Site-specific modification of antibody-based constructs for crossing the blood-brain barrier by BBB shuttling

Abstract

Conjugates of antibodies and selected peptide shuttles that cross the blood-brain barrier, optionally also conjugated with active pharmaceutical ingredients, radiotherapeutic agents or diagnostic agents, pharmaceutical compositions containing them, and conjugates for use in medicine and as diagnostic agents.

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application No. 22382746.0, filed August 1, 2022.

[0002] The present invention relates to the field of antibody conjugates and their use in the treatment and diagnosis of diseases that require the antibody to cross the blood-brain barrier. [Background technology]

[0003] Monoclonal antibodies (mAbs) have revolutionized the treatment of a variety of diseases, particularly in the field of oncology. Antibody-based therapies target leukemia and solid tumors in many organs. However, brain tumors remain virtually untreatable using biologic therapeutics and most small molecule drugs.

[0004] One of the major challenges in treating brain tumors is crossing the blood-brain barrier (BBB) ​​and blood-tumor barrier (BTB) at therapeutic doses. Only 0.1–0.2% of a peripherally injected mAb dose reaches the brain parenchyma. The BBB consists of specialized endothelial cells tightly connected and surrounded by astrocyte end-heads and pericytes, which ensure the brain's insulation. At the tumor center, the BBB is replaced by a leaky BTB; however, at the tumor margins and small brain metastases, the BBB may remain intact and significantly impede therapeutic access.

[0005] Brain metastases (BM) are a significant complication in several cancer types, particularly lung cancer, melanoma, and breast cancer. Breast cancer BM is particularly prevalent, affecting 24% of women with stage IV breast cancer. Breast tumors that overexpress the human epidermal growth factor receptor (HER-2) and those known as triple-negative tumors exhibit a high incidence of BM. While the application of mAbs has proven highly effective in these breast cancer types, the application of mAbs to treat BM or primary brain tumors remains challenging, primarily due to the lack of antibody penetration across the BBB and BTB.

[0006] Several approaches have been explored to enhance antibody penetration into the brain. Methods such as direct injection and temporary disruption of the BBB can carry significant risks to patients. For this reason, much effort has been focused on developing ligands that hijack endogenous transport mechanisms across the brain endothelium. These ligands, referred to as BBB shuttles, include antibody derivatives, endogenous proteins, peptides, and small molecules.

[0007] Anthony Regina et al., in "ANG4043, a novel brain-penetrant peptide-mAb conjugate, is efficacious against HER2-positive intracranial tumors in mice" (published online at mct.aacrjournals.org), disclose that the incorporation of Angiopep-2 (Ang2) into an anti-HER2 monoclonal antibody confers increased brain endothelial cell uptake and BBB permeability. Such incorporation is via a linker and uses a non-site-specific methodology. However, limited efficacy of the conjugated shuttle has been observed, which may be due to the peptide's low protease resistance and the heterogeneous mixture obtained by randomly conjugating peptides to surface lysines.

[0008] WO 2015 / 001015(A1) describes certain apamin-derived peptides, including KAPETAL fragments, which are useful as shuttles because they have the ability to cross the BBB and can facilitate the transport into the brain of drugs or other diagnostically useful substances that cannot cross the BBB by themselves. This document describes two constructs of peptides with antibodies as cargo: Example 22 (cetuximab-V3-Ap3-NH2) and Example 23: (cetuximab-V2-Ap 5a-NH2), where the AP5 peptide corresponds to the MiniAp4 peptide, a cyclic peptidomimetic derived from bee venom that is highly resistant to proteolysis and exhibits negligible toxicity and immunogenicity, and V2 and V3 are as follows: [ka]

[0009] In V3, linker bonds 1 and 2 are connected to the side chains of lysines of the antibody. However, all previous methods based on lysine modifications, such as those disclosed in previous documents, do not allow control over where they are located when the peptide is incorporated into the monoclonal antibody.

[0010] Finally, in their abstract "Paving the way towards brain delivery of biotherapeutics: Modification of proteins with blood-brain barrier peptide shuttles" (ECBS / LS-EuCheMS Madrid, Spain), Macarena Sanchez Navarro et al. propose the possibility of attaching a controlled average number of peptides to various parts of proteins to improve their BBB permeability. In particular, they disclose the modification of GFP with MiniAp4 and with a branched version of THRre. However, they do not describe where the peptides are introduced, how this attachment can be performed, or the consequences of transport through the BBB or BTB.

[0011] Thus, from what is known in the art, it can be seen that there remains a need to address the long-standing problem of increasing antibody transport across the BBB for the treatment or diagnosis of brain tumors and other CNS diseases. Summary of the Invention

[0012] The present inventors have developed conjugates of mAb antibodies and selected peptide shuttles linked to the antibodies at specific sites on the antibodies via bromomaleimide linkers, which effectively cross the blood-brain barrier.

[0013] Unlike methods disclosed in the prior art (see WO 2015 / 001015(A1)), the linker used in the present invention allows for a peptide incorporation strategy that allows for controlled and known shuttle peptide incorporation into monoclonal antibodies. This strategy is based on incorporating the peptide via the -SH group of a reduced antibody. Advantageously, this strategy is applicable to any monoclonal antibody and provides homogeneous conjugates through site-specific modification of the antibody, a highly desirable feature for pharmacological products, enhancing the therapeutic index and facilitating product manufacturing and profiling. Furthermore, the incorporation of a selected peptide shuttle is achieved by exploiting the highly reactive cysteines of the interchain disulfide bridge, overcoming the need for genetic engineering to introduce reactive tags and expanding the applicability of this strategy to other mAbs.

[0014] As an example, we prepared a homogeneous antibody-BBB-shuttle conjugate (Tz-MiniAp4) of four copies of trastuzumab and MiniAp4. Trastuzumab (Herceptin®) is an FDA-approved antibody against HER2, which is widely used in clinical practice to treat breast cancer. This conjugate was achieved by reducing the interchain disulfide bridges and recrosslinking them using dibromomaleimide, which allows for a high degree of control over the number of peptides immobilized on each antibody molecule. This conjugate demonstrates enhanced transport across the blood-brain barrier in an in vitro cell model relative to trastuzumab alone or relative to the conjugate of trastuzumab and 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.

[0015] Thus, a first aspect of the present invention relates to an antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof: [ka] It is inserted into the disulfide bond of the antibody in the form of -P-(W)sY and is linked to the linker -[(L1)-(L2)-(L3) m - 1 to 6 peptides of formula P linked to a sulfide via During the ceremony: Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof; the disulfide bond is any disulfide bond that is initially present in the antibody and that is capable of structurally maintaining the sulfide bond in the structure under reducing conditions, the disulfide bond being selected from the group consisting of the antibody's naturally occurring interchain disulfide bonds, the antibody's naturally occurring intrachain disulfide bonds, and disulfide bonds introduced into the antibody by genetic engineering; L1 is L 1a and L 1b is a linker selected from the group consisting of: [ka] q is an integer from 1 to 6: L1 is attached to the -S- of the antibody disulfide bond via bonds a and b, and bond c is attached to linker L2 via an amide bond, ester bond, or thioester bond between the C=O group next to bond c of linker L1 and the NH, O, or S group on the left side of the drawing of LA under linker L2; L2 is a biradical consisting of 2 to 8 biradicals selected from the group consisting of LA, LB, and LC, and has the formula -LA-(LB) u -LC-, LA is a biradical selected from the group consisting of: -NH-(CH) 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-(CH) 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) r’ NH2)-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) r -O-;-C(=O)-(CH2) r’ -S-;-NH-(CH2) r’ -O-;-NH-(CH2) r’ -NH-;-NH-(CH2) r’ -S-; and their combinations; L C is a biradical selected from the group consisting of: -NH-(CH) 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 via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LC; When u=1, LA is attached to the biradical LB through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LB; LB is attached to the biradical LC through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LB and the left functional group of formula LC; when u is greater than 1, LB are equal or different and are attached therebetween via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester; one LB end is attached to LA via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LB; and another LB end is attached to LC via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LB and the left functional group of formula LC; L3 is an amino acid selected from Lys, Orn, Dap, Dab; ​​Glu, and Asp; -C(=O)-(CH2) r -C(=O)-, -C(=O)-(CH2) t -NH-, -C(=O)-(CH2) t and amino acid derivatives selected from Lys, Orn, Dap, and Dab that are derivatized by attaching a biradical selected from the group consisting of -S-, -C(=O)-(CH2)tO- to the amino group of the lateral chain of the amino acid, wherein the attachment to the amino group is through the C=O terminal group on the left side of the biradical: amino acid derivatives selected from Glu and Asp are represented by -NH-(CH2)tr-C(=O)-; -NH-(CH2) t -NH-;-NH-(CH2) t -S-;-NH-(CH2)t derivatized by attaching a biradical selected from the group consisting of -O- to a C=O group of the side chain of an amino acid, the attachment to the C=O group being via an NH group on the left side of the biradical; any of the aforementioned amino acids or amino acid derivatives further attached by a viable bond to CH2CH2NCH2CO2H4 (DOTA) or streptavidin; 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 and is selected from biologically active substances, substances for use in diagnostic methods; and radioligands for radiotherapy; P is a biradical of a single peptide, equal or different, selected from the group consisting of: (a) a peptide comprising the amino acid sequence X1KAPETALX2, with an intrapeptide bond between X1 and X2 that is an amide bond; X1 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 NO:1: [ka] (b) a peptide having a length of 12 to 20 amino acid residues, having at least one intrapeptide bond which is a disulfide bond or a diselenide bond, and comprising an amino acid sequence of X3KAPETALX4AAA; and having at least an intrapeptide disulfide bond or a diselenide bond between X3 and X4, wherein X3 and X4 are equal and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine); i.e., SEQ ID NO:2: [ka] (c) a peptide having a length of 9 to 11 amino acid residues and having at least one intrapeptide bond that is a disulfide bond or a diselenide bond, the peptide consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6, X5KAPETALX6A, and X5KAPETALX6AA, and having at least one intrapeptide disulfide bond or a diselenide bond between X5 and X6, wherein X5 and X6 are equal and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine); i.e., SEQ ID NO:3: [ka] SEQ ID NO:4: [ka] SEQ ID NO:5: [ka] (d) having 16 amino acid residues and the amino acid sequence X7NX8KAPETALX9AAAX 10 Including H, between X7 and X9, and between X8 and X 10 A peptide having an intrapeptide disulfide bond or diselenide bond between X7 to X 10 are independently selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), provided that X7 and X9 are equal and X8 to X9 are equal. 10 is equal to the peptide; i.e., SEQ ID NO:6: [ka] and (e) a peptide comprising the amino acid sequence X1KAPETALX2, wherein X1 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), and is a linear peptide; W is a biradical selected from the group consisting of —NH—(CH)rC(═O)— and —NH—CH((CH)rNH)—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 attached directly to the LC of L2 via an amide bond formed between the C=O terminal group of LC and the amine group of the first amino acid in the peptide sequence P; When m=1, D is present and is attached to a functional group of the lateral chain of an amino acid of linker L3 or to an amino acid derivative of linker L3 via its derivatization, the attachment being via an amide, ester, disulfide, or thioester bond; L3 is attached to LC of L2 via an amide bond formed between the C=O terminal group of the left side of formula LC and the amine group of linker L3; P is attached directly to L3 via an amide bond formed between the C=O terminal group of the right side of formula LC and the amine group of the first amino acid of peptide sequence P; When s=0, P is directly attached to Y via an amide, carboxylic acid or ester bond, the bond being formed between the C=O at the C-terminus of the last amino acid of the sequence P and a radical Y that is -NH2, -OH, -OR3 or -NHR3; When s=1, P is attached to radical W through an amide bond formed with the C=O of the C-terminus of the last amino acid in sequence P, the bond being formed between the functional group on the left side of the drawn formula W and the C-terminus functional group (C=O) of the last amino acid in sequence P on the right side of the drawn formula; and W is attached to Y as follows: -C(=O)-NH-(CH2). r -C(=O)-Y, or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y; n is an integer independently selected from 1 to 6; [ka] indicates the attachment point; S represents sulfide.

[0016] 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 an antibody; b) re-crosslinking the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide peptide of formula (II); and c) optionally carrying out hydrolysis; q, L2, L3, D, P, m, W, s and Y are as defined in the antibody shuttle conjugate of formula (I); [ka]

[0017] A third aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an antibody shuttle conjugate of the present invention together with an appropriate amount of a pharmaceutically acceptable carrier or excipient.

[0018] A fourth aspect of the present invention relates to an antibody shuttle conjugate of the present invention as defined above for use as a medicament.

[0019] A fifth aspect of the present invention relates to an antibody-shuttle conjugate of the invention as defined above for use in the treatment of a central nervous system (CNS) disorder in a mammal, including a human.

[0020] A sixth aspect of the present invention relates to an antibody shuttle conjugate of the present invention as defined above for use as a diagnostic agent. [Brief explanation of the drawings]

[0021] [Figure 1] 1H NMR of DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid. Example 1. [Figure 2] C NMR of DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid). Example 1. [Figure 3] UPLC trace and MS spectrum of Comparative Example 2 and Example 3. UPLC chromatograms are recorded at 220 nm with a 2 min linear gradient from 0 to 100% MeCN (0.036% TFA) in HO (0.045% TFA). [Figure 4] Site-specific trastuzumab-DBM-TTDS-SEQ ID NO: 1 (Example 4) and trastuzumab-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) were produced and characterized. a) ASC synthesis scheme. b) Mass characterization of trastuzumab-DBM-TTDS-SEQ ID NO: 15 (also called Tz-Ang2, Comparative Example 3) (top) and trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also called Tz-MiniAp4, Example 4) (bottom) by LCT-Premier. Deconvoluted spectra are shown. Calculated M for Tz-Ang2 = 159244; Observed M: 79537, 159396; Calculated M for Tz-MiniAp4 = 153684; Observed M: 76918, 153678; c) Coomassie-stained SDS-PAGE of trastuzumab conjugates with DBM-derivatized BBB shuttle peptides: 1: protein marker; 2: trastuzumab (also known as Tz); 3: trastuzumab-DBM-TTDS-SEQ ID NO: 15 (also known as Tz-Ang2, Comparative Example 3); 4: trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also known as Tz-MiniAp4, Example 4). [Figure 5] Mass characterization by LCT-Premier of anti-trastuzumab (anti-idiotype) AlexaFluor® 647-conjugated antibodies (AF647-modified trastuzumab (aka Tz) (a), trastuzumab-DBM-TTDS-SEQ ID NO: 15 (aka Tz-Ang2, Comparative Example 3) (b), and trastuzumab-DBM-TTDS-SEQ ID NO: 1 (aka Tz-MiniAp4, Example 4) (c). The antibodies are deglycosylated. Deconvoluted spectra are shown. [Figure 6]Mass characterization of trastuzumab (also known as Tz) (a), trastuzumab-DBM-TTDS-SEQ ID NO: 15 (also known as Tz-Ang2, Comparative Example 3) (b), and trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also known as Tz-MiniAp4, Example 4) (c) after immunoprecipitation of acceptor wells from HBBBCMTA with LCT-Premier. Raw data (top) and deconvoluted spectra (bottom) are shown. M calculated for Tz: 148212; M observed: 148215; M calculated for Tz-Ang2 = 159244; M observed: 159408; M calculated for Tz-MiniAp4 = 153684; M observed: 76920, 153678. [Figure 7] Stability study in mouse serum of the BBB shuttle peptides present in Example 4 and Comparative Example 3. [Figure 8]Binding of Tz, 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 cells and SKBR3 cells were split and incubated with Tz, Tz-Ang2, or Tz-MiniAp4 for 2 hours at 4°C. Anti-human Dylight650 was used to detect Ig1. The amount of bound antibody was analyzed by flow cytometry. Error bars represent standard deviation (n=3). P values ​​were calculated using one-way analysis of variance 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.Tzp=0.0004;VehicleVs.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-Sequence number 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). [Figure 9]Binding of Cx and Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to MDA-MB-231 cells. There is no significant difference in binding. Activity is conserved. [Figure 10] Binding of Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to BT474 cells. There is no significant difference in binding. Activity is preserved. [Figure 11] Cell cycle arrest analysis of cells treated with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4), and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3). 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 the cell cycle was analyzed by flow cytometry. Error bars represent standard deviation (n=3). [Figure 12] Permeability in a human in vitro BBB cell 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 standard deviation (n=3). P values ​​were calculated using a 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). [Figure 13] Permeability of Cx and Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) (1 μM) in a human in vitro BBB cell model. Error bars represent SEM (n=3). P value was calculated using a two-tailed t-test (Cx vs. Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) p=0.069). [Figure 14]Permeability of Bv and Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6) (1 μM) in a human in vitro BBB cell model. Error bars represent SEM (n=3). P values ​​were calculated using a two-tailed t-test (Bv vs. Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6) p<0.001). [Figure 15] Permeability of Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) (1 μM) in a human in vitro BBB cell model. Error bars represent SEM (n=3). P values ​​were calculated using a two-tailed t-test (Pt vs. Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) p<0.0001). [Figure 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 μM) in a human in vitro BBB cell model. Error bars represent SD (n=3). [Figure 17]Brain concentrations of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4), and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) after iv bolus injection. Results are expressed in nmol per gram of tissue. Error bars represent standard deviation (n=3). P values ​​were calculated using one-way ANOVA (TzVs.Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p=0.7697; TzVs.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. mAb was injected into the tail vein of mice at 10 mg / kg. After 8 hours, serum was collected and systemic saline perfusion was performed. Brains were then removed, and mAb was quantified by ELISA. The results are expressed as the brain / serum ratio of mAb. Error bars represent standard deviation (n=3). P values ​​were calculated using one-way ANOVA (TzVs.Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) p=0.2657; TzVs.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). [Figure 18] Permeability of Cx and Cx-MiniAp4 (1 μM) in a human in vitro BBB cell model. Error bars represent SD (n=3). P values ​​were calculated using one-way ANOVA (Cx vs. Cx-DBM-MiniAp4 p=0.0012; Cx-mal-MiniAp4 vs. Cx-DBM-MiniAp4 p=0.0007). [Figure 19] Papp ratios of Cx modified with SN38-conjugated peptide shuttles (MiniAp4 or Ang2) and Cx modified with naked shuttles (MiniAp4 and Ang2) assayed at 1 μM in a human in vitro BBB cell model. Error bars represent SD (n=3). P values ​​were calculated using a two-tailed t-test (Cx vs. Cx-MiniAp4 p=0.0061). DETAILED DESCRIPTION OF THE INVENTION

[0022] All terms used herein in this application shall be understood in their ordinary meaning as known in the art unless otherwise specified. Other, more specific definitions of certain terms used in this application are set forth below and are intended to be applied uniformly throughout the specification and claims, unless a different, expressly set forth definition provides a broader definition.

[0023] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise specified, definite articles such as "the" used herein also include the plural of the noun.

[0024] For purposes of the present invention, the term "comprise" includes the term "consisting of."

[0025] Unless otherwise specified, amino acids referred to herein are L-amino acids. One-letter and three-letter codes are used interchangeably. The following abbreviations are used for the following amino acids: diaminopropionic acid (Dap), diaminobutyric acid (Dab), selenocysteine ​​(Sec), and penicillamine (Pen). In the context of the present invention, penicillamine encompasses only D-penicillamine. Diaminopropionic acid (Dap) may also be abbreviated as Dpr, and diaminobutyric acid (Dab) may also be abbreviated as Dbu. As stated above, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof is part of the present invention, [ka] It comprises one to six peptides of formula P inserted into the disulfide bonds of the antibody in the form -P-(W)sY and linked to the sulfides via the linker -[(L1)-(L2)-(L3)m-], where Z, L1, L2, L3, D, m, P, W, S, Y and n have the meanings given above.

[0026] In certain embodiments, the antibody shuttle conjugate of the present invention is one in which the disulfide bonds initially present in the antibody are capable of structurally retaining the sulfide bonds in the structure under reducing conditions, and forming new bridges between the sulfide groups of the reduced disulfide bonds of the antibody by reaction with a linker L1, thereby incorporating the following biradical between the sulfide groups of the antibody: [ka]

[0027] The antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof may be in the form of a formulation containing the antibody or its salt and an excipient. For example, suitable excipients for bevacizumab (Avastin) are trehalose dihydrate, sodium phosphate, polysorbate 20, and water for injection; suitable excipients for pertuzumab (Perjeta) are glacial acetic acid, L-histidine, sucrose, polysorbate 20, and water for injection; suitable excipients for cetuximab (Erbitux) are sodium chloride, glycine, polysorbate 80, citric acid monohydrate, sodium hydroxide, and water for injection; and suitable excipients for trastuzumab (Herceptin) are, for example, L-histidine hydrochloride monohydrate, L-histidine, α,α-trehalose, or polysorbate 20 dihydrate.

[0028] In certain embodiments, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof is one in which L1 is attached to the -S- of a disulfide bond of the antibody via bonds a and b, and bond c is attached to linker L2 via an amide bond between the C=O group adjacent to bond c of linker L1 and the NH group of linker L2.

[0029] In another particular embodiment, alone or in combination with any of the above embodiments, the antibody shuttle conjugate of Formula (I) or a pharmaceutically acceptable salt thereof is one in which the linker L2 is a biradical selected from the group consisting of: [ka] [ka]

[0030] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof is one in which n is an integer from 1 to 4. In another specific embodiment, in combination with any of the above embodiments, the antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof is one in which n is 4, i.e., it has four peptides incorporated into the antibody structure. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, all peptides are equal.

[0031] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the disulfide bonds of the antibody are interchain bonds. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the disulfide bonds of the antibody are intrachain bonds. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the disulfide bonds of the antibody are genetically engineered disulfide bonds.

[0032] In another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is one in which P is a biradical of a peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence DapKAPETALD and having an intrapeptide bond between Dap and D which is an amide bond, i.e., SEQ ID NO: 8: [ka] (b) a peptide having a length of 9 to 20 amino acid residues, having at least one intrapeptide bond that is a disulfide bond, and comprising the amino acid sequence CKAPETALCAAA with at least one intrapeptide disulfide bond between cysteines 1 and 9, i.e., SEQ ID NO: 9: [ka] (c) a peptide having a length of 9 to 11 amino acid residues, having at least one intrapeptide disulfide bond that is a disulfide bond, and having at least one intrapeptide bond between cysteines 1 and 9, and consisting of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA, i.e. SEQ ID NO:10: [ka] SEQ ID NO:11: [ka] SEQ ID NO:12: [ka] (d) A peptide having 16 amino acid residues, comprising the amino acid sequence CNCKAPETALCAAACH, and having intrapeptide disulfide bonds between the first and third cysteines (cysteines 1 and 11) and between the second and fourth cysteines (cysteines 3 and 15), i.e., SEQ ID NO: 13: [ka] (e) A peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

[0033] A line between two amino acids in the above or below sequences represents an intrapeptide bond between the side chains of the two amino acids. In certain embodiments, a line between two amino acids in the above or below sequences represents an intrapeptide bond between the side chains of the two amino acids.

[0034] In another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is one in which P is a biradical of a peptide selected from the group consisting of: (a) a peptide having the amino acid sequence DapKAPETALD and having an intrapeptide bond that is an amide bond between Dap and D (SEQ ID NO: 7); (b) a peptide having the amino acid sequence CKAPETALC and having at least one intrapeptide disulfide bond between the cysteines at positions 1 and 9 (SEQ ID NO: 10); and (c) a peptide having the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

[0035] In another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is one in which P is a biradical of the peptide DapKAPETALD, with an intrapeptide bond between Dap and D that is an amide bond (SEQ ID NO: 7).

[0036] In another particular embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is one in which P is a biradical of a peptide having one intrapeptide bond. In another particular embodiment, the antibody shuttle conjugate is one in which P is a biradical of a peptide having two intrapeptide bonds.

[0037] Antibodies generally consist of two heavy chains (HC) and two light chains (LC) that fold into constant and variable domains, although some antibodies, such as camelid antibodies, contain only two heavy chains. They can occur individually as monomers (e.g., IgG) or in multimers of two (e.g., IgA) to five (e.g., IgM) units. They have disulfide interchain bonds. Antibodies for purposes of the present invention may be chimeric, humanized, or fully human. In the present invention, antibodies may be natural or recombinant. In certain embodiments, the antibody is a therapeutic antibody. In another particular embodiment, the antibody is diagnostic. In another particular embodiment, the antibody is an antibody-drug conjugate (ADC).

[0038] An antibody may be an antibody fragment, so long as it contains at least one disulfide bond and maintains the function of the antibody from which it is derived, particularly its therapeutic or diagnostic activity. For example, the antibody may be an antibody fragment such as a Fab, scFV, (Fab)2, diabody, triabody, tetrabody, minibody, or nanobody. For example, disulfide bonds can be incorporated into an antibody or fragment thereof by incorporating cysteines by mutating the antibody's nucleic acid sequence to encode a cysteine-engineered antibody and replacing one or more amino acid residues with cysteines (see Canadian Patent Application Publication No. 2957354(A1)). Antibody fragments may offer several advantages over full-length antibodies. Their small size allows for better tissue penetration into solid tumors, and their shorter half-life is ideal for using antibodies as radioimaging agents. Antibodies are useful for applications that do not require immune system involvement, such as blocking signaling molecules or receptors.

[0039] In a specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate is one in which the antibody comprises a heavy chain constant domain of the IgA, IgD, IgE, IgG, or IgM type. Antibodies of the IgA type can be divided into two isotypes: IgA1 and IgA2. Antibodies of the IgG class can be classified into four isotypes: IgG1, IgG2, IgG3, and IgG4. Any class of antibody is encompassed within the scope of the present invention.

[0040] In another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is one in which the antibody comprises a light chain constant domain, for example of the kappa or lambda type.

[0041] In another specific embodiment, the antibody shuttle conjugate, alone or in combination with any of the embodiments of the present invention, is one in which the antibody or antibody fragment according to the present invention comprises (a) an immunoglobulin constant region, (b) an IgG1 constant region, or (c) a human IgG1 constant region. IgG1 is particularly preferred because it is the most widely used isotype for anti-cancer mAbs and the most effective IgG isotype in mediating ADCC (antibody-dependent cellular cytotoxicity). In more specific embodiments, the antibody is a monoclonal chimeric antibody, a humanized antibody, or a fully human antibody. Antibody humanization can reduce immunogenicity by reducing the mouse content of the monoclonal antibody. This can be achieved, for example, by expressing isolated human variable domain genes in E. coli. Common techniques developed for producing fully human monoclonal antibodies can be used, such as phage display, in which a library of human antibodies is expressed on the surface of phage and then selected and amplified in E. coli, and transgenic mice expressing human antibody repertoires.

[0042] In yet another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is one in which the antibody is multispecific. Multispecific antibodies are a type of engineered antibody and antibody-like protein that combine multiple specific antigen-binding elements in a single structure.

[0043] In yet another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is a heterodimeric bispecific antibody in which the antibody is a conventional IgG molecule with one arm targeting one antigen and the other targeting a second antigen.

[0044] In yet another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is a bispecific antibody fusion in which the antibody is a non-canonical IgG molecule, which is extended at the N-terminus of the corresponding heavy and light chains by an additional variable domain of a second antibody.

[0045] In yet another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is one in which the antibody is also a trispecific antibody, a non-canonical IgG molecule. The same techniques used to generate bispecific antibodies can also be combined to generate trispecific antibodies with various valencies.

[0046] In yet another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody shuttle conjugate is an antibody in which the scFv is fused to an IgG via attachment of the scFv to the N-terminus or C-terminus of the heavy or light chain.

[0047] In certain embodiments, the antibody shuttle conjugate of the invention is one in which the antibody is selected from the group consisting of trastuzumab, bevacizumab, cetuximab, pertuzumab, aducanumab, bapineuzumab, nimotuzumab, and necitumumab. In more specific embodiments, the antibody shuttle conjugate of the invention is one in which the antibody is selected from the group consisting of trastuzumab, bevacizumab, cetuximab, and pertuzumab.

[0048] In a specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is trastuzumab. Trastuzumab (Herceptin®) is an FDA-approved antibody against HER2 and is widely used in clinical practice to treat breast cancer. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1), having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid.

[0049] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is cetuximab. Cetuximab (Erbitux®) is an FDA-approved antibody against EGFR and is widely used in clinical practice to treat colorectal cancer and head and neck cancer. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Cx-DBM-TTDS-SEQ ID NO: 1), which has an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid.

[0050] The term "DBM" refers to the linker L 1aThe term "TTDS" corresponds to the linker L 2a In both cases, the terms are used interchangeably. DBM=L 1a has the following formula: [ka] TTDS=L 2a has the following formula: [ka]

[0051] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is bevacizumab. Bevacizumab (Avastin®) is an FDA-approved antibody against VEGF-A and is widely used in clinical practice to treat colorectal cancer, lung cancer, glioblastoma, and renal cell carcinoma. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1), which has an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid.

[0052] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is pertuzumab. Pertuzumab (Perjeta®) is an FDA-approved antibody against HER2 and is widely used in clinical practice to treat breast cancer. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-DBM-TTDS-SEQ ID NO: 1), having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid.

[0053] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is aducanumab, an IgG1 antibody directed against an epitope of β-amyloid protein. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is bapineuzumab, an anti-Aβ-amyloid IgG1 antibody. Both antibodies are effective in treating Alzheimer's disease.

[0054] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is nimotuzumab, an IgG1 anti-EGFR antibody. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody shuttle conjugate of the present invention is one in which the antibody is necitumumab, an IgG1 anti-EGFR antibody. Both antibodies are effective in treating brain metastases.

[0055] In certain embodiments, formulations comprising the antibody shuttle conjugates of the invention, alone or in combination with any of the embodiments of the invention, when D is present, yield antibody drug shuttle conjugates, antibody-radioligand shuttle conjugates, or antibody-diagnostic agent shuttle conjugates.

[0056] In certain embodiments, the biologically active agent is an active pharmaceutical ingredient. In another specific embodiment, the antibody shuttle conjugate of the invention is one in which the antibody is an anti-cancer therapeutic antibody, m=1, and the radical of the active biological agent, D, is the radical of an anti-cancer active pharmaceutical ingredient selected from the group consisting of an auristatin, a duocarmycin, a PBD dimer, a maytansinoid, a calicheamicin, an anthracycline, a camptothecin, alpha-amanitin, a tubulysin, MMAE, T-DM1, and a PROTAC moiety.

[0057] In another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody is an anti-cancer therapeutic antibody, m=1, and D is a chemotherapeutic agent.

[0058] In another specific embodiment, alone or in combination with any of the embodiments of the invention, the antibody-drug shuttle conjugate is one in which the antibody is cetuximab and D is SN38. In another specific embodiment, the antibody-drug shuttle conjugate according to the invention is one in which the antibody is cetuximab, the drug is SN38, the peptide is MiniAp4, and the linker is TTDS-DBM.

[0059] In another specific embodiment, alone or in combination with any of the embodiments of the present invention, the antibody-containing formulation is an anti-cancer therapeutic antibody, m=1, and D is a radioligand. This formulation is useful for radioimmunotherapy. In another specific embodiment, alone or in combination with any of the embodiments of the present invention, D is biotin-yttrium-90 or biotin-iodine-131 or 123. These can be linked to the antibody shuttle conjugate of the present invention via streptavidin, which is attached to the lateral chain of an amino acid of the biradical L3 when present in the antibody shuttle conjugate of the present invention.

[0060] In another specific embodiment, alone or in combination with any of the embodiments of the invention, D is a contrast agent for magnetic resonance imaging (MRI), such as gadolinium, particularly Gd-DTPA (Magnevist®). Gadolinium can be conjugated to DOTA, which, when present in the antibody-shuttle conjugate of the invention, is attached to the lateral chain of the amino acid of the biradical L3 or to an amino acid derivative by a viable bond.

[0061] In another specific embodiment, the antibody shuttle conjugate of the invention is one in which the antibody is an anti-neurodegenerative therapeutic antibody, m=1, and the radical of the active pharmaceutical ingredient is the radical of an anti-neurodegenerative active pharmaceutical ingredient.

[0062] The antibody shuttle conjugate of the present invention can be prepared by a process comprising: a) reducing the disulfide bridges of an antibody; and b) re-crosslinking 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), where the dibromomaleimide is attached to the peptide via its N-terminus, and DBM, P, W, Y, and s are as defined for the antibody shuttle conjugate of formula (I). The attachment of the DBM peptide to the antibody is possible due to the reactivity of cysteines to form disulfide bridges. This method allows for a high degree of control over the number of peptides immobilized on each antibody molecule. In a specific embodiment, the antibody is trastuzumab.

[0063] The antibody shuttle conjugates of the present invention can be defined by the process for their preparation. Thus, antibody shuttle conjugates obtainable by the above-defined process are also considered to be part of the present invention.

[0064] In particular, the antibody shuttle conjugates defined above can be obtained by reacting the hydrosulfide groups of the reduced disulfide bonds of the corresponding antibody or fragment thereof with the dibromomaleimide peptide of formula (II) defined above, re-bridging the disulfide bonds with the following biradical incorporated between the disulfide bridges of the antibody: [ka]

[0065] In another particular embodiment, the antibody shuttle conjugate defined above may be obtained by a process comprising: a) reducing the disulfide bridges of the antibody; b) re-crosslinking the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide peptide of formula (II); and c) optionally, carrying out hydrolysis, wherein q, L2, L3, D, P, m, W, s, and Y are as defined for the antibody shuttle conjugate of formula (I). [ka]

[0066] In certain embodiments of this process, the disulfide bond is an interchain bond. All embodiments defined above as products of the conjugate of formula (I) itself are also embodiments of the preparation process thereof.

[0067] The shuttle peptides used in the conjugates of the invention may be produced in whole or in part 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 easily prepared, for example, by solution phase synthesis, preferably solid phase peptide synthesis, and several procedures for this method have been published (see M. Amblard, et al., "Methods and protocols of modern solid-phase peptide synthesis." Molecular Biotechnology 2006, Vol. 33, pp. 239-254). The compounds of formula (I) can also be prepared by any combination of solution phase synthesis and / or solid phase synthesis, for example, by synthesizing the peptide P itself by solid phase synthesis and then removing the protecting groups in solution. The linker dibromomaleimide can be attached to the linker in solid phase or in solution. The linker construct can also be prepared by any combination of solution phase synthesis and / or solid phase synthesis. The linker used is 3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid or a similar derivative thereof.

[0068] Compounds of formula (II) can be prepared by a process comprising reacting a linker-derivatized peptide with 3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid by solid phase peptide synthesis or in solution to give the DBM peptide. 1b Compounds of formula (II) having the linker L 1aThe hydrolysis can be carried out under basic conditions at mild pH.

[0069] In certain embodiments, the DBM peptide of formula (II) has the formula: [ka]

[0070] In another particular embodiment, the DBM peptide of formula (II) has the formula: formula: [ka]

[0071] In certain embodiments, the antibody shuttle conjugates of the invention comprise a linker L1 But, L 1b This conjugate contains the linker L 1a can be prepared from the corresponding antibody shuttle conjugate having the formula: by hydrolysis under mildly basic pH conditions.

[0072] The antibody shuttle conjugates of the present invention may be in the form of their pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salts" encompasses any salt formed from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids or bases. There are no limitations on the salts, but when used for therapeutic purposes, they must be pharmaceutically acceptable. Since 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. Examples of such acids include hydrochloric acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like.

[0073] Examples of antibodies in the form of a salt according to the present invention include bevacizumab (Avastin) in the form of a salt with trehalose dihydrate, sodium phosphate, a salt of polysorbate 20, 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 with sodium chloride, glycine, polysorbate 80, citric acid monohydrate, or sodium hydroxide, and trastuzumab (Herceptin) in the form of a salt with L-histidine hydrochloride monohydrate, L-histidine, α,α-trehalose, or polysorbate 20 dihydrate.

[0074] The preparation of pharmaceutically acceptable salts of compounds of formula (I) can be carried out by methods known in the art. For example, they can be prepared from conjugates containing basic or acidic moieties by conventional chemical methods. Generally, such salts are prepared by, for example, reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate pharmaceutically acceptable base or acid in water or an organic solvent or a mixture thereof.

[0075] Pharmaceutical compositions comprising a therapeutically effective amount of an antibody shuttle conjugate as defined above together with an appropriate amount of a pharmaceutically acceptable carrier or excipient are also part of the present invention.

[0076] The term "pharmaceutical composition" refers to a mixture of a compound described herein with other chemical components, such as a diluent or carrier. A pharmaceutical composition facilitates administration of a compound to an organism. The term "pharmaceutically acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other components of the pharmaceutical composition. It must also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0077] As used herein, the phrase "therapeutically effective amount" refers to an amount of a compound that, when administered, is sufficient to prevent or alleviate to some extent one or more symptoms of the disease being addressed. The specific dose of a compound administered in accordance with the present 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 similar considerations.

[0078] The phrase "pharmaceutically acceptable excipient, diluent, or carrier" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical composition. It must also be suitable for use in contact with the tissues or organs 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.

[0079] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. Except insofar as any conventional excipient medium is incompatible with the substance or its derivatives, such as by producing some undesirable biological effect or by otherwise interacting in a deleterious manner with any of the other component(s) of the pharmaceutical composition, its use is intended to be within the scope of the present invention.

[0080] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the characteristics, size, and / or condition of the subject being treated, as well as the route by which the composition is administered.

[0081] The compositions of the present invention may be administered in parenteral forms suitable for injection, such as intravenous bolus injection, intravenous infusion, implantation into the body, oral, intrathecal, or intranasal.

[0082] Antibody shuttle conjugates as defined above for use as pharmaceuticals are also part of the present invention, which includes antibody shuttle conjugates and antibody-drug shuttle conjugates according to the invention.

[0083] As used herein, the term "pharmaceutical" is synonymous with a widely accepted medical or veterinary drug (also called a drug, medication, or simply drug) used to cure, treat, or prevent disease in animals, including humans. Drugs are classified in various ways. One important distinction is between traditional small molecule drugs, which are typically obtained through chemical synthesis, and biopharmaceuticals, which include recombinant proteins, vaccines, therapeutically used blood products (e.g., IVIG), gene therapy, monoclonal antibodies, and cell therapy (e.g., stem cell therapy).

[0084] An antibody shuttle conjugate as defined above for use in the treatment of a CNS disorder in a mammal, including a human, is also part of the present invention. This aspect can also be formulated as the use of an antibody shuttle conjugate as defined above for the preparation of a medicament for the treatment of a CNS disorder in a mammal, including a human. The present invention also relates to a method of treating a mammal, including a human, suffering from or likely to suffer from a central nervous system disorder, said method comprising administering to the patient a therapeutically effective amount of an antibody shuttle conjugate as defined above, together with a pharmaceutically acceptable excipient or carrier.

[0085] In a particular embodiment, the antibody shuttle conjugate is for use in a CNS disorder that is cancer.

[0086] In another particular embodiment, the antibody shuttle conjugates of the invention are for use in the treatment of primary brain tumors. In another particular embodiment, the antibody shuttle conjugates of the invention are for use in the treatment of brain metastases (BM), which are a major complication in several types of cancer, particularly lung cancer, melanoma, and breast cancer.

[0087] The antibody shuttle conjugates of the present invention, both the antibody shuttle conjugates according to the present invention and the antibody-drug shuttle conjugates, can be used in combination with other treatments, similar to other known chemotherapeutic agents, i.e., either simultaneously or sequentially, depending on the condition being treated. They may be used alone or in combination with other suitable biologically active compounds. Thus, the antibody shuttle conjugates of the present invention are intended for use in the treatment of cancer in mammals, including humans, in combination with chemotherapeutic agents. Antibody-drug shuttle conjugates in combination with additional chemotherapeutic agents can be used, for example, in certain treatment regimens.

[0088] In another specific embodiment, the antibody shuttle conjugates of the present invention are for use in combination with radioimmunotherapy. Both the antibody shuttle conjugates and antibody-radioligand shuttle conjugates of the present invention can be used. For example, a combination of radiotherapy and immunotherapy can be used to treat other types of cancer, including non-Hodgkin's lymphoma and brain tumors. The antibody-radioligand shuttle conjugates of the present invention can bind to cancer cells and deliver high doses of radiation directly to the tumor.

[0089] An antibody shuttle conjugate as defined above for use as a diagnostic agent is also part of the present invention. In a particular embodiment, the antibody shuttle conjugate according to the present invention is for use in a method for the diagnosis of a CNS disorder.

[0090] Throughout the description and claims, the word "comprise" and variations of that word are not intended to exclude other technical features, additives, components, or steps. Furthermore, the term "comprise" includes the term "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the detailed description or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to limit the invention. Reference signs placed in parentheses in connection with the drawings and in the claims are intended only to enhance the completeness of the claims and shall not be construed as limiting the scope of the claims. Furthermore, the present invention includes all possible combinations of the specific preferred embodiments described herein.

[0091] Example Protected amino acids, handles, and resins were supplied by: Luxembourg Industries (Tel-Aviv, Israel), Neosystem (Strasbourg, France), Calbiochem Novabiochem AG (Laufelfingen, Switzerland), Bachem AG (Bubendorf, Switzerland), or Iris Biotech (Marktredwitz, Germany). Other reagents and solvents used are summarized in Table 1.

[0092] Table 1. Suppliers and reagents used. DCM was passed through an Al2O3 column. DMF was stored over molecular sieves 4A and nitrogen bubbling was performed to remove volatiles.

[0093] 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). Other chemicals used were obtained from Aldrich (Milwaukee, WI) and were of the highest purity commercially available.

[0094] Cell culture-treated plates and flasks were purchased from Corning Costar. Culture medium was obtained from Lonza. XTT cell proliferation kit was purchased from Biological Industries (Cromwell, CT). Pierce® iodinated beads were obtained from Pierce. Desalting columns (MiniTrap and MidiTrap G-25) were obtained from GE-Healthcare. [Table 1]

[0095] General Methods for the Preparation of Conjugates of the Invention General considerations for manual synthesis: Solid-phase peptide elongation and other solid-phase manipulations were performed manually with polypropylene syringes fitted with porous polyethylene discs. Solvents and soluble reagents were removed by aspiration. Washing between different synthesis steps was performed with dimethylformamide (DMF) (5x30 s) and dichloromethane (DCM) (5x30 s), using 10 mL of solvent per g of resin each time.

[0096] General considerations for microwave-assisted synthesis: Microwave-assisted solid-phase peptide synthesis was performed on a LibertyBlue automated microwave peptide synthesizer using H-Rink amidoprotide resin (loading: 0.56 mmol / g). Linear peptides were synthesized on a 0.5 mmol scale using a 5-fold excess of Fmoc amino acids (0.2 M) relative to the resin.

[0097] Identification tests: The tests used for identification and control of the synthesis were: A) Kaiser colorimetric assay for the detection of primary amines bound to a solid phase (E. Kaiser et al., Anal. Biochem. 1970, vol. 34, pp. 595-598); B) p-nitrophenyl ester test for secondary amines bound to a solid phase (A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791).

[0098] Protocols used during manual synthesis of compounds: Compounds were synthesized on a 100 μmol scale using the following method and protocol: Resins for manual synthesis were selected depending on the Y group: if Y is OH, the terminus is COOH and 2-chlorotrityl chloride resin is selected among other available resins; if Y is NH2, the terminus is CONH2 and Rink amide MBHA resin is selected among other available resins.

[0099] Initial resin conditioning: The resin was conditioned by washing with MeOH (5x30 sec), DMF (5x30 sec), DCM (5x30 sec), 1% TFA / DCM (1x30 sec and 2x10 min), DCM (5x30 sec), DMF (5x30 sec), DCM (5x30 sec), 5% DIEA / DCM (1x30 sec, 2x10 min), DCM (5x30 sec), DMF (5x30 sec).

[0100] Removal of the Fmoc group: Removal of the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was achieved with 20% (v / v) piperidine in DMF using a 30 s treatment followed by two treatments of 10 min each. Two additional treatments (2 x 5 min) with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) were performed to ensure removal of the Fmoc group from the secondary amine (proline).

[0101] Coupling method described for 100 μmol scale: Coupling Method 1: The protected amino acid (4 equiv., 400 μmol) and TBTU (4 equiv., 400 μmol, 128 mg) dissolved in DMF (1–3 mL / g resin) were added sequentially to the resin, followed by DIEA (8 equiv., 800 μmol, 136 μl). The mixture was allowed to react for 1 h with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 × 30 s) and DCM (5 × 30 s). The extent of coupling was confirmed by Kaiser colorimetry. The Fmoc group was removed with a 20% solution (v / v) of piperidine in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2 x 5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0102] Coupling Method 2: Protected amino acid (4 equiv., 400 μmol), PyBOP (4 equiv., 400 μmol, 208 mg), and HOAt (12 equiv., 1.2 mmol, 163 mg) dissolved in DMF (1–3 mL / g resin) were added sequentially to the resin, followed by DIEA (12 equiv., 1.2 mmol, 204 μL). The mixture was reacted for 1 h with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 × 30 s) and DCM (5 × 30 s). The coupling reaction was carried out twice under the same conditions. The extent of coupling was confirmed by Kaiser colorimetry. The Fmoc group was removed with a 20% solution (v / v) of piperidine in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2 x 5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0103] Coupling Method 3: Protected amino acid (4 equiv., 400 μmol), PyBOP (4 equiv., 400 μmol, 208 mg), and HOBt (12 equiv., 1.2 mmol, 162 mg) dissolved in DMF (1–3 mL / g resin) were added sequentially to the resin, followed by DIEA (12 equiv., 1.2 mmol, 204 μL). The mixture was reacted for 1 h with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 × 30 s) and DCM (5 × 30 s). The coupling reaction was carried out twice under the same conditions. The extent of coupling was confirmed by Kaiser colorimetry. The Fmoc group was removed with a 20% solution (v / v) of piperidine in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, an additional treatment (2 x 5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) was applied to ensure removal of the Fmoc group.

[0104] Coupling method 4, scale 100 μmol: Protected amino acid (3 equiv., 300 μmol), DIC (3 equiv., 300 μmol, 46 μL), and oxima (3 equiv., 300 μmol, 43 mg) in DCM / DMF (1:1). The mixture was reacted for 45 min with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). The extent of coupling was confirmed by Kaiser colorimetry. The Fmoc group was removed with a 20% solution (v / v) of piperidine in DMF using a 30 s treatment and two 10 min treatments. When the amino acid to be deprotected was proline, additional treatments (2 x 5 min) with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) were applied to ensure complete removal of the Fmoc group.

[0105] Coupling method 5, scale 100 μmol: Protected amino acid (3 equiv., 300 μmol), DIC (3 equiv., 300 μmol, 46 μL), and HOBt (3 equiv., 300 μmol, 41 mg) in DCM / DMF (1:1). The mixture was reacted for 45 min with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). The extent of coupling was confirmed by Kaiser colorimetry. The Fmoc group was removed with a 20% solution (v / v) of piperidine in DMF using a 30 s treatment and two 10 min treatments. When the amino acid to be deprotected was proline, additional treatments (2 x 5 min) with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) were applied to ensure complete removal of the Fmoc group.

[0106] Protocol used during microwave-assisted automated synthesis: Compounds were synthesized on a 500 μmol scale using the following method and protocol. Resins were selected for microwave-assisted automated synthesis depending on the Y group: if Y is OH, the terminus is COOH, and Cl-TCP(Cl)ProTide resin was selected among other available resins; if Y is NH2, the terminus is CONH, and Rink Amide ProTide resin was selected among other available resins.

[0107] Initial resin conditioning: The resin was conditioned by washing with MeOH (5x30 sec), DMF (5x30 sec), DCM (5x30 sec), 1% TFA in DCM (1x30 sec and 2x10 min), DCM (5x30 sec), DMF (5x30 sec), DCM (5x30 sec), 5% DIEA in DCM (1x30 sec, 2x10 min), DCM (5x30 sec), DMF (5x30 sec).

[0108] Coupling and deprotection conditions for microwave-assisted automated peptide synthesis: Coupling conditions: [Table 2] [Table 3]

[0109] Deprotection conditions: [Table 4] [Table 5]

[0110] Methods for cyclization of peptide sequence P: Cyclization Method 1: Disulfide or Diselenide Bond: Cyclization was performed in solution after cleavage from the resin or on the resin after selective deprotection of Cys, Sec, or Pen residues. The product was dissolved at a concentration of 100 μM in 10 mM aqueous ammonium bicarbonate buffer at pH 8.0. The solution was vigorously stirred at room temperature for 24 hours. The product was then acidified with TFA to pH 2-3, frozen, and lyophilized.

[0111] Cyclization Method 2: Amide Bond: Cyclization was performed on the resin. The Fmoc group was removed with a 20% solution of piperidine in DMF (v / v) using a 30-second treatment and two 10-minute treatments. The N-terminal amine was protected with a Boc protecting group using BocO (3 equiv., 1000 μmol, 56 mg) and DIEA (30 equiv., 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 10 μM, 12 mg) and phenylsilane (10 equiv., 1000 μmol, 123 mg) in DCM (3 × 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 × 5 min). Coupling of the amino group of Dap with the carboxylate group of aspartic acid was then achieved by adding PyBOP (4 equiv., 400 μmol, 208 mg), HOAt (12 equiv., 1.2 mmol, 163 mg), DMF (1–3 mL / g resin), and DIEA (12 equiv., 1.2 mmol, 204 μL). The coupling was allowed to stand for 1.5 h and then repeated overnight.

[0112] Cyclization Method 3: Amide Bond: Cyclization was performed on the resin. The Fmoc group was removed with a 20% solution of piperidine in DMF (v / v) using a 30-second treatment and two 10-minute treatments. The N-terminal amine was protected with a Boc protecting group using BocO (3 equiv., 1000 μmol, 56 mg) and DIEA (30 equiv., 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 10 μM, 12 mg) and phenylsilane (10 equiv., 1000 μmol, 123 mg) in DCM (3 × 15 min). The resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3 × 5 min). Coupling of the amino group of Dap with the carboxylate group of aspartic acid was then achieved by two 30-min cycles of 4 equivalents of oxime (400 μmol, 57 mg) and 4 equivalents of N,N'-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL).

[0113] Cyclization Method 4: Amide Bond: Cyclization was performed on the resin. The Fmoc group was removed with a 20% solution of piperidine in DMF (v / v) using a 30-second treatment and two 10-minute treatments. The N-terminal amine was protected with a Boc protecting group using BocO (3 equiv., 1000 μmol, 56 mg) and DIEA (30 equiv., 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 10 μM, 12 mg) and phenylsilane (10 equiv., 1000 μmol, 123 mg) in DCM (3 × 15 min). The resin was washed with 0.02 M sodium diethyldithiocarbamate in DCM (3 × 5 min). Coupling of the amino group of Dap with the carboxylate group of aspartic acid was then achieved by two 1 h cycles of 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 μmol, 54 mg).

[0114] General Method for Linker Construction General method for disulfide formation: Disulfide bonds can be achieved by the reaction of two thiols. The thiols are dissolved in aqueous ammonium bicarbonate buffer (10 mM, pH 8.0) at a concentration of 100 μM, and the solution is vigorously stirred at room temperature for 24 hours. The solution is then acidified with TFA to pH 2-3, frozen, and lyophilized.

[0115] General method for the formation of thioethers: Thioether bonds are achieved by reaction of the N-terminal bromoacetyl group with cysteine ​​thiol as described in PL Barker et al. J. Med. Chem., 1992, vol 35, pp. 2040-2048.

[0116] General Methods for Formation of Ethers: Ether formation can be achieved by reaction of a hydroxyl group with a haloalkyl 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.

[0117] General methods for the formation of esters: Ester formation can be achieved by reacting a hydroxyl group with a carboxylic acid using typical esterification conditions such as Fischer esterification in the presence of an acid catalyst, or by the reaction of a hydroxyl group with the corresponding acid chloride, as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter GMWuts. 2014 John Wiley & Sons, Inc. pp. 271-279.

[0118] General method for the formation of thioesters: Thioester bonds are achieved by the reaction of thiols with carboxylic acids as described in M. Kazemi et al., Journal of Sulfur Chemistry, 2015, vol. 36:6, pp. 613-623.

[0119] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equiv.) was achieved by two cycles of 4 equiv. of oxime and 4 equiv. of N,N'-diisopropylcarbodiimide (DIC) in DMF for 30 min or 4 equiv. of DIC and 4 equiv. of HOBt in DCM for 2 h. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was then removed with 20% (v / v) piperidine in DMF using two cycles of 30 s followed by 10 min.

[0120] Coupling of 5-hexynoic acids: Coupling of 5-hexynoic acids (2 equiv., 200 μmol, 23 mg) was achieved by either 4 equiv. oxime (400 μmol, 57 mg) and 4 equiv. N,N'-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL) in DMF:DCM (1:1) for 30 min, or 4 equiv. DIC (400 μmol, 61 μL) and 4 equiv. HOBt (400 μmol, 54 mg) in DCM (DMF:DCM 1:1) for 4 h, or two cycles of 2 equiv. PyBOP (400 μmol, 208 mg) in DMF (DMF:DCM 1:1), 6 equiv. HOAt (600 μmol, 81.5 mg), and 6 equiv. DIEA (600 μmol, 102 μL) for 1.5 h. The solvent was removed by aspiration and the resin was washed with DMF (5 x 30 seconds) and DCM (5 x 30 seconds). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.

[0121] Coupling of diglycolic anhydride: Coupling of diglycolic anhydride (10 equiv., 1000 μmol, 116 mg) was achieved by two 60-min cycles of 10 equiv. of DIEA (1000 μmol, 174 μL) in DMF. The solvent was removed by aspiration, and the resin was washed with DMF (5×30 sec) and DCM (5×30 sec). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay.

[0122] Method for Alkyne-Azide Cycloaddition Reaction: Alkyne-azide cycloaddition (click reaction) coupling was carried out in solution using the protocol described by SFM van Dongen et al.; Bioconjugate Chem. 2009, vol. 20, pp. 20-23.

[0123] General method for cleavage from resin: Final cleavage of the resin and deprotection of the side chains: This was performed by treating the resin with TFA (95%), HO (2.5%), and TIS (2.5%) (2 h). tert-Butyl methyl ether was added to the resulting product, and the mixture was centrifuged (3 x 8 min). The supernatant was discarded, and the pellet was resuspended in a mixture of HO, MeCN, and TFA (1000:1000:1). The product was filtered and frozen.

[0124] General method for compound characterization: Compounds were characterized by UPLC (Acquity High Class system (PDA detector, Sample Manager FNT, and Quaternary Solvent Manager), AcquityBEHC18 (50x2mmx1.7µm) column, 0.61mL / min, and MeCN (0.036% TFA) and HO (0.045% TFA) as solvents). In all cases, a 2-minute linear gradient was used, followed by UPLC-MS spectroscopy (Waters High Class (PDA detector, Sample Manager FNT, and Quaternary Solvent Manager)) with an electrospray ion source ESI-MS Micromass The analysis was performed using MassLynx 4.1 software (Waters, Milford, MA) connected to a ZQ. A BEHC18 column (50 x 2.1 mm x 1.7 μm, Waters) was used. 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 by positive ionization: the ion spray voltage was 30 V and the capillary temperature was 1 kV. Accurate mass was obtained using a mass spectrometer: LTQ-FT Ultra (Thermo Scientific), and samples were introduced by direct infusion (automated nanoelectrospray). A NanoMate (Advion BioSciences, Ithaca, NY, USA) was used to measure the amount of protein in a 384-well plate (protein ion beam) using disposable conductive pipette tips. Samples were aspirated from a Lobind and injected through a nanoESI chip (consisting of 400 nozzles in a 20x20 array) into the mass spectrometer. The spray voltage was 1.70 kV, the discharge pressure was 0.50 psi, and the ionization was NanoESI, positive.

[0125] NMR experiments were performed on a Bruker Avance III 600 MHz spectrometer equipped with a TCI cryoprobe. Samples were prepared by dissolving the compounds at 3-4 mM in 90% H2O / 10% D2O, and the pH was adjusted to 2-3. Chemical shifts were referenced to internal sodium-3-(trimethylsilyl)propanesulfonate (DSS). Suppression of water signals was achieved by excitation sculpting. Residue-specific assignments were obtained from 2D total correlation spectroscopy (TOCSY) and correlation spectroscopy (COSY) experiments, while 2D nuclear Overhauser effect spectroscopy (NOESY) enabled sequence-specific assignments. 13C resonances were assigned from 2D 1H13C HSQC spectra. All experiments were performed at 298 K, except for the NOESY spectrum, which was obtained at 278 K. Proton temperature coefficients of amides were determined from a series of 1D spectra acquired between 278 K and 308 K. The mixing times for TOCSY and NOESY were 70 ms and 250 ms, respectively.

[0126] Amino acid analysis: Amino acid analysis was performed to assess the amino acids present and the amounts obtained for each peptide. For this purpose, ion-exchange chromatography analysis after acid hydrolysis was performed. Samples were hydrolyzed with 6 M HCl at 110 °C for 16 h. They were then evaporated to dryness under reduced pressure and dissolved in 20 mM aqueous HCl. Finally, the amino acids were modified using Waters' AccQ Tag protocol and analyzed by ion-exchange HPLC. For amino acid analysis, 100 μL of peptide (1 mg / mL) was added to 100 μL of 12 M HCl and 20 μL of aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization reagent. The mixture was left overnight at 110 °C. The liquid was completely evaporated, and 200 μL of 20 mM HCl was added, followed by the Waters AccQ-Tag protocol.

[0127] General method for peptide purification and characterization: Crude products were purified by RP-HPLC on a semi-preparative scale and characterized by UPLC (Acquity high-end system (PDA detector, Sample Manager FNT and Quaternary Solvent Manager, AcquityBEHC18 (50x2mmx1.7µm) column, 0.61mL / min, and MeCN (0.036% TFA) and HO (0.045% TFA) as solvents). In all cases, a 2-minute linear gradient was used and UPLC-MS spectroscopy (Waters high-end system (PDA detector, Sample Manager FNT and Quaternary Solvent Manager) was used with an electrospray ion source ESI-MS Micromass The analysis was performed using MassLynx 4.1 software (Waters, Milford, MA) connected to a ZQ. A BEHC18 column (50 x 2.1 mm x 1.7 μm, Waters) was used. 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 by positive ionization: the ion spray voltage was 30 V and the capillary temperature was 1 kV. Accurate mass was obtained using a mass spectrometer: LTQ-FT Ultra (Thermo Scientific), and samples were introduced by direct infusion (automated nanoelectrospray). A NanoMate (Advion BioSciences, Ithaca, NY, USA) was used to measure the amount of protein in a 384-well plate (protein ion beam) using disposable conductive pipette tips. Samples were aspirated from a Lobind and injected through a nanoESI chip (consisting of 400 nozzles in a 20x20 array) into the mass spectrometer. The spray voltage was 1.70 kV, the ejection pressure was 0.50 psi, and the ionization was NanoESI, positive. All peptides were obtained with purity greater than 95%.

[0128] General Methods for Antibody Conditioning: All monoclonal antibody experiments were performed in microcentrifuge tubes (1.5, 2, or 5 mL) at room temperature unless otherwise noted. All buffer solutions were prepared with MilliQ water. Borate-buffered saline (BBS) refers to 50 mM sodium borate, 50 mM NaCl, and 5 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.5. Phosphate-buffered saline (PBS) refers to 10 mM sodium phosphate, 137 mM sodium chloride, and 2.7 mM potassium chloride at pH 7.4. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution, 10 mM (2.87 mg mL), was prepared in BBS immediately before use.

[0129] Concentration was performed using Amicon Ultra-15 low-binding cellulose filters with a 10 kDa MWCO. Centrifugation was performed in a Beckman Coulter Allegra 21K centrifuge operating at 3500 rcf at 4°C.

[0130] The following acronyms are used to describe antibody fragments based on their constituent heavy and light chains: heavy-heavy-light (HHL), heavy-heavy (HH), heavy-light (HL), heavy (H) C ), and light chains (L C ).

[0131] General method for antibody characterization: The LC-MS system settings were as follows: 8 μL of sample was automatically injected onto a BioSuite pPhenyl 1000 (Waters, 10 μm RPC 2.0 x 75 mm) column at a flow rate of 100 μL / min using an Acquity UPLC system (Waters Corporation) equipped 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-PremierXE mass spectrometer (TOF). The capillary and cone voltages were set to 3000 V and 100 V, respectively. The desolvation and source temperatures were set to 350 °C and 120 °C, respectively. The cone gas flow rate and desolvation gas flow rate were set at 50 L / h and 600 L / h, respectively. The mass spectrometer was operated in positive polarity mode to acquire a full MS scan (400–4000 m / z).

[0132] Data were acquired using MassLynx software, V4.1.SCN704 (Waters Inc.). MS spectra corresponding to chromatographic peaks were summed. Charged protein species in the resulting spectra were deconvoluted to their zero-charged average mass using an integrated MaxEnt1 (maximum entropy) algorithm.

[0133] The output parameters were as follows: mass range 5000-70000, resolution 1 Da / channel. A uniform Gaussian model was used with the corresponding peak width at half height.

[0134] Example 1: Preparation of (3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (L1, DBM): [ka]

[0135] DBM ((3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid) was prepared as described in Mol. Pharm. 12, 3986-3998 (2015). Briefly, 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 minutes until all 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). The pure fractions were concentrated to give 1.08 g (3.4 mmol, 89% yield) of the dibromomaleimide derivative, 2-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid. 1 H NMR(400MHz,CH3OD):δ4.32(s,2H). 13 C NMR(101MHz,CH3OD):δ170,164,129,40.m / z:309.81,311.84,313.87[MH] - .

[0136] 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 the Dap side chain amino group and the Asp side chain carboxylic acid. [Table 6]

[0137] For the manual coupling of the first protected amino acid to the resin, coupling method 4 was applied using Fmoc-Asp(OAl)-OH (118.5 mg). Subsequent amino acids were coupled sequentially using coupling method 4 as follows:

[0138] 46 μL of DIC and 43 mg of Oxyma in DMF / DCM (1:1) were used. The mixture was allowed to react for 45 minutes with intermittent manual stirring. After each coupling, the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was removed with 20% (v / v) piperidine in DMF using a 30-second treatment followed by two 10-minute treatments. Two additional treatments with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) (2 x 5 minutes) were performed to ensure removal of the Fmoc group from the secondary amine (proline). Cyclization was performed on the resin according to Cyclization Method 2. The Fmoc group was removed with 20% (v / v) piperidine in DMF using a 30-second treatment followed by two 10-minute treatments. The N-terminal amine was protected with a Boc protecting group using BocO (3 equiv., 1000 μmol, 56 mg) and DIEA (30 equiv., 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 10 μM, 12 mg), phenylsilane (10 equiv., 1000 μmol, 123 mg) in DCM (3 × 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 × 5 min). Coupling of the amino group of Dap with the carboxylate group of aspartic acid was then achieved by adding PyBOP (4 equiv., 400 μmol, 208 mg), HOAt (12 equiv., 1.2 mmol, 163 mg), DMF (1–3 mL / g resin), and DIEA (12 equiv., 1.2 mmol, 204 μL). The coupling was allowed to stand for 1.5 h and then repeated overnight.

[0139] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equiv., 200 μmol, 108.53 mg) was achieved with 4 equiv. of DIC (400 μmol, 61 μL) and 4 equiv. of HOBt (400 μmol, 54 mg) in DCM for 2 h. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was then removed with 20% (v / v) piperidine in DMF using 30 s treatments followed by 10 min treatments.

[0140] Example 3: Preparation of DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH (DBM-TTDS-SEQ ID NO: 1) with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid. Starting with 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 the conjugate DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1).

[0141] DBM (4 equiv., 400 μmol, 125 mg), PyBOP (4 equiv., 400 μmol, 208 mg), and HOAt (12 equiv., 1.2 mmol, 163 mg) in DMF (1-3 mL / g resin) were added sequentially to the resin, followed by 12 equiv. of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 hours with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 seconds) and DCM (5 x 30 seconds). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay. Molecular formula: C 59 H 94 Br2N 14 O 22. Cal MW(Da):1508.5034. Observed MW(Da):1508.4992.t R UPLC (min): 1.374. Purity: >95%. Yield: 5%.

[0142] Comparative Example 1: Preparation of NH2-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-NH2 (NH2-TTDS-SEQ ID NO: 15) For the manual coupling of the first protected amino acid to the resin, coupling method 4 was applied using Fmoc-L-Tyr(tBu)-OH (137.7 mg). Subsequent amino acids were coupled sequentially using coupling method 4 as follows: [Table 7]

[0143] 46 μL of DIC and 43 mg of Oxyma in DMF / DCM (1:1) were used. The mixture was allowed to react for 45 minutes with intermittent manual stirring. After each coupling, the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was removed with 20% (v / v) piperidine in DMF using a 30-second treatment followed by two 10-minute treatments. Two additional treatments with DBU, toluene, piperidine, and DMF (5%, 5%, 20%, 70%) (2 x 5 minutes) were performed to ensure removal of the Fmoc group from the secondary amine (proline). Cyclization was performed on the resin according to Cyclization Method 2. The Fmoc group was removed with a 20% solution (v / v) of piperidine in DMF using a 30-second treatment followed by two 10-minute treatments. The N-terminal amine was protected with a Boc protecting group using BocO (3 equiv., 1000 μmol, 56 mg) and DIEA (30 equiv., 3000 μmol, 240 μL). The OAl and Alloc groups were first deprotected by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equiv., 10 μM, 12 mg), phenylsilane (10 equiv., 1000 μmol, 123 mg) in DCM (3 × 15 min). The resin was washed with 0.02 M sodium diethylcarbamate in DCM (3 × 5 min). Coupling of the amino group of Dap with the carboxylate group of aspartic acid was then achieved by adding PyBOP (4 equiv., 400 μmol, 208 mg), HOAt (12 equiv., 1.2 mmol, 163 mg), DMF (1–3 mL / g resin), and DIEA (12 equiv., 1.2 mmol, 204 μL). The coupling was allowed to stand for 1.5 h and then repeated overnight.

[0144] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 equiv., 200 μmol, 108.53 mg) was achieved with 4 equiv. DIC (400 μmol, 61 μL) and 4 equiv. HOBt (400 μmol, 54 mg) in DCM for 2 h. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group was then removed with 20% (v / v) piperidine in DMF using a 30 s treatment followed by two 10 min treatments.

[0145] Comparative Example 2: Preparation of DBM-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr2 (DBM-TTDS-SEQ ID NO: 15) Starting with 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 2 method was used to achieve the 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).

[0146] DBM (4 equiv., 400 μmol, 125 mg), PyBOP (4 equiv., 400 μmol, 208 mg), and HOAt (12 equiv., 1.2 mmol, 163 mg) in DMF (1-3 mL / g resin) were added sequentially to the resin, followed by 12 equiv. of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 hours with intermittent manual stirring. The solvent was removed by aspiration, and the resin was washed with DMF (5 x 30 seconds) and DCM (5 x 30 seconds). The coupling was repeated under the same conditions. The extent of coupling was monitored using the Kaiser colorimetric assay. Molecular formula: C 124 H 176 Br2N 32 O39. Cal MW(Da):2895.1139. Observed MW(Da):2895.1296.t R UPLC (min): 1.415. Purity: >95%. Yield: 10%.

[0147] Example 4. Preparation of trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH (Tz-DBM-TTDS-SEQ ID NO: 1) with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid. Starting with DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in Example 3, trastuzumab after general methods for monoclonal antibody conditioning was conjugated using the following protocol to achieve a compound of formula Tz-DBM-TTDS-SEQ ID NO: 1.

[0148] Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL of sterile water, and completely buffer exchanged into BBS pH 8.5 using a PD10g25 column (GE Healthcare), following common methods for monoclonal antibody conditioning. Concentration was determined by UV / Vis absorbance (using ε = 215380 M cm for trastuzumab mAb), and the protein was stored in flash-frozen aliquots at -20°C. For experiments, aliquots were thawed and used immediately.

[0149] The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4). Briefly, trastuzumab (111 μM, 4.9 mL, 544 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 μM. A fresh solution of TCEP (10 mM, 332.2 μL, 3.26 μmol, 6 equiv.) was added, and the reaction was incubated at 37°C for 2 hours with gentle agitation. TCEP was removed by SEC using a PD10G25 column with BBS as the buffer according to the manufacturer's instructions. Next, DBM peptide (Example 3) (10 mM, 247 μL, 4.35 μmol, 8 equiv.) in dry DMF was added to the reduced trastuzumab, and the reaction was left at room temperature for 30 minutes. Excess reagent was then removed by SEC using a PD10 G25 column with PBS. The final conjugate was characterized by LC-MS to confirm the integrity of the antibody after peptide conjugation.

[0150] Comparative Example 3. Preparation of Trastuzumab-DBM-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-NH2 (Tz-DBM-TTDS-SEQ ID NO: 15) Starting with DBM-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-NH (DBM-TTDS-SEQ ID NO: 15) prepared as in Comparative Example 2, trastuzumab after general methods for monoclonal antibody conditioning was conjugated using the following protocol to achieve a compound of formula Tz-DBM-TTDS-SEQ ID NO: 15.

[0151] Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL of sterile water, and completely buffer exchanged into BBS pH 8.5 using a PD10g25 column (GE Healthcare), following common methods for monoclonal antibody conditioning. Concentration was determined by UV / Vis absorbance (using ε = 215380 M cm for trastuzumab mAb), and the protein was stored in flash-frozen aliquots at -20°C. For experiments, aliquots were thawed and used immediately.

[0152] The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) Briefly, trastuzumab (111 μM, 4.9 mL, 544 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 μM. A fresh solution of TCEP (10 mM, 332.2 μL, 3.26 μmol, 6 equiv.) was added, and the reaction was incubated at 37 °C for 2 h with gentle agitation. TCEP was removed by SEC using a PD10G25 column with BBS as the buffer according to the manufacturer's instructions. Next, DBM peptide (Comparative Example 2) (10 mM, 247 μL, 4.35 μmol, 8 equiv.) in dry DMF was added to the reduced trastuzumab, and the reaction was left at room temperature for 30 min. Excess reagent was then removed by SEC using a PD10 G25 column with PBS. The final conjugate was characterized by LC-MS to confirm the integrity of the antibody after peptide conjugation.

[0153] Example 5. Preparation of cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH (Cx-DBM-TTDS-SEQ ID NO: 1) bearing an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid. Starting with DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in Example 3, cetuximab after general methods for monoclonal antibody conditioning was conjugated using the following protocol to achieve a compound of formula Cx-DBM-TTDS-SEQ ID NO: 1.

[0154] Cetuximab was obtained in its clinical form (SelleckChem, lyophilized), resuspended in sterile water, and completely buffer exchanged into BBS pH 8.5 using a PD10g25 column (GE Healthcare), following common methods for monoclonal antibody conditioning. Concentration was determined by UV / Vis absorbance (using ε = 215380 M cm for cetuximab mAb), and the protein was stored in flash-frozen aliquots at -20°C. For experiments, aliquots were thawed and used immediately.

[0155] The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017) (4). Briefly, cetuximab (1 mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 μM. A fresh solution of DTT (10 mM, 4 μL, 2.64 μmol, 6 equivalents) was added, and the reaction was incubated at 37°C for 2 hours with gentle agitation. DTT was removed by SEC using a PD10 G25 column with BBS as the buffer according to the manufacturer's instructions. Next, DBM peptide (Example 3) (10 mM, 5.3 μL, 3.52 μmol, 8 equivalents) in dry DMF was added to the reduced cetuximab, and the reaction was left at room temperature for 30 minutes. Excess reagent was then removed by SEC using a PD10 G25 column with PBS. The final conjugates were characterized by LC-MS to confirm the integrity of the antibody after peptide conjugation. The mass characterization of cetuximab and cetuximab-DBM-TTDS-SEQ ID NO: 1 after PNGaseF deglycosylation was 148182 and 153654, respectively.

[0156] Example 6. Preparation of bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH (Bv-DBM-TTDS-SEQ ID NO: 1) with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid. Starting with DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in Example 3, bevacizumab after general methods for monoclonal antibody conditioning was conjugated using the following protocol to achieve a compound of formula Bv-DBM-TTDS-SEQ ID NO: 1.

[0157] Bevacizumab was obtained in its clinical form (HSJD, lyophilized), resuspended in sterile water, and completely buffer exchanged into BBS pH 8.5 on a PD10g25 column (GE Healthcare) according to common methods for monoclonal antibody conditioning. The concentration was determined by UV / Vis absorbance (ε = 215380 M for bevacizumab mAb). -1 cm -1 Protein was measured by ELISA (using a ELISA kit) and stored in flash-frozen aliquots at −20° C. For experiments, aliquots were thawed and used immediately.

[0158] The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4). Briefly, bevacizumab (1 mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 μM. A fresh solution of DTT (10 mM, 4 μL, 2.64 μmol, 6 equivalents) was added, and the reaction was incubated at 37 °C for 2 hours with gentle agitation. DTT was removed by SEC using a PD10 G25 column with BBS as the buffer according to the manufacturer's instructions. Next, DBM peptide (Example 3) (10 mM, 5.3 μL, 3.52 μmol, 8 equivalents) in dry DMF was added to the reduced bevacizumab, and the reaction was left at room temperature for 30 minutes. Excess reagent was then removed by SEC using a PD10 G25 column with PBS. The final conjugates were characterized by LC-MS to confirm the integrity of the antibody after peptide conjugation. The mass characterization of bevacizumab and bevacizumab-DBM-TTDS-SEQ ID NO: 1 after PNGase F deglycosylation was 146322 and 151794, respectively.

[0159] Example 7. Preparation of pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH (Pt-DBM-TTDS-SEQ ID NO: 1) bearing an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid. Starting with DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH (DBM-TTDS-SEQ ID NO: 1) prepared as in Example 3, Pertuzumab after general methods for monoclonal antibody conditioning was conjugated using the following protocol to achieve a compound of formula Pt-DBM-TTDS-SEQ ID NO: 1.

[0160] Pertuzumab was obtained in its clinical form (SelleckChem, lyophilized), resuspended in sterile water, and completely buffer exchanged into BBS pH 8.5 using a PD10g25 column (GE Healthcare), following common methods for monoclonal antibody conditioning. Concentration was determined by UV / Vis absorbance (using ε = 215380 M cm for Pertuzumab mAb), and the protein was stored in flash-frozen aliquots at -20°C. For experiments, aliquots were thawed and used immediately.

[0161] The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4). Briefly, pertuzumab (1 mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 μM. A fresh solution of DTT (10 mM, 4 μL, 2.64 μmol, 6 equivalents) was added, and the reaction was incubated at 37°C for 2 hours with gentle agitation. DTT was removed by SEC using a PD10 G25 column with BBS as the buffer according to the manufacturer's instructions. Next, DBM peptide (Example 3) (10 mM, 5.3 μL, 3.52 μmol, 8 equivalents) in dry DMF was added to the reduced pertuzumab, and the reaction was left at room temperature for 30 minutes. Excess reagent was then removed by SEC using a PD10 G25 column with PBS. The final conjugates were characterized by LC-MS to confirm the integrity of the antibody after peptide conjugation. The mass characterization of Pertuzumab and Pertuzumab-DBM-TTDS-SEQ ID NO: 1 after PNGase F deglycosylation was 145214 and 150686, respectively.

[0162] Example 8: Stability of the shuttle in mouse serum. One of the main advantages of the shuttles of the present invention is that, unlike the majority of peptides composed only of L amino acids (which are rapidly metabolized by a series of enzymes present in the blood serum, thus limiting their therapeutic effect), they are made of D amino acids and therefore are not recognized by metabolic enzymes present in the serum, thereby significantly increasing their half-life in serum.

[0163] For mouse serum stability studies of the BBB shuttle peptides present in Example 4 and Comparative Example 3, they were incubated at a concentration of 150 μM in HBSS buffer at 37° C. in the presence of 90% mouse serum. At various time points, 50 μL aliquots were taken, to which methanol was added to precipitate serum proteins. The samples were centrifuged, filtered, and analyzed by HPLC to determine the extent of degradation. Figure 7 shows the mouse serum stability studies of the BBB shuttle peptides present in Example 4 and Comparative Example 3.

[0164] 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 DMEM supplemented with FBS. Cells in suspension were washed with ice-cold PBS, counted, and aliquoted into individual 1.5 mL tubes (10 cells). 6 pieces / tube).

[0165] 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 at increasing concentrations in ice-cold PBS for 30 minutes at 4°C. Cells were then washed and incubated with anti-human Dylight650 secondary antibody (Abcamplc) 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). Figure 8 shows the binding of Tz, 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.

[0166] 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 the flask with trypsin, which was neutralized with DMEM supplemented with FBS. Cells in suspension were washed with ice-cold PBS, counted, and aliquoted into individual 1.5 mL tubes (10 cells). 6 pieces / tube).

[0167] Binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) was performed at increasing concentrations in ice-cold PBS for 30 minutes at 4°C. Cells were then washed and incubated with anti-human Dylight488 secondary antibody (Abcamplc) 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). Figure 9 shows the binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to EGFR-positive MDA-MB-231 breast cancer cells.

[0168] 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 DMEM supplemented with FBS. Cells in suspension were washed with ice-cold PBS, counted, and aliquoted into individual 1.5 mL tubes (10 cells). 6 pieces / tube).

[0169] Binding of Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) was performed at increasing concentrations in ice-cold PBS for 30 minutes at 4°C. Cells were then washed and incubated with anti-human Dylight488 secondary antibody (Abcamplc) 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). Figure 10 shows the binding of Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to HER-2-overexpressing cells.

[0170] Example 12: Cell cycle arrest of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) in breast cancer cells. Cells were grown in monolayers in 12-well plates to 50% confluence and serum-starved overnight. Cells were then treated with PG (100 nM) and / or Tz (10 μg / ml). 24 hours after stimulation, cells were trypsinized, washed twice with ice-cold PBS, fixed in 70% ethanol at -20°C for 15 minutes, resuspended in RNase A 1 mg / ml (EUR X Ltd., Gdansk, Poland), and stained with propidium iodide (2.5 μg / ml). Cell cycle analysis was performed using a BD LSR II flow cytometer (BD Biosciences).

[0171] Cell cycle arrest analysis of cells treated with Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4), and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3). 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 the cell cycle was analyzed by flow cytometry. The results are shown in Figure 11.

[0172] Example 13. Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) 125 I labeling and quantification Pierce™ iodinated beads (Life Technologies) were used to radiolabel 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 μL of reaction buffer (50 mM NaPi, pH 6.5) and dried on filter paper. In a glass vial, the beads were added with a calculated amount of carrier-free Na in 200 μL of reaction buffer. 125 I (1 mCi / mg protein) was added. The reaction was incubated for 5 minutes. Protein was then added, and the reaction was allowed to proceed for 15 minutes 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 Healthcare) pre-equilibrated with PBS. The iodinated protein was dialyzed overnight against PBS (Slide-A-Lyzer® mini dialysis device, 20 KDa, 0.5 mL) to remove unincorporated iodized protein. 125 I was further removed. The radioactivity of 10 μL fractions was measured using a Packard Cobra II gamma counter for 2 min, and protein concentrations were determined using BCA analysis (Thermo Scientific). Samples were diluted with Ringer Hepes to a final concentration of 100 nM.

[0173] Example 14. AlexaFluor 488-NHS Labeling and Quantitation 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) Briefly, 0.2 μL of AlexaFluor 488 (10 mg mL in DMSO) was added. -1 Two aliquots of 100 μL of the selected mAb (0.25 mg, 82 nmol, NaPi pH 8) were added every 15 min, and the sample was mixed for 1 h in the dark. Excess dye was removed by SEC using a PDmini G25 column with PBS as the buffer according to the manufacturer's instructions.

[0174] Example 15. Permeability assay in an in vitro human BBB cell model These experiments were performed using a model developed in Professor R. Cecchelli's laboratory (5). Briefly, pluripotent stem cell-derived endothelial cells and bovine pericytes were thawed 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 hours, endothelial cells were seeded (8,000 cells / well) into 12-well transwell inserts, and pericytes were seeded (50,000 cells / well) into 12-well plates precoated with Matrigel and gelatin, respectively. sECM medium was used for both cell lines and was changed every 2–3 days. Assays were performed 7–8 days after seeding by placing the endothelial cell-containing inserts into new wells without pericytes.

[0175] To perform the assay, 500 μL of unlabeled or 125I-labeled mAb (5 μM or 100 nM, respectively) was added to the donor compartment, and 1500 μL of ECM medium or Ringer HEPES was introduced into the acceptor compartment. Lucifer Yellow (25 μM) was added to monitor barrier integrity (Papp < ​​15 10 -6 For unlabeled compounds, plates were incubated for 16 hours, but after 2 hours 500 μL of the acceptor compartment was removed and replaced with fresh medium for analysis. 125 For I-labeled mAbs, plates were incubated at 37°C for 2 hours, and solutions from both compartments were collected and analyzed. For AlexaFluor488-labeled mAbs, plates were incubated at 37°C for 2 hours, and solutions from both compartments were collected and analyzed by fluorescence. Samples were evaluated in triplicate. The amount of protein was quantified using a gamma counter, and the apparent permeability was calculated using the following formula:

number

[0176] For MS analysis, proteins from the acceptor compartment were purified by immunoprecipitation with protein A magnetic beads according to the manufacturer's instructions. Briefly, 25 μL of beads were placed in a 1.5 microcentrifuge tube, diluted with PBST, and gently mixed. The tube was placed in a magnetic stand to facilitate supernatant removal. 500 μL of PBS solution was added to the tube to wash the beads. After mixing, the solution was removed and the beads were then collected using the magnetic stand. This procedure was repeated three times. 1 mL of acceptor solution was added and left to mix with the beads overnight at 4°C. The supernatant was then discarded, and the beads were washed (3 x 500 μL PBST and 3 x 500 μL PBS). The mAb was eluted with 50 μL of 0.1 M glycine pH 2, and the solution was neutralized with 8 μL of 3 M Tris pH 8.5. The sample was analyzed by LCT-MS. Figure 12 shows the permeability results in a human in vitro BBB cell model for Tz, Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), and Tz-DBM-TTDS-SEQ ID NO:15 (Comparative Example 3). Figures 13, 14, and 15 show the permeability results in a human in vitro BBB cell model for Cx, Cx-DBM-TTDS-SEQ ID NO:1 (Example 5); Bv, Bv-DBM-TTDS-SEQ ID NO:1 (Example 6), and Pt, Pt-DBM-TTDS-SEQ ID NO:1 (Example 7), respectively. Figure 16 shows a comparison of the permeability of the conjugates.

[0177] Example 16. Biodistribution study 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 the ChemPartner animal facility according to protocols approved by the ChemPartner Laboratory Animal Care and Use Committee (IACUC) in accordance with the AAALAC guidelines. CD-1 male mice (6-8 weeks old) 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. Eight hours after injection, blood was collected for serum production, and brains were harvested terminally. Antibody levels in brain tissue and serum were determined by ELISA (goat anti-human IgG F(c) antibody, Sigma, catalog no. 609-101-017, anti-human IgG (Fab specific)-peroxidase, Sigma, catalog no. A0293). Figure 17 shows the biodistribution study in mice.

[0178] Example 17: Conjugation of DBM peptide to IgG1 mAb at pH 8.5 The conjugation protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). Briefly, the selected monoclonal antibody (mAb) (1 mg, 660 nmol) was diluted with BBS (pH 8.5) to a final concentration of 22.9 μM. Fresh DTT solution (10 mM, 4 μL, 2.64 μmol, 6 equiv.) was added, and the reaction was incubated at 37°C for 2 hours with gentle agitation. DTT was removed by SEC using a PDmini G25 column with BBS as the buffer according to the manufacturer's instructions. Next, DBM peptide (10 mM, 5.3 μL, 3.52 μmol, 8 equiv.) in dry DMF was added to the reduced mAb, and the reaction was left at room temperature for 30 minutes. Excess reagent was then removed by SEC using a PDmini G25 column with PBS. The final conjugate was characterized by LC-MS. DBM peptides used: DBM-TTDS-MiniAp4 ([M+H+] theoretical value: 1509,269 Da; [M+H+] experimental value: 1508,517 Da) [ka]

[0179] DBM-TTDS-SN38-MiniAp4 (M+H+) theoretical value: 2125,900 Da; [M+H+] experimental value: 2126,710 Da [ka] DBM-TDS-Ang2 ([M+H+] theoretical value: 2898,765 Da; [M+2H+ / 2] experimental value: 1449,571 Da) [ka] DBM-TTDS-SN38-Ang2 ([M+H+] theoretical value: 3516,380 Da; [M+2H+ / 2] experimental value: 1758,172 Da) [ka]

[0180] Example 18: Labeling of conjugates with AlexaFluor488-NHS All antibodies were labeled with AlexaFluor® 488 (λex: 485 / λem: 535 nm) to perform the transport assay. Antibodies were assayed at 500 nm and quantification was performed using a fluorimeter.

[0181] Briefly, 0.2 μL of AlexaFluor 488 (10 mg mL in DMSO) was added. -1 Two aliquots of 100 μL of the selected mAb (0.25 mg, 82 nmol, NaPi pH 8) were added every 15 min, and the sample was mixed for 1 h in the dark. Excess dye was removed by SEC using a PDmini G25 column with PBS as the buffer according to the manufacturer's instructions.

[0182] Example 19: Permeability assay in an in vitro human BBB cell model These experiments were performed using a model developed in Professor R. Cecchelli's laboratory. (5) Briefly, pluripotent stem cell-derived endothelial cells and bovine pericytes were thawed 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 hours, endothelial cells were seeded (8,000 cells / well) into 12-well transwell inserts, and pericytes were seeded (50,000 cells / well) into 12-well plates pre-coated with Matrigel and gelatin, respectively. sECM medium was used for both cell lines and was changed every 2–3 days. Assays were performed 7–8 days after seeding by placing the inserts containing endothelial cells into new wells without pericytes.

[0183] To perform the assay, 500 μL of fluorescent mAb (1 μM) in Ringer HEPES was added to the donor compartment, and 1500 μL of Ringer HEPES was introduced into the acceptor compartment. Lucifer Yellow (25 μM) was added to assess barrier integrity (Papp < ​​15 10 -6 The mAb was treated for 2 hours at 37°C, and the solution from both compartments was collected and the fluorescence (λ ex :485;λ em The proteins were analyzed by FTIR (535 nm). Samples were evaluated in triplicate. The amount of protein was quantified using a gamma counter and the apparent permeability was calculated using the following formula:

number

[0184] The results are shown in Figure 18. These results demonstrate that the permeability of the antibody modified according to the present invention (Cx-DBM_MiniAp4) is higher than that of the antibody modified with maleimide MiniAp4 (Cx-mal-MiniAp4) as in Example 23 of WO 2015 / 001015 A1.

[0185] Meanwhile, Figure 19 shows the Papp ratios of Cx modified with SN38-conjugated peptide shuttles (MiniAp4 or Ang2) and naked shuttles (MiniAp4 and Ang2) assayed at 1 μM in a human in vitro BBB cell model. These results indicate that the MiniAp4 conjugate increases SN38 transport more efficiently than the Ang2 conjugate.

[0186] Additional notes For reasons of completeness, various aspects of the invention are described in the following numbered clauses:

[0187] (Appendix 1) An antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof; [ka] It is inserted into the disulfide bond of the antibody in the form of -P-(W)sY and incorporated into the linker -[(L1)-(L2)-(L3) m- comprising 1 to 6 peptides of formula P linked to a sulfide via During the ceremony: Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof; The disulfide bond is any disulfide bond that can structurally retain its structure under reducing conditions and is selected from the group consisting of naturally occurring interchain disulfide bonds of antibodies, naturally occurring intrachain disulfide bonds of antibodies, and disulfide bonds introduced into antibodies by genetic engineering; L1 is L 1a and L 1bis a linker selected from the group consisting of: [ka] q is an integer from 1 to 6: L1 is attached to the -S- of the antibody disulfide bond via bonds a and b, and bond c is attached to linker L2 via an amide bond, ester bond, or thioester bond between the C=O group next to bond c of linker L1 and the NH, O, or S group on the left side of the drawing of LA under linker L2; L2 is a biradical consisting of 2 to 8 biradicals selected from the group consisting of LA, LB, and 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-(CH) 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) r’ NH2)-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) r -O-;-C(=O)-(CH2) r’ -S-;-NH-(CH2) r’ -O-;-NH-(CH2) r’ -NH-;-NH-(CH2) r’ -S-; and their combinations; L C is a biradical selected from the group consisting of: -NH-(CH) 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 via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LC; When u=1, LA is attached to the biradical LB through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LB; LB is attached to the biradical LC through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LB and the left functional group of formula LC; when u is greater than 1, LB are equal or different and are attached therebetween via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester; one LB end is attached to LA via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LB; and another LB end is attached to LC via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LB and the left functional group of formula LC; L3 is an amino acid selected from Lys, Orn, Dap, Dab; ​​Glu, and Asp: C(=O)-(CH2) r -C(=O)-;-C(=O)-(CH2) t -NH-;-C(=O)-(CH2) t -S-; an amino acid derivative selected from Lys, Orn, Dap, and Dab that is derivatized by attaching a biradical selected from the group consisting of -C(=O)-(CH2)tO- to the amino group of the side chain of the amino acid, wherein the attachment to the amino group is through the C=O terminal group on the left side of the biradical; an amino acid derivative selected from Glu and Asp that is derivatized by attaching a biradical selected from the group consisting of -NH-(CH2)tr-C(=O)-; -NH-(CH2) t -NH-;-NH-(CH2) t -S-;-NH-(CH2) t derivatized by attaching a biradical selected from the group consisting of -O- to a C=O group of the side chain of an amino acid, the attachment to the C=O group being via an NH group on the left side of the biradical; any of the aforementioned amino acids or amino acid derivatives further having CH2CH2NCH2CO2H4 (DOTA) or streptavidin attached by a viable 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 from biologically active substances, substances for use in diagnostic methods, and radioligands for radiotherapy; P is an equal or different peptide biradical selected from the group consisting of: (a) a peptide comprising the amino acid sequence X1KAPETALX2, having an intrapeptide bond between X1 and X2 that is an amide bond, wherein X1 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 NO:1: [ka] (b) a peptide having a length of 12 to 20 amino acid residues, having at least one intrapeptide bond which is a disulfide bond or a diselenide bond, and comprising an amino acid sequence of X3KAPETALX4AAA; a peptide having at least an intrapeptide disulfide bond or a diselenide bond between X3 and X4, wherein X3 and X4 are equal and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine); i.e., SEQ ID NO:2: [ka] (c) A peptide having a length of 9 to 11 amino acid residues and having at least one intrapeptide bond that is a disulfide bond or a diselenide bond, the peptide consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6, X5KAPETALX6A, and X5KAPETALX6AA, and having at least one intrapeptide disulfide bond or a diselenide bond between X5 and X6, wherein X5 and X6 are equal and selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), i.e., SEQ ID NO:3: [ka] SEQ ID NO:4: [ka] SEQ ID NO:5: [ka] (d) having 16 amino acid residues and the amino acid sequence X7NX8KAPETALX9AAAX 10 Including H, between X7 and X9, and between X8 and X 10 A peptide having an intrapeptide disulfide bond or diselenide bond between X7 to X 10 are independently selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine), provided that X7 and X9 are equal and X8 to X9 are equal. 10 is equal to the peptide; i.e., SEQ ID NO:6: [ka] and (e) a peptide comprising the amino acid sequence X1KAPETALX2, wherein X1 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), and is a linear peptide; W is -NH-(CH2) r -C(=O)-, and -NH-CH((CH2) r is a biradical selected from the group consisting of: (NH2)—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 attached directly to the LC of L2 via an amide bond formed between the C=O terminal group of LC and the amine group of the first amino acid in the peptide sequence P; When m=1, D is present and is attached to a functional group of the lateral chain of an amino acid of the linker L3 or to an amino acid derivative of the linker L3 via its derivatization, the attachment being via an amide, ester, disulfide, or thioester bond; L3 is attached to LC of L2 via an amide bond formed between the C=O terminal group of the left side of the formula LC and the amine group of the linker L3; P is attached directly to L3 via an amide bond formed between the C=O terminal group of the right side of the formula LC and the amine group of the first amino acid of the peptide sequence P; When s=0, P is directly attached to Y via an amide, carboxylic acid or ester bond, the bond being formed between the C=O at the C-terminus of the last amino acid of the sequence P and a radical Y that is -NH2, -OH, -OR3 or -NHR3; When s=1, P is attached to radical W through an amide bond formed with the C=O of the C-terminus of the last amino acid in sequence P, the bond being formed between the functional group on the left side of the drawn formula W and the C-terminus functional group (C=O) of the last amino acid in sequence P on the right side of the drawn formula; and W is attached to Y as follows: -C(=O)-NH-(CH2). r -C(=O)-Y, or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y; n is an integer independently selected from 1 to 6; [ka] indicates the attachment point; In formula (I), S represents a sulfide.

[0188] (Appendix 2) An antibody shuttle conjugate according to appendix 1, having four copies of peptide P, all of which are equal.

[0189] (Supplementary Item 3) An antibody shuttle conjugate according to Supplementary Item 1 or 2, wherein the disulfide bond is an interchain bond.

[0190] (Appendix 4) The antibody shuttle conjugate according to any one of Appendices 1 to 3, wherein P is a biradical of a peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence DapKAPETALD and having an intrapeptide bond between Dap and D which is an amide bond, i.e., SEQ ID NO: 8: [ka] (b) having a length of 9 to 20 amino acid residues and having at least one intrapeptide bond that is a disulfide bond; and A peptide comprising the amino acid sequence CKAPETALCAAA with at least one intrapeptide disulfide bond between cysteines 1 and 9, i.e., SEQ ID NO: 9: [ka] (c) a peptide having a length of 9 to 11 amino acid residues, having at least one intrapeptide disulfide bond that is a disulfide bond, and having at least one intrapeptide bond between cysteines 1 and 9, and consisting of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA, i.e. SEQ ID NO:10: [ka] SEQ ID NO: 11 [ka] SEQ ID NO: 12) [ka] and (d) A peptide having 16 amino acid residues, comprising the amino acid sequence CNCKAPETALCAAACH, and having intrapeptide disulfide bonds between the first and third cysteines (cysteines 1 and 11) and between the second and fourth cysteines (cysteines 3 and 15), i.e., SEQ ID NO: 13: [ka] (e) A peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

[0191] (Supplementary Item 5) The antibody shuttle conjugate of Supplementary Item 4, wherein P is a biradical of a peptide selected from the group consisting of: (a) a peptide having the amino acid sequence DapKAPETALD and having an intrapeptide bond that is an amide bond between Dap and D (SEQ ID NO: 7); (b) a peptide having the amino acid sequence CKAPETALC and having at least one intrapeptide disulfide bond between the cysteines at positions 1 and 9 (SEQ ID NO: 10); (c) A peptide having the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

[0192] (Supplementary Item 6) The antibody-shuttle conjugate according to any one of Supplementary Items 1 to 5, wherein the antibody-shuttle conjugate is selected from the group consisting of trastuzumab, bevacizumab, cetuximab, pertuzumab, aducanumab, bapineuzumab, nimotuzumab, and necitumumab.

[0193] (Supplementary Item 7) The antibody shuttle conjugate of Supplementary Item 1, 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) having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid; cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Cx- bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1) having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid (Bv-DBM-TTDS-SEQ ID NO: 1); pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-DBM-TTDS-SEQ ID NO: 1) having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid (Pt-DBM-TTDS-SEQ ID NO: 1), where DBM is a linker L 1a and TTDS is the linker L 2a is.

[0194] (Additional note 8) a) the antibody is an anti-cancer therapeutic antibody, m=1, and D is a radical of an anti-cancer active pharmaceutical ingredient selected from the group consisting of an auristatin, a duocarmycin, a PBD dimer, a maytansinoid, a calicheamicin, an anthracycline, a camptothecin, alpha-amanitin, a tubulysin, MMAE, T-DM1, and a PROTAC moiety; or b) the antibody is an anti-neurodegenerative therapeutic antibody, m=1, and D is the radical of an anti-neurodegenerative active pharmaceutical ingredient; An antibody shuttle conjugate according to appendix 1.

[0195] (Supplementary Item 9) A process for preparing an antibody shuttle conjugate as defined in any one of Supplementary Items 1 to 8, comprising: a) reducing disulfide bonds of the antibody; b) re-crosslinking disulfide bridges by reacting -SH groups of the antibody with the dibromomaleimide peptide of formula (II); [ka] c) optionally, carrying out hydrolysis; Including, wherein q, L2, L3, D, P, m, W, s, and Y are as defined in the antibody shuttle conjugate of formula (I).

[0196] (Appendix 10) A pharmaceutical composition comprising a therapeutically effective amount of an antibody shuttle conjugate as defined in any of Appendices 1 to 8 together with an appropriate amount of a pharmaceutically acceptable carrier or excipient.

[0197] (Appendix 11) An antibody shuttle conjugate as defined in any one of Appendices 1 to 8 for use as a pharmaceutical.

[0198] (Appendix 12) An antibody-shuttle conjugate as defined in any of Appendices 1 to 8 for use in treating a CNS disorder in a mammal, including a human.

[0199] (Appendix 13) An antibody shuttle conjugate for use according to appendix 12, wherein the CNS disorder is cancer.

[0200] (Appendix 14) An antibody shuttle conjugate for use according to appendix 12 or 13, which is for use in combination therapy with conventional chemotherapy or radiation therapy.

[0201] (Appendix 15) An antibody shuttle conjugate as defined in any one of Appendices 1 to 8, for use as a diagnostic agent or a radioligand for radiotherapy.

[0202] Reference List Patent documents International Publication No. 2015 / 001015(A1) 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 - P.L.Barker et al.J. Med.Chem., 1992. vol 35, pp. 2040-2048. -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 - M. Kazemi et al.,Journal of Sulfur Chemistry,2015, vol. 36:6,pp. 613-623. - M. T.Nguyen et al., J. Org.Chem.1998, 63, vol. 20, pp. 6878-6885. - F. David et al., Org.Process Res.Dev.2010, 14, 4, pp. 999-1007. - https: / / www.cliffsnotes.com / study-guides / chemistry / organic-chemistry-ii / aldehydes-and-ketones / reactions-of-aldehydes-and-ketones. - Eur J Pharm Biopharm, 2017, 115, 149-158or triphosgene as described inJ. Med.Chem.2008, 51, 21,6916-6926. - Dongen et al.;Bioconjugate Chem.2009, vol. 20, pp. 20-23. - CA2957354A1

Claims

1. An antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof; 【Chemical 1】 wherein -P-(W)sY is inserted into the disulfide bond of the antibody and a linker -[(L １ )-(L ２ )-(L ３ ) ｍ - 1 to 6 peptides of formula P linked to said sulfide via During the ceremony: Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof; the disulfide bond is any disulfide bond that is originally present in the antibody and that can structurally maintain the sulfide bond in the structure under reducing conditions; the disulfide bond is selected from the group consisting of a naturally occurring interchain disulfide bond of the antibody, a naturally occurring intrachain disulfide bond of the antibody, and a disulfide bond introduced into the antibody by genetic engineering; L １ Is, L １ａ and L １ｂ is a linker selected from the group consisting of: 【Chemistry 2】 q is an integer from 1 to 6: L １ is attached to the -S- of the disulfide bond of the antibody via bonds a and b, and bond c is attached to the linker L １ The C═O group adjacent to the bond c and the linker L ２ The linker L is connected to the NH, O, or S group on the left side of the drawing of LA below by an amide bond, an ester bond, or a thioester bond. ２ Attached to; L ２ is a biradical consisting of 2 to 8 biradicals selected from the group consisting of LA, LB, and LC and having the formula -LA-(LB) ｕ -LC-, LA is -NH-(CH ２ ) ｒ’ -C(=O)-, -S-(CH ２ ) ｒ’ -C(=O)-;-O-(CH ２ ) ｒ’ -C(=O)-;-NH-(CH ２ ) ｒ’ -;-S-(CH ２ ) ｒ’ -; -O-(CH ２ ) ｒ’ -; -NH-(CH ２ ) ｒ’ -O-;-NH-(CH ２ ) ｒ’ -NH- and -NH-(CH ２ ) ｒ’ is a biradical selected from the group consisting of: -S-; LB is -NH-(CH ２ ) ｒ’ -C(=O)-;-C(=O)-(CH ２ ) ｒ’ -C(=O)-;-S-(CH ２ ) ｒ’ -C(=O)-;-O-(CH ２ ) ｒ’ -C(=O)-;-NH-(CH ２ ) ｒ’ -;-C(=O)-(CH ２ ) ｒ’ -;-S-(CH ２ ) ｒ’ -; -O-(CH ２ ) ｒ’ -;-NH-CH-((CH ２ ) ｒ’ NH ２ )-C(=O)-;-S-CH ２ -CH(NH ２ )-C(=O)-;-(CH ２ ) ｒ’ -C(=O)-;-(CH ２ ) ｒ’ -O-; -(CH ２ ) ｒ’ -NH-; -(CH ２ ) ｒ’ -S-;-C(=O)-(CH ２ ) ｒ’ -NH-;-C(=O)-(CH ２ ) ｒ -O-;-C(=O)-(CH ２ ) ｒ’ -S-;-NH-(CH ２ ) ｒ’ -O-;-NH-(CH ２ ) ｒ’ -NH-;-NH-(CH ２ ) ｒ’ -S-; and combinations thereof; LC is -NH-(CH ２ ) ｒ’ -C(=O)-;-NH-CH-((CH ２ ) ｒ’ -NH ２ )-C(=O)-;-C(=O)-(CH ２ ) ｒ’ -C(=O)-;-S-(CH ２ ) ｒ’ -C(=O)-;-S-CH ２ -CH(NH ２ )-C(=O)-;-O-(CH ２ ) ｒ’ -C(=O)-;-(CH ２ ) ｒ’ is a biradical selected from the group consisting of: —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 selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LC; When u=1, LA is attached to biradical LB through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LB; and LB is attached to biradical LC through a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LB and the left functional group of formula LC; When u is greater than 1, LB are equal or different and are attached between them via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester; one LB end is attached to LA via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LA and the left functional group of formula LB; and another LB end is attached to LC via a chemically feasible bond selected from the group consisting of amine, amide, ether, thioether, disulfide, ester, and thioester, the bond being formed between the right functional group of formula LB and the left functional group of formula LC; L ３ is an amino acid selected from Lys, Orn, Dap, Dab; ​​Glu, and Asp; an amino acid derivative selected from Lys, Orn, and Dap, and Dap is —C(═O)—(CH ２ )r-C(=O)-, -C(=O)-(CH ２ )t-NH-, -C(=O)-(CH ２ )t-S-, -C(=O)-(CH ２ )t-O-, wherein the attachment to the amino group is through the C=O terminal group on the left side of the biradical, and the amino acid derivative: -NH-(CH ２ )tr-C(=O)-;-NH-(CH ２ ) ｔ -NH-;-NH-(CH ２ ) ｔ -S-;-NH-(CH ２ ) ｔ an amino acid derivative selected from Glu and Asp that is derivatized by attaching a biradical selected from the group consisting of —O— to a C═O group on the side chain of the amino acid, the attachment to the C═O group being through the NH group on the left side of the biradical; and ２ CH ２ NCH ２ CO ２ H) ４ (DOTA) or any of the foregoing amino acids or amino acid derivatives to which streptavidin is further attached; t is an integer from 1 to 5; m is an integer selected from 0 or 1; D is a linker L selected from biologically active substances, substances for use in diagnostic methods; and radioligands for radiotherapy. ３ a substance to be attached to the P is an equal or different biradical of a single peptide, (a) amino acid sequence X １ KAPETALX ２ X, which is an amide bond １ and X ２ A peptide having an intrapeptide bond between: １ is selected from the group consisting of Dap (2,3-diaminopropionic acid) and Dab (2,4-diaminobutanoic acid), and X ２ is selected from the group consisting of D (aspartic acid) and E (glutamic acid); That is, SEQ ID NO: 1: 【Chemistry 3】 (b) having a length of 12 to 20 amino acid residues and at least one intrapeptide bond that is a disulfide bond or a diselenide bond, and X ３ KAPETALX ４ AAA; X ３ and X ４ A peptide having at least one intrapeptide disulfide or diselenide bond between X ３ and X ４ are equal and are selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine); i.e., SEQ ID NO:2: 【Chemistry 4】 (c) having a length of 9 to 11 amino acid residues and at least one intrapeptide bond that is a disulfide bond or a diselenide bond, and X ５ KAPETALX ６ , X ５ KAPETALX ６ A and X ５ KAPETALX ６ X consists of an amino acid sequence selected from the group consisting of AA ５ and X ６ and X has at least one intrapeptide disulfide bond or diselenide bond between them; ５ and X ６ are equal and are selected from the group consisting of C (cysteine), Sec (selenocysteine), Pen (penicillamine), i.e. SEQ ID NO:3: 【Chemistry 5】 SEQ ID NO: 4: 【Chemistry 6】 SEQ ID NO:5: 【Chemistry 7】 (d) having 16 amino acid residues and the amino acid sequence X ７ NX ８ KAPETALX ９ AAAX １０ Including H and X ７ and X ９ Between and X ８ and X １０ A peptide having an intrapeptide disulfide bond or diselenide bond between: ７ ~X １０ are independently selected from the group consisting of C (cysteine), Sec (selenocysteine), and Pen (penicillamine); ７ and X ９ are equal, and X ８ ~X １０ is equal to, peptide; That is, SEQ ID NO: 6: 【Chemistry 8】 and (e) amino acid sequence X １ KAPETALX ２ A peptide comprising X １ is selected from the group consisting of Dap and Dab; ​​and X ２ is a linear peptide selected from the group consisting of D (aspartic acid) and E (glutamic acid) (SEQ ID NO: 7); selected from the group consisting of: W is -NH-(CH ２ ) ｒ -C(=O)-, and -NH-CH((CH ２ ) ｒ NH ２ )—C(═O)—; r is an integer independently selected from 1 to 5; s is an integer independently selected from 0 to 1; Y is -NH ２ , —OH, —OR ３ , and -NHR ３ is a radical selected from the group consisting of: When m=0, L ３ and D is absent, and P is connected to L 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. ２ directly attached to the LC of When m=1, D is present and is a linker L ３ to the functional groups of the side chains of the amino acids of the linker L or via derivatization thereof ３ attached to an amino acid derivative of, wherein the attachment is via an amide, ester, disulfide, or thioester bond; ３ represents the C═O terminal group on the left side of formula LC and the linker L ３ through an amide bond formed between the amine groups of ２ and P is attached to L through an amide bond formed between the C=O terminal group on the right side of formula LC and the amine group of the first amino acid of the peptide sequence P. ３ directly attached to the When s=0, P is directly attached to Y via an amide, carboxylic acid or ester bond, said bond being connected to the C═O of the C-terminus of the last amino acid of said sequence P and to —NH ２ , —OH, —OR ３ , or -NHR ３ and When s=1, P is attached to the radical W through an amide bond formed with the C=O of the C-terminus of the last amino acid of said sequence P, said bond being formed between the functional group on the left side of the drawn formula W and the C-terminus functional group (C=O) of the last amino acid of said sequence P on the right side of the drawn sequence; and W is attached to Y as follows: -C(=O)-NH-(CH ２ ) ｒ -C(=O)-Y, or -C(=O)-NH-CH((CH ２ ) ｒ NH ２ )-C(=O)-Y; n is an integer independently selected from 1 to 6; 【Chemistry 9】 indicates the point of attachment; and S in formula (I) represents sulfide. An antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof:

2. The disulfide bond initially present in the antibody is structurally maintained in the structure under reduction conditions, and the sulfide group of the reduced disulfide bond of the antibody and the linker L are bonded to each other to incorporate a biradical between the sulfide groups of the antibody. １ 2. The antibody shuttle conjugate of claim 1, wherein new bridges can be formed between them by the reaction: 【Chemistry 10】

3. 3. The antibody shuttle conjugate of claim 1 or 2, having four copies of said peptide P, and all said peptides being equal.

4. The antibody shuttle conjugate of any one of claims 1 to 3, wherein the disulfide bond is an interchain bond.

5. P, (a) a peptide comprising the amino acid sequence DapKAPETALD and having an intrapeptide bond between Dap and D that is an amide bond, i.e., SEQ ID NO: 8: 【Chemistry 11】 (b) a peptide having a length of 9 to 20 amino acid residues, having at least one intrapeptide bond that is a disulfide bond, and comprising the amino acid sequence CKAPETALCAAA, and having at least one intrapeptide disulfide bond between cysteines 1 and 9, i.e., SEQ ID NO: 9: 【Chemistry 12】 (c) a peptide having a length of 9 to 11 amino acid residues, having at least one intrapeptide bond that is a disulfide bond, and consisting of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA, and having at least one intrapeptide disulfide bond between cysteines 1 and 9, i.e. SEQ ID NO: 10: 【Chemistry 13】 SEQ ID NO: 11 【Chemistry 14】 SEQ ID NO: 12) 【Chemistry 15】 and (d) a peptide having 16 amino acid residues and comprising the amino acid sequence CNCKAPETALCAAACH, with intrapeptide disulfide bonds between the first and third cysteines, i.e., cysteines 1 and 11, and between the second and fourth cysteines, i.e., cysteines 3 and 15; SEQ ID NO: 13: 【Chemistry 16】 (e) a peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14) 5. The antibody shuttle conjugate of claim 1, which is a biradical of a peptide selected from the group consisting of:

6. P, (a) a peptide having the amino acid sequence DapKAPETALD and an intrapeptide bond between Dap and D that is an amide bond (SEQ ID NO: 7); (b) a peptide having the amino acid sequence CKAPETALC and at least one intrapeptide disulfide bond between the cysteines at positions 1 and 9 (SEQ ID NO: 10); (c) a peptide having the amino acid sequence DapKAPETALD (SEQ ID NO: 14) 6. The antibody shuttle conjugate of claim 5, which is a biradical of a peptide selected from the group consisting of:

7. 6. The antibody shuttle conjugate of any one of claims 1 to 5, wherein the antibody is selected from the group consisting of trastuzumab, bevacizumab, cetuximab, pertuzumab, aducanumab, bapineuzumab, nimotuzumab, and necitumumab.

8. Trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid ２ (Tz-DBM-TTDS-SEQ ID NO: 1); Cetuximab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid ２ (Cx-DBM-TTDS-SEQ ID NO: 1), bevacizumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid ２ (Bv-DBM-TTDS-SEQ ID NO: 1); and Pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid. ２ (Pt-DBM-TTDS-SEQ ID NO: 1): wherein DBM is selected from the group consisting of a linker L １ａ and TTDS is a linker L ２ａ 2. The antibody shuttle conjugate of claim 1, wherein: 【Chemistry 17】 【Chemistry 18】

9. a) the antibody is an anti-cancer therapeutic antibody, m=1 and D is a radical of an anti-cancer active pharmaceutical ingredient selected from the group consisting of an auristatin, a duocarmycin, a PBD dimer, a maytansinoid, a calicheamicin, an anthracycline, a camptothecin, alpha-amanitin, a tubulysin, MMAE, T-DM1, and a PROTAC moiety; or b) the antibody is an anti-neurodegenerative therapeutic antibody, m=1, and D is a radical of an anti-neurodegenerative active pharmaceutical ingredient; The antibody shuttle conjugate of claim 1.

10. 2. The antibody shuttle conjugate of 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 one of claims 1 to 10, comprising: a) reducing disulfide bonds of the antibody; b) re-crosslinking disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide peptide of formula (II); 【Chemistry 19】 c) optionally, carrying out hydrolysis; Including, 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 of any one of claims 1 to 10 together with a suitable amount of a pharmaceutically acceptable carrier or excipient.

13. An antibody shuttle conjugate as defined in any one of claims 1 to 10 for use as a medicament.

14. 11. An antibody shuttle conjugate as defined in any one of claims 1 to 10 for use in the treatment of a CNS disorder in a mammal, including a human.

15. 15. The antibody-shuttle conjugate for use according to claim 14, wherein the CNS disorder is cancer.

16. 16. An antibody shuttle conjugate for use according to claim 14 or 15, for use in combination therapy with conventional chemotherapy or radiotherapy.

17. An antibody shuttle conjugate as defined in any one of claims 1 to 10 for use as a diagnostic agent or a radioligand for radiotherapy.

Images