to the present invention, can be used in the same manner as other known chemotherapeutic agents, i.e. , in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. They may be used alone or in combination with other suitable bioactive compounds. Thus, the antibody shuttle conjugates of the present invention are for use in the treatment of cancer in a mammal, including a human in combination therapy with a chemotherapeutic agent. The antibody-drug shuttle conjugates in combination therapy with a further chemotherapeutic agent can be used for instance for specific treatment regimens.
In another particular embodiment, the antibody shuttle conjugate of the present invention is for use in combination with radioimmunotherapy. Both the antibody shuttle conjugates as the antibody-radioligand shuttle conjugates according to the invention can be used. For instance, the combination of radiation therapy and immunotherapy can be used to treat non-Hodgkin lymphoma and other types of cancer including brain tumors. The antibodyradioligand shuttle conjugates according to the invention can bind to cancer cells and deliver a high dose of radiation directly to the tumor.
An antibody shuttle conjugate as defined above for use as a diagnostic agent is also part of the invention. In a particular embodiment the antibody shuttle conjugate according to the invention is for use in a method of diagnostic of CNS disorders.
Throughout the description and claims the word “comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. Examples
Protected amino acids, handles and resins were supplied by: Luxembourg Industries (Tel- Aviv, Israel), Neosystem (Strasbourg, France), CalbiochemNovabiochem AG (Laufelfingen, Switzerland), Bachem AG (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). Other reagents and solvents used are summarized in Table 1. Table 1 Commercials suppliers and reagents used. DCM passed through an AI2O3 column. DMF is stored on molecular sieves 4 , and nitrogen is bubbled in order to eliminate volatile agents.
Protected amino acids were supplied by Iris Biotech (Marktredwitz, Germany). ChemMatrix resin was purchased from PCAS BioMatrix (QC, Canada). Diisopropylethylamine (DIEA), N,N’-diisopropylcarbodiimide (DIC) and ninhydrin were supplied by Fluka Chemika (Buchs, Switzerland). Solvents for peptide synthesis and liquid chromatography were provided by SDS (Barcelona, Spain). Trifluoroacetic acid (TFA) was purchased from Scharlau (Barcelona, Spain). The other chemicals used were obtained from Aldrich (Milwaukee, Wl) and were of the highest purity commercially available.
Cell culture-treated plates and flasks were purchased from Corning Costar. Culture medium was acquired from Lonza. XTT cell proliferation kit was purchased from Biological Industries (Cromwell, CT). Pierce® iodination beads were obtained from Pierce. Desalting columns (MiniTrap and MidiTrap G-25) were obtained from GE-Healthcare.
Figure imgf000030_0001
Figure imgf000031_0003
General methods for the preparation of the
Figure imgf000031_0001
of the present invention
General considerations about the manual
Figure imgf000031_0002
Solid-phase peptide elongation and other solid-phase manipulations were carried out manually in polypropylene syringes fitted with a porous polyethylene disk. Solvents and soluble reagents were removed by suction. Washings between different synthetical steps were carried out with dimethylformamide (DMF) (5 x 30 s) and dichloromethane (DCM) (5 x 30 s) using 10 mL of solvent/g of resin each time.
General considerations about the microwave assisted synthesis: Microwave assisted solid-phase peptide synthesis was carried out on a Liberty Blue Automated Microwave Peptide Synthesizer using H-Rink amide Protide resin (loading: 0.56 mmols/g). Linear peptide was synthesized on a 0.5 mmol scale using a 5 excess of Fmoc-amino acid (0.2M) relative to the resin.
Identification tests The test used for the identification and control of the synthesis was the following: A) Kaiser colorimetric assay for the detection of solid-phase bound primary amines (E. Kaiser et al., Anal. Biochem. 1970, vol. 34, pp. 595-598); B) p-nitro phenyl ester test for secondary amines bound to solid-phase (A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791). Protocols used durinq the manual is of the com
Figure imgf000032_0001
The compounds were synthesized at a 100 pmol scale using the following methods and protocols: The resin for the manual synthesis was selected depending on the group Y: If Y is a OH, the terminus will be COOH, 2-chlorotrytil chloride resin will be chosen among others available. If Y is a NH2, the terminus will be CONH2, Rink amide MBHA resin will be chosen among others available.
Resin initial
Figure imgf000032_0002
: The resin was conditioned by washing with MeOH (5 x 30 s),
DMF (5 x 30 s), DCM (5 x 30 s), 1% TFA in DCM (1 x 30 s and 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30s), 5 % DIEA in DCM (1 x 30 s, 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s).
Fmoc removal Removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was done with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each. Two additional treatments with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) (2 x 5 min) were performed to ensure the removal of the Fmoc group from secondary amines (proline).
Figure imgf000032_0003
scale: method 1 The protected amino acid (4 eq 400 pmols), TBTU (4 eq., 400 pmols, 128 mg) dissolved in DMF (1-3 mL/g resin) were added sequentially to the resin, subsequently DIEA was added (8 eq, 800 pmols, 136 pl). The mixture was allowed to react with intermittent manual stirring for 1 h. The solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The extent of coupling was checked by the Kaiser colorimetric assay. The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes. If the amino acid to be deprotected was a proline, an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group. method 2 The protected amino acid (4 eq 400 pmols) PyBOP (4 eq 400 pmols, 208 mg), HOAt (12 eq., 1.2 mmols, 163 mg) dissolved in DMF (1-3 mL/g resin) were added sequentially to the resin, subsequently DIEA was added (12 eq., 1.2 mmols, 204 pL). The mixture was allowed to react with intermittent manual stirring for 1 h. The solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling reaction was carried out twice under the same conditions. The extent of coupling was checked by the Kaiser colorimetric assay. The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using 30 s treatments and two treatments of 10 minutes. If the amino acid to be deprotected was a proline, an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group. method 3 The protected amino acid (4 eq 400 pmols), PyBOP (4 eq., 400 pmols, 208 mg), HOBt (12 eq., 1.2 mmols, 162 mg) dissolved in DMF (1-3 mL/g resin) were added sequentially to the resin, subsequently DIEA was added (12 eq., 1.2 mmols, 204 p L). The mixture was allowed to react with intermittent manual stirring for 1 h. The solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling reaction was carried out twice under the same conditions. The extent of coupling was checked by the Kaiser colorimetric assay. The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using 30 s treatments and two treatments of 10 minutes. If the amino acid to be deprotected was a proline, an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group. method 4 scale 100 pmols: The protected amino acid (3 eq 300 pmols)), DIG
(3 eq., 300 pmols, 46 pL) and Oxyma (3 eq., 300 pmols, 43 mg) in DCM/DMF (1 :1). The mixture was allowed to react with intermittent manual stirring for 45 min. The solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The extent of coupling was checked by the Kaiser colorimetric assay. The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using 30 s treatments and two treatments of 10 minutes. If the amino acid to be deprotected was a proline, an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group. method 5 scale 100 pmols: The protected amino acid (3 eq 300 pmols)), DIC
(3 eq., 300 pmols, 46 pL) and HOBt (3 eq., 300 pmols, 41 mg) in DCM/DMF (1:1). The mixture was allowed to react with intermittent manual stirring for 45 min. The solvent was removed by suction and the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The extent of coupling was checked by the Kaiser colorimetric assay. The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using 30 s treatments and two treatments of 10 minutes. If the amino acid to be deprotected was a proline, an additional treatment with DBU, toluene, piperidine, DMF (5%, 5 %, 20%, 70%) (2 x 5 min) was applied to ensure the removal of the Fmoc group.
Protocols used during the microwave assisted automated
Figure imgf000033_0001
is: The compounds were synthesized at a 500 pmol scale using the following methods and protocols: The resin for the microwave assisted automated synthesis was selected depending on the group Y: If Y is a OH, the terminus will be COOH, CI-TCP(CI) ProTide resin will be chosen among others available. If Y is a NH2, the terminus will be CONH2, Rink amide ProTide resin will be chosen among others available.
Resin initial conditioning: The resin was conditioned by washing with MeOH (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30 s), 1% TFA in DCM (1 x 30 s and 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s), DCM (5 x 30s), 5 % DIEA in DCM (1 x 30 s, 2 x 10 min), DCM (5 x 30 s), DMF (5 x 30 s).
Coupling and deprotection conditions for the microwave assisted automated peptide synthesis:
Coupling conditions:
Figure imgf000034_0001
Figure imgf000034_0002
Deprotection conditions:
Figure imgf000034_0003
Figure imgf000034_0004
Methods for the
Figure imgf000035_0001
of the
Figure imgf000035_0002
P: method 1 disulfide or diselenide bond The cyclization was performed in solution after the cleavage from the resin or on resin after the selective deprotection of the Cys, Sec or Pen residues. The peptide was dissolved at a concentration of 100 pM in aqueous ammonium bicarbonate buffer 10 mM and pH 8.0. The solution was intensely stirred for 24 h at room temperature. After that, the product was acidified with TFA to pH 2-3, frozen and lyophilized. method 2 amide bond The cyclization was performed on resin The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes. The /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq, 1000 pmol, 56 mg) and DIEA (30 eq, 3000 pmol, 240 pL). The OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq, 10 pM, 12 mg), phenyl silane (10 eq, 1000 pmol, 123 mg) in DCM (3 x 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 x 5 min). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by addition of PyBOP (4 eq, 400 pmols, 208 mg), HOAt (12 eq, 1.2 mmol, 163 mg), DMF (1-3 mL/g resin) and DIEA (12 eq, 1.2 mmol, 204 p L). The coupling was left 1.5 h and repeated overnight. method 3 amide bond The cyclization was performed on resin The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes. The /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq, 1000 pmol, 56 mg) and DIEA (30 eq, 3000 pmol, 240 pL). The OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq, 10 pM, 12 mg), phenyl silane (10 eq, 1000 pmol, 123 mg) in DCM (3 x 15 min). The resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3 x 5 min). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by_2 cycles of 30 min of 4 equivalents of Oxyma (400 pmols, 57 mg) and 4 of N,N’-Diisopropylcarbodiimide (DIC) (400 pmols, 61 pL ). method 4 amide bond The cyclization was performed on resin The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes. The /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq., 1000 pmols, 56 mg) and DIEA (30 eq., 3000 pmols, 240 pL). The OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 pM, 12 mg), phenyl silane (10 eq., 1000 pmols, 123 mg) in DCM (3 x 15 min). The resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3 x 5 min). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by_2 cycles of 1 hour of 4 equivalents of DIC (400 pmols, 61 pL) and 4 of HOBt (400 pmols, 54 mg).
General methods for the construction of the linker
General method for the formation of disulfides the disulfide bond can be accomplished by reaction of two thiols. The thiols are dissolved at a concentration of 100 pM in aqueous ammonium bicarbonate buffer 10 mM and pH 8.0 and the solution is intensely stirred for 24 h at room temperature. After that, the solution is acidified with TFA to pH 2-3, frozen and lyophilized.
General methods for the formation of thioethers The thioether bond is accomplished by reaction of an /V-terminal bromoacetyl group with a cysteine thiol as described in P.L.
Barker et a/. J. Med. Chem., 1992. vol 35, pp. 2040-2048.
General methods for the formation of ethers Ether formation can be accomplished by reaction of a hydroxyl group with a halo alkyl compound, preferably under basic conditions as described in Greene’s Protective Groups in Organic Synthesis, Fifth Edition. Peter G. M. Wuts. 2014 John Wiley & Sons, Inc. pp. 26-29.
General methods for the formation of esters Ester formation can be accomplished by reaction of a hydroxyl group and a carboxylic acid using typical esterification conditions, such as Fischer esterification in the presence of acid catalysis, or alternatively with the reaction of the hydroxyl group with the corresponding acid chloride, as described in Greene’s Protective Groups in Organic Synthesis, Fifth Edition. Peter G. M. Wuts. 2014 John Wiley & Sons, Inc. pp. 271-279
General methods for the formation of thioesters The thioester bond is accomplished by reaction of a thiol with a carboxylic acid as described in M. Kazemi et al., Journal of Sulfur Chemistry, 2015, vol. 36:6, pp. 613-623. of Fmoc-TTDS-OH The coupling of the Fmoc-TTDS-OH (2 equivalents), was achieved by either 2 cycles of 30 min of 4 equivalents of oxyma and 4 of N,N’- Diisopropylcarbodiimide (DIC) in DMF or 4 equivalents of DIC and 4 of HOBt in DCM during 2 h. Followed by removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each.
Figure imgf000037_0001
ic acid The coupling of the 5-hexynoic acid (2 eq 200 nmols, 23 mg), was achieved by either 2 cycles of 30 min of 4 equivalents of Oxyma (400 nmols, 57 mg) and 4 of N,N’-diisopropylcarbodiimide (DIG) (400 pLmols, 61 pL ) in DMF:DCM (1 :1) or 4 equivalents of DIG (400 pL mols, 61 pL) and 4 of HOBt (400 pmols, 54 mg) in DMF:DCM 1 :1 in DOM during 4 h or 2 equivalents of PyBOP (400 pmols, 208 mg ) in DMF:DCM 1 :1 , 6 equivalents of HOAt (600 pmols, 81.5 mg) and 6 equivalents of DIEA
(600 pmols, 102 pL) for 1.5 h in DMF. The solvent was removed by suction, the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.
Figure imgf000037_0002
ic anhidride The coupling of diglycolic anhydride (10 eq 1000 pmols,
116 mg), was achieved by_2 cycles of 60 min of 10 equivalents of DIEA (1000 pmols, 174 pL) in DMF. The solvent was removed by suction, the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.
Methods for the alkyne-azide
Figure imgf000037_0003
: The alkyneazide cycloaddition (Click reaction) coupling was performed in solution using the protocol described in S.F.M. van Dongen et al , Bioconjugate Chem. 2009, vol. 20, pp. 20-23.
General methods for the
Figure imgf000037_0004
from the resin Final cleavage of the resin and sidechain deprotection: It was carried out by treating resin with TFA (95%), H2O (2.5%) and
TIS (2.5%) (2 h). Tert-butyl methyl ether was added to the obtained product and the mixture was centrifuged (3 x 8 min). The supernatant was discarded, and the pellet was resuspended in a mixture of H2O, MeCN and TFA (1000:1000:1). The product was filtered out and frozen.
General methods for the characterization of the com
Figure imgf000037_0005
The compounds were characterized by LIPLC (Acquity high-class system (PDA detector, sample manager FNT and Quaternary solvent manager, Acquity BEH C18 (50 x 2 mm x 1.7 pm) column, 0.61 mL/min and MeCN (0.036% TFA) and H2O (0.045% TFA) were used as solvents. In all cases, 2-min linear gradients were used) and UPLC-MS spectrometry (Waters high class (PDA detector, sample manager FNT and Quaternary solvent manager) coupled to an electrospray ion source ESI-MS Micromass ZQ and using the MassLynx 4.1 software (Waters, Milford, MA). Using a BEH C18 column (50 x 2.1 mm x 1.7 pm, Waters). The flow rate was 0.6 mL/min, and MeCN (0.07% formic acid) and H2O (0.1% formic acid) were used as solvents. Samples were analyzed with positive ionization: the ion spay voltage was 30 V and the capillary temperature was 1 kV). The exact mass was obtained by Mass Spectrometer: LTQ-FT Ultra (Thermo Scientific) with sample introduction in Direct infusion (Automated Nanoelectrospray). The NanoMate (Advion BioSciences, Ithaca, NY, USA) aspirated the samples from a 384-well plate (protein Lobind) with disposable, conductive pipette tips, and infused the samples through the nanoESI Chip (which consists of 400 nozzles in a 20 x 20 array) towards the mass spectrometer. Spray voltage was 1.70 kV and delivery pressure were 0.50 psi; the ionization was NanoESI, positive ionization.
NMR experiments were carried out on a Bruker Avance III 600 MHz spectrometer equipped with a TCI cryoprobe. Samples were prepared by dissolving compounds in 90% H2O/10% D2O at 3-4 mM and pH was adjusted to 2-3. Chemical shifts were referenced to internal sodium-3-(trimethylsilyl)propane sulfonate (DSS). Suppression of the water signal was achieved by excitation sculpting. Residue specific assignments were obtained from 2D total correlated spectroscopy (TOCSY) and correlation spectroscopy (COSY) experiments, while 2D nuclear Overhauser effect spectroscopy (NOESY) permitted sequence specific assignments. 13C resonances were assigned from 2D 1 H13C HSQC spectra. All experiments were performed at 298 K except NOESY spectra that were acquired at 278 K. Amide proton temperature coefficients were determined from a series of one dimensional spectra acquired between 278 and 308K. The TOCSY and NOESY mixing times were 70 and 250 ms, respectively.
Amino Acid Analysis: Amino acid analysis was performed to assess the amino acids present and the amount obtained for each peptide. To this end, ion exchange chromatographic analysis after acid hydrolysis was performed. The samples were hydrolyzed with 6 M HCI at 110 °C for 16 h. They were then evaporated to dryness at reduced pressure and dissolved in 20 mM aqueous HCI. Finally, the amino acids were modified using the AccQ Tag protocol from Waters and analyzed by ion exchange HPLC. For amino acid analysis, 100 pL of peptide (1 mg/mL) was added to 100 pL HCI (12 M) and 20 pL aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization reagent. This mixture was left overnight at 110°C. The liquid was fully evaporated and 200 pL 20 mM HCI was added before the Waters AccQ-Tag protocol was performed.
General method for purification and characterization of peptides: The crude was purified by RP-HPLC at semi-preparative scale and characterized by UPLC (Acquity high-class system (PDA detector, sample manager FNT and Quaternary solvent manager, Acquity BEH C18 (50 x 2 mm x 1.7 pm) column, 0.61 mL/min and MeCN (0.036% TFA) and H2O (0.045% TFA) were used as solvents. In all cases, 2-min linear gradients were used) and UPLC-MS spectrometry (Waters high class (PDA detector, sample manager FNT and Quaternary solvent manager) coupled to an electrospray ion source ESI-MS Micromass ZQ and using the MassLynx 4.1 software (Waters, Milford, MA). Using a BEH C18 column (50 x 2.1 mm x 1.7 pm, Waters). The flow rate was 0.6 mL/min, and MeCN (0.07% formic acid) and H2O (0.1% formic acid) were used as solvents. Samples were analyzed with positive ionization: the ion spay voltage was 30 V and the capillary temperature was 1 kV). The exact mass was obtained by Mass Spectrometer: LTQ-FT Ultra (Thermo Scientific) with sample introduction in Direct infusion (Automated Nanoelectrospray). The NanoMate (Advion BioSciences, Ithaca, NY, USA) aspirated the samples from a 384-well plate (protein Lobind) with disposable, conductive pipette tips, and infused the samples through the nanoESI Chip (which consists of 400 nozzles in a 20 x 20 array) towards the mass spectrometer. Spray voltage was 1.70 kV and delivery pressure were 0.50 psi; the ionization was NanoESI, positive ionization. All peptides were obtained with a purity higher than 95%.
General Methods for
Figure imgf000039_0001
monoclonal antibodies experiments were performed in microcentrifuge tubes (1.5, 2 or 5 mL) at r. t. unless otherwise noticed with mixing. All buffer solutions were prepared with MilliQ water. Borate buffered saline (BBS) stands for 50 mM sodium borate, 50 mM NaCI and 5 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.5. Phosphate-buffered saline (PBS) stands for 10 mM sodium phosphate, 137 mM sodium chloride and 2.7 mM potassium chloride at pH 7.4. Tris(2- carboxyethyl)phosphine hydrochloride (TCEP) solutions 10 mM (2.87 mgmL-1) were prepared in BBS immediately before use.
Concentration was carried out by in Amicon Ultra-15 low binding cellulose filters with 10 kDa MWCO. Centrifugation was carried out on a Beckman Coulter Allegra 21 K centrifuge operating at 3500 ref at 4 °C.
The following acronyms are used to describe antibody fragments based on their constituent heavy and light chains: heavy-heavy-light (HHL), heavy-heavy (HH), heavylight (HL), heavy chain (He) and light chain (Lc).
General Methods for characterization of antibodies The LC-MS system set up was as follows. 8 pL of sample was injected automatically to a BioSuite pPhenyl 1000 (Waters, 10 pm RPC 2.0x75 mm) column at a flow rate of 100 pL/min using an Acquity UPLC system (Waters Corporation) provided with a binary solvent manager and an automatic autosampler. Intact protein was eluted using a linear gradient from 5% to 80% B in 60 min (A= 0.1 % formic acid (FA) in water, B= 0.1% FA in CH3CN). The column outlet was directly introduced into the electrospray ionization (ESI) source of a Waters LCT-Premier XE mass spectrometer (TOF). Capillary voltage and cone voltage were set to 3000 V and 100 V respectively. Desolvation and source temperatures were set to 350°C and 120°C, respectively. Cone and desolvation gas flow were set to 50 L/h and 600 L/h, respectively. The mass spectrometer acquired full MS scans (400-4000 m/z) working in positive polarity mode.
Data were acquired with MassLynx software, V4.1.SCN704 (Waters Inc.). MS spectra corresponding to the chromatographic peak were summed. Charged protein species in the resulting spectrum were deconvoluted to their zero charged average masses using the integrated MaxEntl (maximum entropy) algorithm.
Output parameters were as follows: mass range 5000-70000 and resolution 1 Da/channel. A uniform Gaussian model was used, with the corresponding peak widths at half height.
Example 1: Preparation of (3,4-dibromo-2,5-dioxo-2,5-dihvdro-1H-pyrrol-1-yl)acetic acid (L1 , DBM):
Figure imgf000040_0001
DBM ((3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetic acid) was prepared as described in Mol. Pharm. 12, 3986-3998 (2015). (1) In brief, glycine (0.294 mg, 3.91 mmol) was added to a solution of 3, 4-dibromofuran-2, 5-dione (1 g, 3.91 mmol) in acetic acid (20 mL), and the solution was stirred at room temperature for 10 min until all the solids dissolved. The reaction mixture was heated to 100 °C overnight. The solution was concentrated under vacuum and purified by silica gel chromatography (eluent DCM/MeOH 9:1). Concentration of pure fractions afforded 1.08 g (3.4 mmol, 89% yield) of dibromomaleimide derivative, 2-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)acetic acid. 1H NMR (400 MHz, CH3OD): 54.32 (s, 2H).13C NMR (101 MHz, CH3OD): 5 170, 164, 129, 40. m/z: 309.81, 311.84, 313.87 [M - H]’.
Example 2: Preparation of NH2-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (NH2- TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
Figure imgf000041_0001
1 D Fmoc-L-Asp(OAI) -OH 395.4 118.5 3
2 L Fmoc-L-Leu-OH 353.4 105.9 3
3 A Fmoc-Ala-OH H2O 329.3 98.7 3
4 T Fmoc-L-Thr(tBu)-OH 397.5 119.1 3
5 E Fmoc-Glu(OtBu)-OH H2O 443.5 132.9 3
6 P Fmoc-L-Pro-OH H2O 355.4 106.5 3
7 A Fmoc-Ala-OH H2O 329.3 98.7 3
8 K Fmoc-Lys(Boc)-OH 468.5 140.4 3
9 Dap Fmoc-L-Dap(Alloc)-OH 410.4 123 3
For the manual coupling of the first protected amino acid to the resin, the coupling method
4 was applied using Fmoc-Asp(OAI)-OH (118.5 mg). The subsequent amino acids were coupled sequentially as follows using coupling method 4:
Using 46 pL DIC and 43 mg Oxyma in DMF/DCM (1:1). The mixture was allowed to react with intermittent manual stirring for 45 min. After each coupling removal of the 9- fluorenylmethyloxycarbonyl (Fmoc) protecting group was done with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each. Two additional treatments with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) (2 x 5 min) were performed to ensure the removal of the Fmoc group from secondary amines (proline). The cyclization was performed on resin following cyclization method 2: The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes. The /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq., 1000 pmols, 56 mg) and DIEA (30 eq., 3000 pmols, 240 pL). The OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 pM, 12 mg), phenyl silane (10 eq., 1000 pmols, 123 mg) in DCM (3 x 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 x 5 min). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by addition of PyBOP (4 eq., 400 pmols, 208 mg), HOAt (12 eq., 1.2 mmols, 163 mg), DMF (1-3 mL/g resin) and DIEA (12 eq., 1.2 mmols, 204 p L). The coupling was left 1.5 h and repeated overnight.
Coupling of Fmoc-TTDS-OH: The coupling of the Fmoc-TTDS-OH (2 equivalents, 200 pmols, 108.53 mg), was achieved by_4 equivalents of DIC (400 pmols, 61 pL) and 4 of HOBt (400 pmols, 54 mg) in DCM during 2 h._Followed by removal of the 9- fluorenylmethyloxycarbonyl (Fmoc) protecting group with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each.
Example 3: Preparation of DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM- TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
Starting from NH2-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (NH2-TTDS-SEQ ID NO: 1) prepared as in example 2, and using DBM prepared as in example 1, coupling method 2 was used to achieve compound DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- Asp-NH2 (DBM-TTDS-SEQ ID NO: 1).
DBM (4 eq., 400 pmols, 125 mg) in DMF (1-3 mL/g resin), PyBOP (4 eq., 400 pmols, 208 mg) and HOAt (12 eq., 1.2 mmols, 163 mg) were sequentially added to the resin followed by the addition of 12 eq. of DIEA (1.2 mmols, 204 pL). The mixture was allowed to react with intermittent manual stirring for 1.5 h. The solvent was removed by suction, the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay. Molecular formula: C59H94Br2Ni4O22. Cal MW (Da): 1508.5034. Found MW (Da): 1508.4992. tR UPLC (min): 1.374. Purity: >95%.Yield:5%.
Comparative Example 1 : Preparation of NH2-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg- Glv-Lvs-Arg-Asn-Asn-Phe-Lvs-Thr-Glu-Glu-Tyr-NH2 (NH2-TTDS-SEQ ID NO: 15)
For the manual coupling of the first protected amino acid to the resin, the coupling method 4 was applied using Fmoc-L-Tyr(tBu)-OH (137.7 mg). The subsequent amino acids were coupled sequentially as follows using coupling method 4:
Figure imgf000042_0001
Figure imgf000043_0001
Using 46 pL DIC and 43 mg Oxyma in DMF/DCM (1:1). The mixture was allowed to react with intermittent manual stirring for 45 min. After each coupling removal of the 9- fluorenylmethyloxycarbonyl (Fmoc) protecting group was done with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each. Two additional treatments with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) (2 x 5 min) were performed to ensure the removal of the Fmoc group from secondary amines (proline). The cyclization was performed on resin following cyclization method 2:The Fmoc group was removed with a 20% solution of piperidine in DMF (v/v) using a 30 s treatment and two treatments of 10 minutes. The /V-terminal amine was protected with a Boc protecting group using BOC2O (3 eq., 1000 pmols, 56 mg) and DIEA (30 eq., 3000 pmols, 240 pL). The OAI and Alloc groups were first deprotected by addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 pM, 12 mg), phenyl silane (10 eq., 1000 pmols, 123 mg) in DCM (3 x 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 x 5 min). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by addition of PyBOP (4 eq., 400 pmols, 208 mg), HOAt (12 eq., 1.2 mmols, 163 mg), DMF (1-3 mL/g resin) and DIEA (12 eq., 1.2 mmols, 204 p L). The coupling was left 1.5 h and repeated overnight.
Coupling of Fmoc-TTDS-OH: The coupling of the Fmoc-TTDS-OH (2 equivalents, 200 pmols, 108.53 mg), was achieved by_4 equivalents of DIC (400 pmols, 61 pL) and 4 of HOBt (400 pmols, 54 mg) in DCM during 2 h._Followed by removal of the 9- fluorenylmethyloxycarbonyl (Fmoc) protecting group with 20% (v/v) piperidine in DMF using a treatment of 30 s followed by two treatments of 10 minutes each.
Comparative Example 2: Preparation of DBM-TTDS- Thr-Phe-Phe-Tyr-GIv-GIv-Ser-Arg- Glv-Lvs-Arg-Asn-Asn-Phe-Lvs-Thr-Glu-Glu-Tyr (DBM-TTDS-SEQ ID NO: 15)
Starting from NH2-TTDS- Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe- Lys-Thr-Glu-Glu-Tyr (NH2-TTDS-SEQ ID NO: 15) prepared as in Comparative Example 1 , and using DBM prepared as in example 1 , coupling method 2 was used to achieve compound DBM-TTDS- Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys- Thr-Glu-Glu-Tyr (DBM-TTDS-SEQ ID NO: 15.
DBM (4 eq., 400 pmols, 125 mg) in DMF (1-3 mL/g resin), PyBOP (4 eq., 400 pmols, 208 mg) and HOAt (12 eq., 1.2 mmols, 163 mg) were sequentially added to the resin followed by the addition of 12 eq. of DIEA (1.2 mmols, 204 pL). The mixture was allowed to react with intermittent manual stirring for 1.5 h. The solvent was removed by suction, the resin washed with DMF (5 x 30 s) and DCM (5 x 30 s). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay. Molecular formula: Ci24Hi76Br2N32Os9. Cal MW (Da): 2895.1139. Found MW (Da): 2895.1296. tR UPLC (min): 1.415. Purity: >95%.Yield: 10%.
Example 4. Preparation of Trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- ASP-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid. Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in example 3 and Trastuzumab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Tz-DBM-TTDS-SEQ ID NO: 1.
Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
Concentration was determined by UV/Vis absorbance (using s280 =215380 M'1 cm-1 for trastuzumab mAb), and the protein was stored in flash frozen aliquots at -20 °C. For experiments, aliquots were thawed and used immediately.
The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) In brief, trastuzumab (111 pM, 4.9 mL, 544 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM. A fresh solution of TCEP was added (10 mM, 332.2 pL, 3.26 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. TCEP was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions. Next, the DBM peptide (Example 3) in dry DMF (10 mM, 247 pL, 4.35 pmol, 8 eq.) was added to the reduced trastuzumab and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PD10 G25 columns with PBS. The final conjugates were characterized by LC-MS confirming the integrity of the antibody after peptide conjugation.
Comparative Example 3. Preparation of Trastuzumab-DBM-TTDS- Thr-Phe-Phe-Tyr-GIv- Gly-Ser-Ara-GIv-Lvs-Arg-Asn-Asn-Phe-Lvs-Thr-Glu-Glu-Tyr-NHz (Tz-DBM-TTDS-SEQ ID NO: 15).
Starting from DBM-TTDS- Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe- Lys-Thr-Glu-Glu-Tyr-NH2 (DBM-TTDS-SEQ ID NO: 15) prepared as in Comparative Example 2 and Trastuzumab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Tz- DBM-TTDS-SEQ ID NO: 15.
Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
Concentration was determined by UV/Vis absorbance (using s280 =215380 M-1 cm-1 for trastuzumab mAb), and the protein was stored in flash frozen aliquots at -20 °C. For experiments, aliquots were thawed and used immediately.
The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) In brief, trastuzumab (111 pM, 4.9 mL, 544 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM. A fresh solution of TCEP was added (10 mM, 332.2 pL, 3.26 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. TCEP was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions. Next, the DBM peptide (Comparative Example 2) in dry DMF (10 mM, 247 pL, 4.35 pmol, 8 eq.) was added to the reduced trastuzumab and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PD10 G25 columns with PBS. The final conjugates were characterized by LC-MS confirming the integrity of the antibody after peptide conjugation.
Example 5. Preparation of Cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp- NH2 (Cx-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino and Asp side-chain carboxylic acid.
Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in example 3 and Cetuximab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Cx-DBM-TTDS-SEQ ID NO: 1.
Cetuximab was obtained in its clinical form (SelleckChem, lyophilized), resuspended in sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
Concentration was determined by UV/Vis absorbance (using s280 =215380 M-1 cm-1 for Cetuximab mAb), and the protein was stored in flash frozen aliquots at -20 °C. For experiments, aliquots were thawed and used immediately.
The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) In brief, Cetuximab (1mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM. A fresh solution of DTT was added (10 mM, 4 pL, 2.64 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. DTT was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions. Next, the DBM peptide (Example 3) in dry DMF (10 mM, 5.3 pL, 3.52 pmol, 8 eq.) was added to the reduced Cetuximab and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PD10 G25 columns with PBS. The final conjugates were characterized by LC-MS confirming the integrity of the antibody after peptide conjugation. Mass characterization of Cetuximab and Cetuximab-DBM-TTDS-SEQ ID NO: 1 by deglicosylating with PNGase F were 148182 and 153654, respectively.
Example 6. Preparation of Bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- ASP-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in example 3 and Bevacizumab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Bv-DBM-TTDS-SEQ ID NO: 1.
Bevacizumab was obtained in its clinical form (HSJD, lyophilized), resuspended in sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning.
Concentration was determined by UV/Vis absorbance (using s280 =215380 M'1 cm-1 for Bevacizumab mAb), and the protein was stored in flash frozen aliquots at -20 °C. For experiments, aliquots were thawed and used immediately.
The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) In brief, Bevacizumab (1mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM. A fresh solution of DTT was added (10 mM, 4 pL, 2.64 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. DTT was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions. Next, the DBM peptide (Example 3) in dry DMF (10 mM, 5.3 pL, 3.52 pmol, 8 eq.) was added to the reduced Bevacizumab and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PD10 G25 columns with PBS. The final conjugates were characterized by LC-MS confirming the integrity of the antibody after peptide conjugation. Mass characterization of Bevacizumab and Bevacizumab-DBM- TTDS-SEQ ID NO: 1 by deglicosylating with PNGase F were 146322 and 151794, respectively.
Example 7. Preparation of Pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu- ASP-NH2 (Pt-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid.
Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ
ID NO: 1) prepared as in example 3 and Pertuzumab after general method for monoclonal antibody conditioning were conjugated using the following protocol to achieve the compound of formula Pt-DBM-TTDS-SEQ ID NO: 1.
Pertuzumab was obtained in its clinical form (SelleckChem, lyophilized), resuspended in sterile water and the buffer exchanged completely for BBS pH 8.5 with PD10 g25 columns (GE Healthcare), following General Methods for monoclonal antibody conditioning. Concentration was determined by UV/Vis absorbance (using s280 =215380 M'1 cm-1 for Pertuzumab mAb), and the protein was stored in flash frozen aliquots at -20 °C. For experiments, aliquots were thawed and used immediately.
The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) In brief, Pertuzumab (1mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM. A fresh solution of DTT was added (10 mM, 4 pL, 2.64 pmol, 6 eq.) and the reaction was incubated at 37 °C for 2 h under mild agitation. DTT was removed by SEC using PD10 G25 columns with BBS as buffer, following manufacturer's instructions. Next, the DBM peptide (Example 3) in dry DMF (10 mM, 5.3 pL, 3.52 pmol, 8 eq.) was added to the reduced Pertuzumab and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PD10 G25 columns with PBS. The final conjugates were characterized by LC-MS confirming the integrity of the antibody after peptide conjugation. Mass characterization of Pertuzumab and Pertuzumab-DBM- TTDS-SEQ ID NO: 1 by deglicosylating with PNGase F were 145214 and 150686, respectively.
Example 8: Stability of the shuttle in mouse serum.
One of the major advantages of the shuttles of this invention is that unlike the vast majority of peptides composed exclusively of L-amino acids (which are rapidly metabolized by a series of enzymes present in the serum of the blood, thus limiting their therapeutic effects), these are made with D-amino acids, thus are not recognized by the metabolic enzymes present in the serum, thereby significantly increasing their half-life in serum.
Regarding to the stability studies in mouse serum of the BBB-shuttles peptides present in Example 4 and Comparative Example 3, these were incubated at a concentration of 150 pM in buffer HBSS at 37°C, in the presence of 90% mouse serum. At a range of times, 50 pL aliquots were collected to which methanol was added in order to precipitate the serum proteins. The samples were centrifuged, filtered and analyzed by HPLC in order to determine the degree of degradation. FIG. 7 shows stability studies in mouse serum of the BBB-shuttles peptides present in Example 4 and Comparative Example 3 Example 9: Binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS- SEQ ID NO: 15 (Comparative Example 3) to breast cancer cells.
In vitro binding to HER2-positive BT-474 and SKBR-3 breast cancer cells was determined by flow cytometry. Confluent cells were detached from flasks with trypsin which was neutralized with FBS supplemented DM EM. Cells in suspension were washed in ice-cold PBS counted and separated into individual 1.5 mL tubes (106 cells per tube).
Binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) was performed with increasing concentrations in ice-cold PBS for 30 minutes at 4°C. Cells were then washed and incubated with an anti-human- Dylight 650 secondary antibody (Abeam pic) in ice-cold PBS for 30 minutes at 4°C. Cells were washed with ice-cold PBS and analyzed by flow cytometry (10,000 gated events per condition). FIG. 8 shows binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz- DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) to HER-2 overexpressing cells.
Example 10: Binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to breast cancer cells.
In vitro binding to EGFR-positive MDA-MB-231 breast cancer cells was determined by flow cytometry. Confluent cells were detached from flasks with trypsin which was neutralized with FBS supplemented DM EM. Cells in suspension were washed in ice-cold PBS counted and separated into individual 1.5 mL tubes (106 cells per tube).
Binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) was performed with increasing concentrations in ice-cold PBS for 30 minutes at 4°C. Cells were then washed and incubated with an anti-human-Dylight 488 secondary antibody (Abeam pic) in ice-cold PBS for 30 minutes at 4°C. Cells were washed with ice-cold PBS and analyzed by flow cytometry (2,000 gated events per condition). FIG. 9 shows binding of Cx, Cx-DBM- TTDS-SEQ ID NO: 1 (Example 5) to EGFR-positive MDA-MB-231 breast cancer cells.
Example 11 : Binding of Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to breast cancer cells.
In vitro binding to HER2-positive BT-474 breast cancer cells was determined by flow cytometry. Confluent cells were detached from flasks with trypsin which was neutralized with FBS supplemented DM EM. Cells in suspension were washed in ice-cold PBS counted and separated into individual 1.5 mL tubes (106 cells per tube). Binding of Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) was performed with increasing concentrations in ice-cold PBS for 30 minutes at 4°C. Cells were then washed and incubated with an anti-human-Dylight 488 secondary antibody (Abeam pic) in ice-cold PBS for 30 minutes at 4°C. Cells were washed with ice-cold PBS and analyzed by flow cytometry (2,000 gated events per condition). FIG. 10 shows binding of Pt, Pt-DBM- TTDS-SEQ ID NO: 1 (Example 7) to HER-2 overexpressing cells. and Tz-
Figure imgf000050_0001
Cells were grown in 12-well plates in the monolayer up to 50% of confluence and serum starved overnight. Then cells were treated with PG (100 nM) and/or Tz (10 pg/ml), 24 h after stimulation, cells were trypsinized, washed twice with ice-cold PBS, fixed in 70% ethanol at -20°C for 15 min, resuspended in RNaseA 1 mg/ml (ELIRX Ltd. Gdansk, Poland) and stained with propidium iodide (2,5 pg/ml). Cell cycle was analyzed with BD LSR II flow cytometer (BD Biosciences).
Cell cycle arrest analysis of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM- TTDS-SEQ ID NO: 15 (Comparative Example 3) treated cells. SKBR3, BT-474 or MDA- MB-231 cells were serum starved and stimulated with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) (100 nM) for 5 days. Cells were stained with propidium iodide and cell cycle was analyzed by flow cytometry. Results are shown in FiG. 11.
13. 125l labelling and quantification of Tz, Tz-DBM-TTDS-SEQ ID NO: 1
Figure imgf000050_0002
PierceTM Iodination Beads (Life Technologies) were used to radiolabel the Tz, Tz-DBM- TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3). Briefly, two beads per protein were washed with 500 pL of reaction buffer (50 mM NaPi, pH 6.5) and dried on filter paper. In a glass vial, the beads were added with the calculated amount of carrier-free Na125l (1 mCi/mg protein) in 200 pL of reaction buffer. The reaction was incubated for 5 min. The proteins were then added, and the reaction was carried out for 15 min with occasional mixing. The reaction was stopped by removing the solution from the reaction vessel and adding it to a PD MiniTrap G-25 column (GE Healtcare) previously equilibrated with PBS. The iodinated protein was dialyzed (Slide-A- Lyzer® minidialysis devices, 20 KDa, 0.5 mL) overnight against PBS to further remove the unincorporated 125l. The radioactivity of 10pL fractions was measured for 2 min using a Packard Cobra II Gamma Counter, and the protein concentration was determined using BCA analysis (Thermo Scientific). The samples were diluted with Ringer Hepes to a final concentration of 100 nM.
Example 14. AlexaFluor 488-NHS labelling and quantification of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5); Bv, Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6), Pt, Pt-DBM-TTDS- SEQ ID NO: 1 (Example 7)
In brief, two aliquots of 0.2 pL of AlexaFluor 488 (10mgmL-1 in DMSO) were added every 15 minutes to a 100 pL of the selected mAb (0.25 mg, 82 nmol, NaPi pH8), the samples were mixed under dark during an hour. The excess of dye was removed by SEC using PDmini G25 columns with PBS as buffer, following manufacturer's instructions.
Example 15. Permeability assays in the in vitro human BBB cellular model
These experiments were performed using the model developed in Prof. R. Cecchelli’s laboratory. (5) In brief, endothelial cells derived from pluripotent stem cells and bovine pericytes were defrosted in gelatin-coated Petri dishes (Corning). Pericytes were cultured in DMEM pH 6.8 while endothelial cells were cultured in supplemented endothelial cell growth medium (sECM) (Sciencells). After 48 h, endothelial cells were seeded in 12-well Transwell inserts (8000 cell/well) and pericytes were plated in 12-well plates (50000 cells/well) previously coated with Matrigel and gelatin, respectively. sECM medium was used for both cell lines and changed every 2-3 days. The assays were performed 7-8 days after seeding by placing inserts containing the endothelial cells into new wells without pericytes.
To perform the assay, 500 pL of the unlabeled or 125l labelled mAbs following example 8 (5 pM or 100 nM, respectively) in ECM media or Ringer HEPES was added to the donor compartment and 1500 pL of ECM media or Ringer HEPES was introduced into the acceptor compartment. Lucifer Yellow (25 pM) was added as a control of barrier integrity (Papp < 15- 10-6 cm/s). In the case of the unlabeled compounds the plates were incubated for 16 h but after 2 hours 500 pL of the acceptor compartment were removed for analysis and replaced with fresh media. In the case of the 125l labelled mAb the plates were incubated for 2 h at 37°C, and the solutions from both compartments were recovered and analyzed. In the case of the AlexaFluor 488 labelled mAb the plates were incubated for 2 h at 37°C, and the solutions from both compartments were recovered and analyzed by fluorescence. The samples were evaluated in triplicates. The amount of protein was quantified using a gamma counter and the apparent permeability using the following formula:
Figure imgf000052_0001
where Papp is obtained in cm/s, QA(t) is the amount of compound at the time t in the acceptor well, VD is the volume in the donor well, t is time in seconds, A is the area of the membrane in cm and Qo(to) is the amount of compound in the donor compartment at the beginning of the experiment.
For MS analysis proteins from the acceptor compartment were purified by immunoprecipitation with Protein A magnetic beads following manufacturer’s instructions. In brief, 25 pL of beads were placed into a 1.5 microcentrifuge tube, diluted with PBST and gently mixed. The tube was placed into a magnetic stand to facilitate the supernatant removal. 500 pL were added of PBS solution were added to the tube to wash the beads. After mixing the solution was removed after collecting the beads with the magnetic stand. This operation was repeated 3 times. 1 mL of acceptor solution was added and left mixing with the beads o/n at 4°C. Then the supernatant was discarded, and the beads were washed (3 x 500 pL PBST and 3 x 500 pL PBS). mAbs were eluted with 50 pL of 0.1 M glycine pH 2 and the solution was neutralized with 8 pL of 3M Tris, pH 8.5. Samples were analysis by LCT-MS. FIG. 12 shows permeability results in the human in vitro BBB cellular model for Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) and FIG 13, FIG 14, FIG 15 show permeability results in the human in vitro BBB cellular model for Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5); Bv, Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6), Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7), respectively. FIG 16 shows comparison of permeability of the conjugates.
Example 16. Biodistribution studies of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) in mice.
Biodistribution studies in mice were performed by ChemPartner animal facility according to protocols approved by ChemPartner Institutional Animal Care and Use Committee (IACUC) following Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. CD-1 male mice (6-8 weeks) were injected with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3). (10 mg/kg) via tail vein injection. 8h after injection blood was collected for serum generation and brains were terminally collected. Amount of antibodies in brain tissue and serum was determined by ELISA (Goat anti-human IgG F(c) Antibody, Sigma, Cat#609-101-017, Anti-Human IgG (Fab specific)-Peroxidase, Sigma, Cat#A0293). FIG. 17 shows biodistribution studies in mice. Example 17: Conjugation of DBM peptides to lgG1 mAb at pH 8.5
The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). In brief, the selected monoclonal antibody (mAb) (1 mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 pM. A fresh solution of DTT was added (10 mM, 4 pL,
2.64 pmol, 6 eq) and the reaction was incubated at 37 °C for 2 h under mild agitation. DTT was removed by SEC using PDmini G25 columns with BBS as buffer, following manufacturer's instructions. Next, the DBM peptide in dry DMF (10 mM, 5.3 pL, 3.52 pmol, 8 eq.) was added to the reduced mAb and the reaction was left at r.t. for 30 min. Afterwards, excess reagents were removed by SEC using PDmini G25 columns with PBS. The final conjugates were characterized by LC-MS. DBM peptides used:
DBM-TTDS-MiniAp4 ([M+H+] theorical: 1509,269 Da; [M+H+] experimental: 1508,517 Da
Figure imgf000053_0001
DBM-TDS-Ang2 ( [M+H+] theorical: 2898,765 Da; [M+2H+/2] experimental: 1449,571 Da)
Figure imgf000054_0001
Example 18: Labelling of conjugates with AlexaFluor 488-NHS
All antibodies were labeled with AlexaFluor ® 488 (Aex:485/Aem:535nm) to conduct transport assay, where antibodies were assayed at 500 nm and quantification was carried out using a fluorometer.
In brief, two aliquots of 0.2 pL of AlexaFluor 488 (lOmgmL-1 in DMSO) were added every
15 minutes to a 100 pL of the selected mAb (0.25 mg, 82 nmol, NaPi pH8), the samples were mixed under dark during an hour. The excess of dye was removed by SEC using PDmini G25 columns with PBS as buffer, following manufacturer's instructions.
Example 19: Permeability assays in the in vitro human BBB cellular model
These experiments were performed using the model developed in Prof. R. Cecchelli’s laboratory. (5) In brief, endothelial cells derived from pluripotent stem cells and bovine pericytes were defrosted in gelatin-coated Petri dishes (Corning). Pericytes were cultured in DMEM pH 6.8 while endothelial cells were cultured in supplemented endothelial cell growth medium (sECM) (Sciencells). After 48 h, endothelial cells were seeded in 12-well Transwell inserts (8000 cell/well) and pericytes were plated in 12-well plates (50000 cells/well) previously coated with Matrigel and gelatin, respectively. sECM medium was used for both cell lines and changed every 2-3 days. The assays were performed 7-8 days after seeding by placing inserts containing the endothelial cells into new wells without pericytes.
To perform the assay, 500 iL of the fluorescently mAb (1 pM ) in Ringer HEPES was added to the donor compartment and 1500 iL of Ringer HEPES was introduced into the acceptor compartment. Lucifer Yellow (25 pM) was evaluated in a control of barrier integrity (Papp < 15- 106 cm/s). The mAb were for 2 h at 37°C, and the solutions from both compartments were recovered and analysed by fluorescence (Aex:485;Aem:535nm). The samples were evaluated in triplicates. The amount of protein was quantified using a gamma counter and the apparent permeability using the following formula:
Figure imgf000055_0001
where Papp is obtained in cm/s, QA(Q is the amount of compound at the time t in the acceptor well, VD is the volume in the donor well, t is time in seconds, A is the area of the membrane in cm and Qo(to) is the amount of compound in the donor compartment at the beginning of the experiment.
The results are shown in FIG. 18. The results show that the permeability of the modified antibody according to the present invention (Cx-DBM_MiniAp4) is higher than the permeability of an antibody modified with a maleimide MiniAp4 as in Example 23 of WO2015/001015A1 (Cx-mal-MiniAp4).
On the other hand, FIG 19 shows the ratio of Papp of Cx modified with a SN38 conjugated peptide shuttle (MiniAp4 or Ang2) and Cx modified with the naked shuttle (MiniAp4 and Ang2) assayed at 1 pM in the human in vitro BBB cellular model. The results show that the MiniAp4 conjugate increases the transport of SN38 more efficiently than the Ang2 conjugates.
Clauses
For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:
Clause 1. A antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof,
Figure imgf000056_0001
which comprises from 1-6 peptides of formula P incorporated inserted in the disulfide bonds of the antibody in the form of a -P-(W)s-Y and joined to the sulfides through a linker -[(Ll)-(L2)-(L3)m-; wherein:
Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof; the disulfide bonds are any disulfide bond that is capable of structurally retaining its structure upon reduction conditions and is selected from the group consisting of a naturally present interchain disulfide bond of the antibody, a naturally present intrachain disulfide bond of the antibody, and a disulfide bond introduced in the antibody by genetic engineering;
Li is a linker selected from the group consisting of Lia and Lit>;
Figure imgf000057_0001
Li is attached through the bonds a and b to the -S- of the disulfide bonds of the antibody and the bond c is attached to the linker L2 through an amide bond, an ester bond, or an thioester bond between the C=O group next to the bond c of the linker Li with an NH, O, or S group of the on the left side of the draw of LA below of linker L2;
L2 is a biradical composed from 2 to 8 biradicals selected from the group consisting of LA, LB, LC and has the formula -LA-(LB)U-LC-
LA is a biradical selected from the group consisting of: -NH-(CH2)r-C(=O)-, -S-(CH2)r-C(=O)-; -O-(CH2)r-C(=O)-; -NH-(CH2)r-; -S-(CH2)r-; -O- (CH2)r-; -NH-(CH2)r-O-; -NH-(CH2)r-NH- and -NH-(CH2)r-S-;
LB is a biradical independently selected from the group consisting of: -NH-(CH2)r-C(=O)-; -C(=O)-(CH2)r-C(=O)-; -S-(CH2)r-C(=O)-; -O-(CH2)r-C(=O)-; -NH-(CH2)r-; -C(=O)-(CH2)r- ;-S-(CH2)r-; -O-(CH2)r-; -NH-CH-((CH2)rNH2)-C(=O)-; -S-CH2-CH(NH2)-C(=O)-; -(CH2)r- C(=O)-; -(CH2)r-O-; -(CH2)r-NH-; -(CH2)r-S-; -C(=O)-(CH2)r-NH-; -C(=O)-(CH2)rO-;-C(=O)- (CH2)r-S-; -NH-(CH2)r-O-; -NH-(CH2)r-NH-; -NH-(CH2)r-S-; and combinations thereof;
LC is a biradical selected from the group consisting of: -NH-(CH2)r-C(=O)-; -NH-CH- ((CH2)r-NH2)-C(=O)-; -C(=O)-(CH2)r-C(=O)-; -S-(CH2)r-C(=O)-; -S-CH2-CH(NH2)-C(=O)-; - O-(CH2)r-C(=O)-; -(CH2)r-C(=O)-; u is an integer from 0-6; r’ is an integer from 1 to 5; when u=0, LA is attached to the biradical LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional group on the right side of the LA formulas and the functional groups of the left side of the LC formulas; when u=1 , LA is attached to the biradical LB through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LA formulas and the functional groups on the left side of the LB formulas; and LB is attached to the radical LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LB formulas and the functional groups on the left side of the LC formulas; when u is higher than 1 , LB are equal or different and are attached among them through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester; being one LB terminal attached to LA through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LA formulas and the functional groups of the left side of the LB formulas; and being another LB terminal attached to LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional group on the right side of the LB formulas and the functional group on the left side of the LC formulas;
L3 is a biradical selected from the group consisting of: an amino acid selected from Lys, Orn, Dap, Dab; Glu, and Asp; an amino acid derivative selected from Lys, Orn, Dap, and Dab derivatized by having attached to the amino group of the lateral chain of the amino acid a biradical selected from the group consisting of -C(=O)-(CH2)r-C(=O)-; -C(=O)- (CH2)t-NH-; -C(=O)-(CH2)t-S-; -C(=O)-(CH2)t-O-, wherein the attachment to the amino group is through the C=O terminal group on the left side of the biradicals; an amino acid derivative selected from Glu and Asp, derivatized by having attached to the C=O group of the lateral chain of the amino acid a biradical selected from the group consisting of -NH- (CH2)trC(=O)-; -NH-(CH2)t-NH-; -NH-(CH2)t-S-; -NH-(CH2)t-O- wherein the attachment to the C=O group is through the NH group on the left side of the biradicals; and any of the previous amino acids or amino acids derivatives that have further attached CH2CH2NCH2CO2H) 4 (DOTA) or streptavidin by a feasible bond; t is an integer from 1 to 5; m is an integer selected from 0 or 1 ;
D is a radical of a substance selected form a biologically active substance, a substance for use in a diagnostic method; and a radioligand for radiotherapy;
P is a biradical of a peptide, equal or different, selected from the group consisting of:
(a) a peptide which comprises the amino acid sequence X1KAPETALX2 with an intrapeptide bond between the Xi and X2 which is an amide bond; wherein Xi is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4- diaminobutanoic acid); and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid); i.e.
Figure imgf000059_0001
(b) a peptide having 12-20 amino acids residues in length having at least an intrapeptide bond which is a disulfide or diselenide bond, and comprises an amino acid sequence which is: X3KAPETALX4AAA; having at least an intrapeptide disulfide or diselenide bond between X3 and X4, wherein X3 and X4 are equal and are selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines); i.e.
Figure imgf000059_0002
(c) a peptide having 9-11 amino acids residues in length having at least an intrapeptide bond which is a disulfide or diselenide bond and consists of an amino acid sequence selected from the group consisting of XsKAPETALXe; XsKAPETALXeA; and XsKAPETALXeAA having at least an intrapeptide disulfide or diselenide bond between X5 and Xe; wherein X5 and Xe are equal and are selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines), i.e.
Figure imgf000059_0003
(d) a peptide which has 16 amino acid residues and comprises the amino acid sequence XyNXsKAPETALXgAAAX H with an intrapeptide disulfide or diselenide bond between the X7 and Xg, and between Xs and X10; wherein X7-X10 are independently selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines); provided that X7 and Xg are equal, and Xs-Xw are equal; i.e.
Figure imgf000060_0001
and (e) peptide which comprises the amino acid sequence XI KAPETALX2 wherein Xi is selected from the group consisting of Dap and Dab; and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid) (SEQ ID NO:7) being a linear peptide;
W is a biradical selected from the group consisting of -NH-(CH2)r-C(=O)-, and -NH-CH((CH2)rNH2)-C(=O)-; r is an integer independently selected from 1 to 5; s is an integer independently selected from 0 to 1 ;
Y is a radical is selected from the group consisting of -NH2, -OH, -OR3, and -NHR3; when m=0, L3 and D are absent, and P is directly attached to LC of L2 through an amide bond formed between the C=O terminal group of LC and the amine group of the first amino acid of the peptide sequence P; when m=1 , D is present and is attached to the functional group of the lateral chain of the amino acid of linker L3 or to the amino acid derivative of linker L3 through its derivatization, wherein the attachment is through an amide, ester, disulfide, or thioester bond; L3 is attached to LC of L2 through an amide bond formed between the C=O terminal group on the left side of LC formula and the amine group of the linker L3; and P is directly attached to L3 through an amide bond formed between the C=O terminal group on the right side of LC formula and the amine group of the first amino acid of the peptide sequence P; and when s=0, P is directly attached to Y through an amide, carboxylic acid or ester bond, the bond being formed between the C=O of the C-terminal of the last amino acid of the sequence P, and the radical Y which is -NH2, -OH, -OR3, or -NHR3; and when s=1 , P is attached to a radical W through an amide bond formed with a C=O of the C-terminal of the last amino acid of the sequence P, the bond being formed between the functional groups on the left side of the draw W formulas and the functional groups (C=O) of the C-terminal of the last amino acid of the sequence P on the right side of the draw sequence; and W is attached to Y as follows: -C(=O)-NH-(CH2)r-C(=O)-Y, or -C(=O)-NH- CH((CH2)rNH2)-C(=O)-Y; n is an integer independently selected from 1 to 6; indicates the point of attachment; and
S in formula (I) indicates sulfide.
Clause 2. The antibody shuttle conjugate according to clause 1 , which has four copies of the peptide P, and all the peptides are equal.
Clause 3. The antibody shuttle conjugate according to any of the clauses 1-2, wherein the disulfide bond is an interchain bond.
Clause 4. The antibody shuttle conjugate according to any of the clauses 1-3, wherein P is a biradical of a peptide selected from the group consisting of:
(a) a peptide which comprises the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond, that is SEQ ID NO:8: DapKAPETALD
TIHCO ^
(b) a peptide having 9-20 amino acids residues in length having at least an intrapeptide bond which is a disulfide bond, and comprises an amino acid sequence which is:
CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that is SEQ ID NO:9: CKAPETALCAAA
Figure imgf000061_0001
(c) a peptide having 9-11 amino acids residues in length having at least an intrapeptide bond which is a disulfide bond and consists of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that are SEQ ID NO: 10: CKAPETALC
S-S
SEQ ID NO: 11 CKAPETALCA
Figure imgf000062_0001
SEQ ID NO: 12) CKAPETALCAA; and
Figure imgf000062_0002
(d) a peptide which has 16 amino acid residues and comprises the amino acid sequence CNCKAPETALCAAACH with an intrapeptide disulfide bond between the first and third cysteine which are cysteines 1 and 11 , and between the second and the fourth cysteine which are cysteine 3 and 15, that is,
SEQ ID NO: 13: CNCKAPETALCAAACH
Figure imgf000062_0003
(e) a peptide which comprises the amino acid sequence DapKAPETALD (SEQ ID NO: 14).
Clause 5. The antibody shuttle conjugate according to clause 4, wherein, P is a biradical of a peptide selected from the group consisting of:
(a) the peptide having the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond (SEQ ID NO:7);
(b) the peptide having the amino acid sequence CKAPETALC having at least an intrapeptide disulfide bond between cysteines in position 1 and 9 (SEQ ID NO: 10;
(c) the peptide having the amino acid sequence DapKAPETALD (SEQ ID NO:14).
Clause 6. The antibody shuttle conjugate according to any of the clauses 1-5, wherein the antibody is selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab, Pertuzumab, Aducanumab, Bapineuzumab, Nimotuzumab, and Necitumumab.
Clause 7. The antibody shuttle conjugate according to clause 1 , which is selected from the group consisting of: Trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid; Cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr- Ala-Leu-Asp-NH2 (Cx-DBM-TTDS-SEQ ID NO: 1) ) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid, Bevacizumab-DBM-TTDS- Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid; and Pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-DBM-TTDS- SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp sidechain carboxylic acid, wherein DBM is the linker Lia and TTDS is the linker l_2a.
Clause 8. The antibody shuttle conjugate according to clausel, wherein: a) the antibody is an anticancer therapeutical antibody, m=1 and D is a radical of an anticancer active pharmaceutical ingredient selected from the group consisting of auristatins, duocarmycins, PBD dimers, maytansinoids, calicheamicins, anthracyclines, camptothecines, alpha-amanitin, tubulysins, MMAE, T-DM1 , and PROTAC moieties; or alternatively, b) the antibody in an antineurodegenerative therapeutical antibody, m=1, and D is a radical of an antineurodegenerative active pharmaceutical ingredient.
Clause 9. A process for preparing an antibody shuttle conjugate as defined in any of the clauses 1-8, comprising: a) reducing disulfide bridges of the antibody; b) rebridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II),
Figure imgf000063_0001
c) optionally, carrying out a hydrolysis; wherein q; L2, L3, D, P, m, W, s, and Y are as defined in the antibody shuttle conjugate of formula (I).
Clause 10. A pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle conjugate as defined in any of the clauses 1-8, together with appropriate amounts of pharmaceutically acceptable carriers or excipients. 11. A antibody shuttle conjugate as defined in any of the claims 1-8, for use as a medicament.
12. A antibody shuttle conjugate as defined in any of the claims 1-8, for use in the treatment of CNS disorders in a mammal, including a human.
13. The antibody shuttle conjugate for use according to claim 12, wherein the CNS disorder is cancer.
14. The antibody shuttle conjugate for use according to any of the claims 12-13, wherein the antibody shuttle conjugate is for use in combination therapy with convention chemotherapy agents or radiotherapy.
15. A antibody shuttle conjugate as defined in any of the claims 1-8, for use as a diagnostic agent or radioligand for radiotherapy.
Citation List
Patent Literature
WO2015/001015A1
Non Patent Literature
- Anthony Regina et al., in “ANG4043, a novel brain-penetrant Peptide-mAb conjugate, is efficacious against HER2-positive Intracranial Tumors in Mice”, mct.aacrjournals.org
- Abstract Macarena Sanchez Navarro et al.; “Paving the way towards the brain delivery of biotherapeutics: Modification of proteins with blood-brain barrier peptide shuttles” ECBS/LS-EuCheMS Madrid (Spain) (D2)
- M. Amblard, et al., "Methods and protocols of modern solid-phase peptide synthesis. Molecular Biotechnology 2006, Vol. 33, p. 239-254)
- E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595- 598 (1970)
- A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791 2 P.L. Barker et al. J. Med. Chem., 1992. vol 35, pp. 2040-2048.
2 Greene’s Protective Groups in Organic Synthesis, Fifth Edition. Peter G. M. Wuts. 2014 John Wiley & Sons, Inc. pp. 26-29, pp. 69-77, pp. 271-279, pp. 371-374, pp. 456-463
2 M. Kazemi et al., Journal of Sulfur Chemistry, 2015, vol. 36:6, pp. 613-623.
2 M. T. Nguyen et al., J. Org. Chem. 1998, 63, vol. 20, pp. 6878-6885.
2 F. David et al., Org. Process Res. Dev. 2010, 14, 4, pp. 999-1007.
2 https://www.cliffsnotes.com/study-guides/chemistry/organic-chemistry-ii/aldehydes-and- ketones/reactions-of-aldehydes-and-ketones.
2 Eur J Pharm Biopharm, 2017, 115, 149-158 or triphosgene as described in J. Med. Chem. 2008, 51 , 21 ,6916-6926.
- Dongen et al.-, Bioconjugate Chem. 2009, vol. 20, pp. 20-23.
- CA2957354A1

Claims

Claims
1. A antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof,
Figure imgf000066_0001
which comprises from 1-6 peptides of formula P inserted in the disulfide bonds of the antibody in the form of a -P-(W)s-Y and joined to the sulfides through a linker -[(LI)-(L2)- (L3)m-; wherein:
Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof; the disulfide bonds are any disulfide bond initially present in the antibody and that is capable of structurally retaining sulfide bonds in the structure upon reduction conditions; the disulfide bond being selected from the group consisting of a naturally present interchain disulfide bond of the antibody, a naturally present intrachain disulfide bond of the antibody, and a disulfide bond introduced in the antibody by genetic engineering;
Li is a linker selected from the group consisting of Lia and Lit>;
Figure imgf000066_0002
q is an integer from 1 to 6; Li is attached through the bonds a and b to the -S- of the disulfide bonds of the antibody and the bond c is attached to the linker L2 through an amide bond, an ester bond, or an thioester bond between the C=O group next to the bond c of the linker Li with an NH, O, or S group of the on the left side of the draw of LA below of linker L2;
L2 is a biradical composed from 2 to 8 biradicals selected from the group consisting of LA, LB, LC and has the formula -LA-(LB)U-LC-
LA is a biradical selected from the group consisting of:
-NH-(CH2)r-C(=O)-, -S-(CH2)r-C(=O)-; -O-(CH2)r-C(=O)-; -NH-(CH2)r-; -S-(CH2)r-; -O- (CH2)r-; -NH-(CH2)r-O-; -NH-(CH2)r-NH- and -NH-(CH2)r-S-;
LB is a biradical independently selected from the group consisting of: -NH-(CH2)r-C(=O)-; -C(=O)-(CH2)r-C(=O)-; -S-(CH2)r-C(=O)-; -O-(CH2)r-C(=O)-; -NH-(CH2)r-; -C(=O)-(CH2)r- ;-S-(CH2)r-; -O-(CH2)r-; -NH-CH-((CH2)rNH2)-C(=O)-; -S-CH2-CH(NH2)-C(=O)-; -(CH2)r- C(=O)-; -(CH2)r-O-; -(CH2)r-NH-; -(CH2)r-S-; -C(=O)-(CH2)r-NH-; -C(=O)-(CH2)rO-;-C(=O)- (CH2)r-S-; -NH-(CH2)r-O-; -NH-(CH2)r-NH-; -NH-(CH2)r-S-; and combinations thereof;
LC is a biradical selected from the group consisting of: -NH-(CH2)r-C(=O)-; -NH-CH- ((CH2)r-NH2)-C(=O)-; -C(=O)-(CH2)r-C(=O)-; -S-(CH2)r-C(=O)-; -S-CH2-CH(NH2)-C(=O)-; - O-(CH2)r-C(=O)-; -(CH2)r-C(=O)-; u is an integer from 0-6; r’ is an integer from 1 to 5; when u=0, LA is attached to the biradical LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional group on the right side of the LA formulas and the functional groups of the left side of the LC formulas; when u=1 , LA is attached to the biradical LB through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LA formulas and the functional groups on the left side of the LB formulas; and LB is attached to the biradical LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LB formulas and the functional groups on the left side of the LC formulas; when u is higher than 1, LB are equal or different and are attached among them through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester; being one LB terminal attached to LA through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional groups on the right side of the LA formulas and the functional groups of the left side of the LB formulas; and being another LB terminal attached to LC through a chemically feasible bond which is selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the functional group on the right side of the LB formulas and the functional group on the left side of the LC formulas;
L3 is a biradical selected from the group consisting of: an amino acid selected from Lys, Orn, Dap, Dab; Glu, and Asp; an amino acid derivative selected from Lys, Orn, Dap, and Dab derivatized by having attached to the amino group of the lateral chain of the amino acid a biradical selected from the group consisting of -C(=O)-(CH2)r-C(=O)-; -C(=O)- (CH2)t-NH-; -C(=O)-(CH2)t-S-; -C(=O)-(CH2)t-O-, wherein the attachment to the amino group is through the C=O terminal group on the left side of the biradicals; an amino acid derivative selected from Glu and Asp, derivatized by having attached to the C=O group of the lateral chain of the amino acid a biradical selected from the group consisting of -NH- (CH2)trC(=O)-; -NH-(CH2)t-NH-; -NH-(CH2)t-S-; -NH-(CH2)t-O- wherein the attachment to the C=O group is through the NH group on the left side of the biradicals; and any of the previous amino acids or amino acids derivatives that have further attached CH2CH2NCH2CO2H) 4 (DOTA) or streptavidin by a feasible bond; t is an integer from 1 to 5; m is an integer selected from 0 or 1;
D is a substance attached to the linker L3 selected from a biologically active substance, a substance for use in a diagnostic method; and a radioligand for radiotherapy;
P is a biradical of a single peptide, equal or different, which is selected from the group consisting of:
(a) a peptide which comprises the amino acid sequence X1KAPETALX2 with an intrapeptide bond between the Xi and X2 which is an amide bond; wherein Xi is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4- diaminobutanoic acid); and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid); i.e.
Figure imgf000069_0001
(b) a peptide having 12-20 amino acids residues in length having at least an intrapeptide bond which is a disulfide or diselenide bond, and comprises an amino acid sequence which is: X3KAPETALX4AAA; having at least an intrapeptide disulfide or diselenide bond between X3 and X4, wherein X3 and X4 are equal and are selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines); i.e.
Figure imgf000069_0002
(c) a peptide having 9-11 amino acids residues in length having at least an intrapeptide bond which is a disulfide or diselenide bond and consists of an amino acid sequence selected from the group consisting of XsKAPETALXe; XsKAPETALXeA; and XsKAPETALXeAA having at least an intrapeptide disulfide or diselenide bond between X5 and Xe; wherein X5 and Xe are equal and are selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines), i.e.
Figure imgf000069_0003
(d) a peptide which has 16 amino acid residues and comprises the amino acid sequence XyNXsKAPETALXgAAAX H with an intrapeptide disulfide or diselenide bond between the X7 and Xg, and between Xs and X10; wherein X7-X10 are independently selected from the group consisting of C (cysteines), Sec (selenocysteines), and Pen (penicillamines); provided that X7 and Xg are equal, and Xs-Xw are equal; i.e.
Figure imgf000069_0004
and (e) peptide which comprises the amino acid sequence X1KAPETALX2 wherein Xi is selected from the group consisting of Dap and Dab; and X2 is selected from the group consisting of D (aspartic acid) and E (glutamic acid) (SEQ ID NO:7) being a linear peptide; W is a biradical selected from the group consisting of -NH-(CH2)r-C(=O)-, and -NH-CH((CH2)rNH2)-C(=O)-; r is an integer independently selected from 1 to 5; s is an integer independently selected from 0 to 1 ;
Y is a radical selected from the group consisting of -NH2, -OH, -OR3, and -NHR3; when m=0, L3 and D are absent, and P is directly attached to LC of L2 through an amide bond formed between the C=O terminal group of LC and the amine group of the first amino acid of the peptide sequence P; when m=1 , D is present and is attached to the functional group of the lateral chain of the amino acid of linker L3 or to the amino acid derivative of linker L3 through its derivatization, wherein the attachment is through an amide, ester, disulfide, or thioester bond; L3 is attached to LC of L2 through an amide bond formed between the C=O terminal group on the left side of LC formula and the amine group of the linker L3; and P is directly attached to L3 through an amide bond formed between the C=O terminal group on the right side of LC formula and the amine group of the first amino acid of the peptide sequence P; and when s=0, P is directly attached to Y through an amide, carboxylic acid or ester bond, the bond being formed between the C=O of the C-terminal of the last amino acid of the sequence P, and the radical Y which is -NH2, -OH, -OR3, or -NHR3; and when s=1 , P is attached to a radical W through an amide bond formed with a C=O of the C-terminal of the last amino acid of the sequence P, the bond being formed between the functional groups on the left side of the draw W formulas and the functional groups (C=O) of the C-terminal of the last amino acid of the sequence P on the right side of the draw sequence; and W is attached to Y as follows: -C(=O)-NH-(CH2)r-C(=O)-Y, or -C(=O)-NH- CH((CH2)rNH2)-C(=O)-Y; n is an integer independently selected from 1 to 6; indicates the point of attachment; and
S in formula (I) indicates sulfide.
2. The antibody shuttle conjugate according to claim 1 , wherein the disulfide bonds initially present in the antibody are capable of structurally retaining the sulfide bonds in the structure upon reduction conditions and of forming between them new bridges by reaction of the sulfide groups of the reduced disulfide bonds of the antibody with the linker Li , to incorporate the biradical below between the sulfide groups of the antibody.
Figure imgf000071_0001
3. The antibody shuttle conjugate according to any of the claims 1-2, which has four copies of the peptide P, and all the peptides are equal.
4. The antibody shuttle conjugate according to any of the claims 1-3, wherein the disulfide bond is an interchain bond.
5. The antibody shuttle conjugate according to any of the claims 1-4, wherein P is a biradical of a peptide selected from the group consisting of:
(a) a peptide which comprises the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond, that is SEQ ID NO:8:
DapKAPETALD
\NHCQ
(b) a peptide having 9-20 amino acids residues in length having at least an intrapeptide bond which is a disulfide bond, and comprises an amino acid sequence which is:
CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that is SEQ ID NO:9: CKAPETALCAAA
Figure imgf000071_0002
(c) a peptide having 9-11 amino acids residues in length having at least an intrapeptide bond which is a disulfide bond and consists of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA having at least an intrapeptide disulfide bond between cysteines 1 and 9, that are
SEQ ID NO: 10: CKAPETALC ^S-S^
SEQ ID NO: 11 CKAPETALCA
Figure imgf000071_0003
SEQ ID NO: 12) CKAPETALCAA; and S-S
(d) a peptide which has 16 amino acid residues and comprises the amino acid sequence CNCKAPETALCAAACH with an intrapeptide disulfide bond between the first and third cysteine which are cysteines 1 and 11 , and between the second and the fourth cysteine which are cysteine 3 and 15, that is,
SEQ ID NO: 13: CNCKAPETALCAAACH
Figure imgf000072_0001
(e) a peptide which comprises the amino acid sequence DapKAPETALD (SEQ ID NO: 14).
6. The antibody shuttle conjugate according to claim 5, wherein, P is a biradical of a peptide selected from the group consisting of:
(a) the peptide having the amino acid sequence DapKAPETALD with an intrapeptide bond between the Dap and D which is an amide bond (SEQ ID NO:7);
(b) the peptide having the amino acid sequence CKAPETALC having at least an intrapeptide disulfide bond between cysteines in position 1 and 9 (SEQ ID NO: 10;
(c) the peptide having the amino acid sequence DapKAPETALD (SEQ ID NO:14).
7. The antibody shuttle conjugate according to any of the claims 1-5, wherein the antibody is selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab, Pertuzumab, Aducanumab, Bapineuzumab, Nimotuzumab, and Necitumumab.
8. The antibody shuttle conjugate according to claim 1, which is selected from the group consisting of: Trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz- DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid; Cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala- Leu-Asp-NH2 (Cx-DBM-TTDS-SEQ ID NO: 1) ) with an amide bond between Dap sidechain amino group and Asp side-chain carboxylic acid, Bevacizumab-DBM-TTDS-Dap- Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp side-chain carboxylic acid; and Pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-DBM-TTDS- SEQ ID NO: 1) with an amide bond between Dap side-chain amino group and Asp sidechain carboxylic acid, wherein DBM is the linker Lia and TTDS is the linker l_2a.
Figure imgf000073_0001
I— 2a
9. The antibody shuttle conjugate according to claim 1 , wherein: a) the antibody is an anticancer therapeutical antibody, m=1 and D is a radical of an anticancer active pharmaceutical ingredient selected from the group consisting of auristatins, duocarmycins, PBD dimers, maytansinoids, calicheamicins, anthracyclines, camptothecines, alpha-amanitin, tubulysins, MMAE, T-DM1 , and PROTAC moieties; or alternatively, b) the antibody in an antineurodegenerative therapeutical antibody, m=1 , and D is a radical of an antineurodegenerative active pharmaceutical ingredient.
10. The antibody shuttle conjugate according to claim 1 , wherein the antibody is cetuximab, m=1 and D is SN38.
11. A process for preparing an antibody shuttle conjugate as defined in any of the claim 1- 10, comprising: a) reducing disulfide bridges of the antibody; b) rebridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II),
Figure imgf000074_0001
c) optionally, carrying out a hydrolysis; wherein q; L2, L3, D, P, m, W, s, and Y are as defined in the antibody shuttle conjugate of formula (I).
12. A pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle conjugate as defined in any of the claims 1-10, together with appropriate amounts of pharmaceutically acceptable carriers or excipients.
13. A antibody shuttle conjugate as defined in any of the claims 1-10, for use as a medicament.
14. A antibody shuttle conjugate as defined in any of the claims 1-10, for use in the treatment of CNS disorders in a mammal, including a human.
15. The antibody shuttle conjugate for use according to claim 14, wherein the CNS disorder is cancer.
16. The antibody shuttle conjugate for use according to any of the claims 14-15, wherein the antibody shuttle conjugate is for use in combination therapy with convention chemotherapy agents or radiotherapy.
17. A antibody shuttle conjugate as defined in any of the claims 1-10, for use as a diagnostic agent or radioligand for radiotherapy.
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