Site-specific modification of BBB-shuttling agents by antibody-based entities for crossing blood-brain barrier

Abstract

Conjugates of antibodies that penetrate the blood-brain barrier and selected peptide shuttle agents, optionally also conjugated to active pharmaceutical ingredients, radiotherapy or diagnostic agents, pharmaceutical compositions comprising them, and conjugates for use in medicine and as diagnostic agents.

Description

[0001] This application claims the benefit of European patent application EP22382746.0 filed on August 1, 2022. Technical Field

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

[0003] Monoclonal antibodies (mAbs) have revolutionized the treatment of several diseases, especially in oncology. Antibody-based therapies target leukemias and solid tumors in many organs. However, brain tumors remain virtually intractable to treat with both biological therapeutics and most small molecules.

[0004] One of the major challenges in treating brain tumors is overcoming the blood-brain barrier (BBB) ​​and blood-tumor barrier (BTB) with therapeutic doses. Only 0.1% to 0.2% of peripherally injected mAbs reach the brain parenchyma. The BBB, composed of specialized endothelial cells, tightly junctioned and surrounded by astrocyte end-feet and pericytes, ensures brain insulation. Although in the core of the tumor, the BBB is replaced by the more leaky BTB, in the tumor margins and small brain metastases, the BBB may be intact and greatly hinder the entry of therapeutic agents.

[0005] Brain metastases (BM) are a major complication of several cancer types, particularly in 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 show a higher incidence of BM. Although the use of mAbs has proven to be highly effective in these breast cancer types, the use of mAbs to treat BM or primary brain tumors remains elusive, primarily due to lack of antibody penetration across the BBB and BTB.

[0006] Several approaches have been explored to increase brain penetration of antibodies. Approaches such as direct injection and temporary disruption of the blood-brain barrier may pose high risks to patients. For this reason, many efforts have been devoted to the development of ligands that can hijack endogenous transport mechanisms across the brain endothelium. These ligands are called BBB-shuttle agents and 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), disclosed that the introduction of Angiopep-2 (Ang2) into an anti-HER2 monoclonal antibody conferred the properties of increased brain endothelial cell uptake and BBB permeability. This introduction was performed through a linker and using a non-site-specific approach. However, limited effects of conjugated shuttle agents have been observed, which may be due to the low protease resistance of the peptides and the heterogeneous mixture obtained by randomly conjugating the peptides to surface lysines.

[0008] WO2015 / 001015A1 describes peptides specifically derived from apamin containing KAPETAL fragments, which can be used as shuttle agents because they have the ability to cross the BBB and can promote the transport of drugs or other diagnostically useful substances that cannot cross the BBB into the brain. The document discloses two peptide constructs with antibodies as cargo, Example 22 (cetuximab-V3-Ap3-NH2) and Example 23: (cetuximab-V2-Ap 5a -NH2), AP5 peptide corresponds to MiniAp4 peptide, a cyclic peptide mimetic derived from bee venom that shows high resistance to proteolysis and negligible toxicity and immunogenicity, V2 and V3 are as follows:

[0009]

[0010] In V3, Linker Bond 1 and Linker Bond 2 are attached to lysine side chains of the antibody. However, all previous methods based on lysine modification, such as those disclosed in previous literature, do not allow control over the position of the peptide when it is incorporated into a monoclonal antibody.

[0011] Finally, Macarena Sánchez Navarro et al.; in the abstract "Paving the way towards the brain delivery of biotherapeutics: Modification of proteins with blood-brain barrier peptide shuttles" ECBS / LS-EuCheMS madrid (Spain) proposed the possibility of connecting a controlled average number of peptides to different parts of a protein to increase its BBB penetration, in particular, it discloses the modification of GFP with MiniAp4 and with a branched form of THRre. However, there is no mention of where the peptides were introduced, how this connection was made, or the results regarding transport through the BBB or BTB.

[0012] Therefore, from what is known in the art, it can be concluded that there is still a need to address the long-standing challenge of increasing the transport of antibodies across the BBB for the treatment or diagnosis of brain tumors and other CNS diseases. Summary of the invention

[0013] The present inventors have developed conjugates of mAb antibodies and selected peptide shuttle agents that are linked to the antibody via a bromomaleimide linker at a specific site of the antibody and that can efficiently cross the blood-brain barrier.

[0014] Unlike the methods disclosed in the prior art (see WO2015 / 001015A1), the linker used in the present invention allows a peptide incorporation strategy in which the incorporation of the shuttle peptide into the monoclonal antibody is controlled and allows knowing where it is located. The strategy is based on the incorporation of the peptide by reducing the -SH group of the antibody. Advantageously, this strategy can be applied to any monoclonal antibody, providing a uniform conjugate through site-specific modification of the antibody, which is a very desirable feature for pharmacological products, improving the therapeutic index and facilitating the production and analysis of the product. In addition, the incorporation of selected peptide shuttle agents has been achieved by exploiting the higher reactivity of the cysteine ​​of the interchain disulfide bridge, overcoming the need for genetic engineering to introduce reactive tags and extending the applicability of this strategy to other mAbs.

[0015] As an example, the present inventors have prepared a homogenous antibody-BBB-shuttle agent conjugate of trastuzumab and four copies of MiniAp4 (Tz-MiniAp4). It is an FDA-approved antibody against HER2, which is widely used in the clinical treatment of breast cancer. Coupling is achieved by reducing the interchain disulfide bridge and re-bridging with dibromomaleimide, which enables a high degree of control of the number of peptides anchored to each antibody molecule. Relative to trastuzumab alone or relative to trastuzumab with 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, the conjugate shows enhanced trans-blood-brain barrier transport in an in vitro cell model.

[0016] Therefore, the first aspect of the present invention relates to an antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof,

[0017]

[0018] It contains 1 to 6 disulfide bonds inserted into the antibody in the form of -P-(W)sY and connected via a linker -[(L1)-(L2)-(L3) m - a peptide of formula P linked to a sulfide;

[0019] in:

[0020] Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof;

[0021] The disulfide bond is any disulfide bond originally present in the antibody and capable of structurally retaining a sulfide bond in the structure under reducing conditions, the disulfide bond being selected from the group consisting of the naturally occurring interchain disulfide bonds of the antibody, the naturally occurring intrachain disulfide bonds of the antibody, and the disulfide bonds introduced into the antibody by genetic engineering;

[0022] L1 is selected from L 1a and L 1b The connector;

[0023]

[0024] q is an integer from 1 to 6;

[0025] L1 is linked to -S- of the antibody disulfide bond via bonds a and b, and bond c is linked to linker L2 via an amide bond, ester bond, or thioester bond between the C=O group immediately adjacent to bond c of linker L1 and the NH group, O group, or S group on the left side of the drawn LA of linker L2 below;

[0026] L2 is a diradical containing 2 to 8 radicals selected from LA, LB, LC, and having the formula -LA-(LB) u -LC-.

[0027] LA is a diradical selected from the following: -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-;

[0028] LB is a diradical independently selected from the following: -NH-(CH2) r’ -C(=O)-; -C(=O)-(CH2) r’ -C(=O)-; -S-(CH2) r’ -C(=O)-; -O-(CH2) r’ -C(=O)-; -NH-(CH2) r’ -; -C(=O)-(CH2) r’ -;-S-(CH2) r’ -;-O-(CH2) r’ -;-NH-CH-((CH2) 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 combinations thereof;

[0029] LC is a diradical selected from the following: -NH-(CH2) r’-C(=O)-; -NH-CH-((CH2) r’ -NH2)-C(=O)-;-C(=O)-(CH2) r’ -C(=O)-; -S-(CH2) r’ -C(=O)-;-S-CH2-CH(NH2)-C(=O)-;-O-(CH2) r’ -C(=O)-; -(CH2) r’ -C(=O)-;

[0030] u is an integer from 0 to 6;

[0031] r' is an integer from 1 to 5;

[0032] When u=0, LA is linked to the diradical LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LA formula and the functional group on the left side of the LC formula;

[0033] When u=1, LA is linked to the diradical LB via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LA formula and the functional group on the left side of the LB formula; and LB is linked to the diradical LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LB formula and the functional group on the left side of the LC formula;

[0034] When u is greater than 1, LB are the same or different, and they are connected via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester; one end of LB is connected to LA via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, and the bond is formed between the functional group on the right side of the LA formula and the functional group on the left side of the LB formula; and the other end of LB is connected to LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, and the bond is formed between the functional group on the right side of the LB formula and the functional group on the left side of the LC formula;

[0035] L3 is a diradical selected from the following: an amino acid selected from Lys, Orn, Dap, Dab; ​​Glu, and Asp; selected from -C(=O)-(CH2) r -C(=O)-; -C(=O)-(CH2) t -NH-; -C(=O)-(CH2) t -S-; -C(=O)-(CH2) tAmino acid derivatives of Lys, Orn, Dap and Dab derived from a diradical of -O- to an amino group of an amino acid side chain, wherein the diradical is connected to the amino group through a C=O terminal group on the left side of the diradical; selected from the group consisting of -NH-(CH2)t r -C(=O)-; -NH-(CH2) t -NH-; -NH-(CH2) t -S-; -NH-(CH2) t Amino acid derivatives of Glu and Asp derived from the diradical of -O- linked to the C=O group of the amino acid side chain, wherein the diradical is linked to the C=O group via the NH group on the left side of the diradical; and any previous amino acid or amino acid derivative of CH2CH2NCH2CO2H)4(DOTA) or streptavidin is also linked via a feasible bond;

[0036] t is an integer from 1 to 5;

[0037] m is an integer selected from 0 or 1;

[0038] D is a substance connected to the linker L3, which is selected from a biologically active substance, a substance used in a diagnostic method; and a radioligand used in radiotherapy;

[0039] P is a single peptide diradical which is the same or different and is selected from: (a) a peptide comprising the amino acid sequence X1KAPETALX2 having an intrapeptide bond between X1 and X2 which is an amide bond; wherein X1 is selected from Dap (2,3-diaminopropionic acid) and Dab (2,4-diaminobutyric acid); and X2 is selected from D (aspartic acid) and E (glutamic acid); i.e.

[0040]

[0041] (b) a peptide of 12 to 20 amino acid residues in length, having at least an intrapeptide bond that is a disulfide bond or a diselenide bond, and comprising the following amino acid sequence: X3KAPETALX4AAA; having at least an intrapeptide disulfide bond or an intrapeptide diselenide bond between X3 and X4, wherein X3 and X4 are identical and are selected from C (cysteine), Sec (selenocysteine) and Pen (penicillamine); i.e.

[0042]

[0043] (c) a peptide of 9 to 11 amino acid residues in length having an intrapeptide bond that is at least a disulfide bond or a diselenide bond, and consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6; X5KAPETALX6A; and X5KAPETALX6AA having at least an intrapeptide disulfide bond or an intrapeptide diselenide bond between X5 and X6; wherein X5 and X6 are identical and are selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine), i.e.

[0044]

[0045] (d) having 16 amino acid residues and comprising the amino acid sequence X7NX8KAPETALX9AAAX 10 H peptide, which is between X7 and X9 and between X8 and X 10 There is an intra-peptide disulfide bond or an intra-peptide diselenide bond between them; wherein X7 to X 10 are independently selected from C (cysteine), Sec (selenocysteine) and Pen (penicillamine); provided that X7 and X9 are the same, X8 and X 10 the same; that is

[0046]

[0047] and (e) a peptide comprising the amino acid sequence X1KAPETALX2, wherein X1 is selected from Dap and Dab; ​​and X2 is selected from a linear peptide of D (aspartic acid) and E (glutamic acid) (SEQ ID NO: 7);

[0048] W is selected from -NH-(CH2) r -C(=O)- and -NH-CH((CH2) r NH2)-C(=O)- diradical;

[0049] r is an integer independently selected from 1 to 5;

[0050] s is an integer independently selected from 0 to 1;

[0051] Y is a free radical selected from -NH2, -OH, -OR3 and -NHR3;

[0052] When m=0, L3 and D are absent, and P is directly linked to 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 of the peptide sequence P;

[0053] When m=1, D is present and connected to a functional group of an amino acid side chain of linker L3, or to an amino acid derivative of linker L3, by derivatization thereof, wherein the connection is made by an amide, ester, disulfide bond or thioester bond; L3 is connected to the LC of L2 by an amide bond formed between the C=O terminal group on the left side of the LC formula and the amine group of linker L3; and P is directly connected to L3 by an amide bond formed between the C=O terminal group on the right side of the LC formula and the amine group of the first amino acid of the peptide sequence P; and

[0054] When s=0, P is directly linked to Y via an amide bond, a carboxylic acid bond or an 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 free radical Y, the free radical Y being -NH2, -OH, -OR3 or -NHR3; and

[0055] When s=1, P is linked to the free radical W through an amide bond formed between the free radical W and the C=O at the C-terminus of the last amino acid of the sequence P, the bond being formed between the functional group on the left side of the drawn W formula and the functional group (C=O) at the C-terminus of the last amino acid of the sequence P on the right side of the drawn sequence; and W is linked to Y as follows: -C(=O)-NH-(CH2) r- C(=O)-Y, or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y;

[0056] n is an integer independently selected from 1 to 6;

[0057] Represents a connection point; and

[0058] S represents sulfide.

[0059] A second aspect of the present invention relates to a method for preparing an antibody shuttle agent conjugate as defined above, comprising: a) reducing the disulfide bridges of the antibody; b) re-bridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), and c) optionally, performing hydrolysis;

[0060] wherein q; L2, L3, D, P, m, W, s and Y are as defined in the antibody shuttle agent conjugate of formula (I);

[0061]

[0062] The third aspect of the present invention relates to a pharmaceutical composition, which comprises a therapeutically effective amount of the antibody shuttle agent conjugate of the present invention, and an appropriate amount of a pharmaceutically acceptable carrier or excipient.

[0063] A fourth aspect of the present invention relates to the antibody shuttle agent conjugate of the present invention as defined above for use as a medicament.

[0064] A fifth aspect of the present invention relates to the antibody shuttle agent conjugate of the present invention as defined above, for use in treating a central nervous system (CNS) disorder in a mammal including a human.

[0065] A sixth aspect of the present invention relates to the antibody shuttle agent conjugate of the present invention as defined above for use as a diagnostic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 :DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid) 1 H NMR. Example 1.

[0067] Figure 2 :DBM (3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid) 13 C NMR. Example 1.

[0068] Figure 3 : UPLC traces and MS spectra of Comparative Example 2 and Example 3. UPLC chromatograms were recorded at 220 nm with a 2 min linear gradient from 0% to 100% MeCN (0.036% TFA) in H2O (0.045% TFA).

[0069] 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 generated and characterized. a) ASC synthesis scheme; b) Quality characterization of trastuzumab-DBM-TTDS-SEQ ID NO: 15 (also known as Tz-Ang2, Comparative Example 3) (top) and trastuzumab-DBM-TTDS-SEQ ID NO: 1 (also known as Tz-MiniAp4, Example 4) (bottom) by LCT-Premier. Deconvoluted spectra are shown. For Tz-Ang2, M 计算值 =159244;M 发现值 :79537、159396;For Tz-MiniAp4,M 计算值 =153684;M 发现值: 76918, 153678; c) Coomassie-stained SDS-PAGE of trastuzumab conjugated to DBM-derived BBB-shuttle agent peptide, 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).

[0070] Figure 5 : Anti-Trastuzumab (Anti-Idiotype) Alexa via LCT-Premier Mass characterization of 647-conjugated antibodies (AF647-modified 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). The antibodies have been deglycosylated. Deconvoluted spectra are shown.

[0071] Figure 6 : Quality 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 receptor wells from HBBBCMTA by LCT-Premier. Raw data (upper) and deconvoluted spectra (lower) are shown. For Tz, M 计算值 :148212;M 发现值 :148215; for Tz-Ang2, M 计算值 =159244;M 发现值 :159408;For Tz-MiniAp4,M 计算值 =153684;M 发现值 :76920,153678.

[0072] Figure 7 : Study on the stability of the BBB-shuttle agent peptides present in Example 4 and Comparative Example 3 in mouse serum.

[0073] Figure 8: Binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) to Her-2 overexpressing cells. BT474 cells and SKBR3 cells were divided equally and incubated with Tz, Tz-Ang2 or Tz-MiniAp4 at 4°C for 2 hours. Anti-human dylight 650 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 ANOVA multiple comparisons (SKBR3: vehicle versus Tz, p=0.0037; vehicle versus Tz-DBM-TTDS-SEQ ID NO:15 (Comparative Example 3), p=0.0007; vehicle versus Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p=0.0037; Tz versus Tz-DBM-TTDS-SEQ ID NO:x (Comparative Example 3), p=0.5465; Tz versus Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p>0.9999; Tz-DBM-TTDS-SEQ ID NO:2 (Comparative Example 3) versus Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p=0.5373; BT474: vehicle versus Tz, p=0.0004; vehicle versus Tz-DBM-TTDS-SEQ ID NO: NO:2 (Comparative Example 3), p=0.0034; vehicle relative to Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p=0.0004; Tz relative to Tz-DBM-TTDS-SEQ ID NO:x (Comparative Example 3), p=0.5086; Tz relative to Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p=0.9630; Tz-DBM-TTDS-SEQ ID NO:2 (Comparative Example 3) relative to Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p=0.7174; MDA MB 231SKBR3: vehicle relative to Tz, p=0.9894; vehicle relative to Tz-DBM-TTDS-SEQ ID NO:15 (Comparative Example 3), p=0.7054; vehicle relative to Tz-DBM-TTDS-SEQ ID NO: NO:1 (Example 4), p=0.8647; Tz relative to Tz-DBM-TTDS-SEQ ID NO:15 (Comparative Example 3), p=0.8595; Tz relative to Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p=0.9641; Tz-DBM-TTDS-SEQ ID NO:15 (Comparative Example 3) relative to Tz-DBM-TTDS-SEQ ID NO:1 (Example 4), p=0.9882).

[0074] Fig. 9 : Binding of Cx and Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to MDA-MB-231 cells. No significant difference in binding. Activity was retained.

[0075] Fig.10: Binding of Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to BT474 cells. No significant difference in binding. Activity is retained.

[0076] Fig.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 cells, BT-474 cells 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 deviations (n=3).

[0077] Fig.12 : Permeability of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) (100 nM) in a human in vitro BBB cell model. Error bars represent standard deviations (n ​​= 3). P values ​​were calculated using a two-tailed t-test (Tz versus Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3), p < 0.0001; Tz versus Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4), p < 0.0001; Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) versus Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4), p = 0.0002).

[0078] Fig.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 values ​​were calculated using a two-tailed t-test (Cx vs. Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5), p=0.069).

[0079] Fig.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).

[0080] Fig.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 SE) (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).

[0081] Fig.16 : Permeability of Cx and Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5), Bv and Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6), and Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) (1 μM) in a human in vitro BBB cell model. Error bars represent SD (n=3).

[0082] Fig.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 intravenous bolus injection. Results are expressed in nanomoles / gram tissue. Error bars represent standard deviation (n=3). P values ​​were calculated using a one-way ANOVA test (p=0.7697 for Tz vs. Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3); p=0.0323 for Tz vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4); p=0.0486 for Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4). Brain to plasma ratio. mAbs were injected into the tail vein of mice at a dose of 10 mg / kg. After 8 hours, serum was collected and systemic saline perfusion was performed. The brain was 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 a one-way ANOVA test (Tz vs. Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3), p=0.2657; Tz vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4), p=0.0319; Tz-DBM-TTDS-SEQ ID NO: 2 (Comparative Example 3) vs. Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4), p=0.1710).

[0083] Fig.18: Permeability of Cx and Cx-MiniAp4 (1 μM) in a human in vitro blood-brain barrier 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).

[0084] Fig.19 : In the human in vitro blood-brain barrier cell model, the Papp ratio of Cx modified with SN38-coupled peptide shuttles (MiniAp4 or Ang2) and Cx modified with naked shuttles (MiniAp4 and Ang2) at 1 μM was determined. 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

[0085] Unless otherwise specified, all terms used in this application should be understood as having ordinary meanings known in the art. Other more specific definitions of certain terms used in this application are described below and are intended to be uniformly applied throughout the specification and claims, unless otherwise explicitly listed definitions provide a broader definition.

[0086] As used herein, a noun with no quantifier preceding it is synonymous with "at least one" or "one or more than one". Unless otherwise specified, the use herein, for example, "the" also includes the plural form of the noun.

[0087] For the purpose of the present invention, the wording "comprising" includes the phrase "consisting of."

[0088] Unless otherwise indicated, amino acids cited herein are L-amino acids. 1-letter codes and 3-letter codes are used indiscriminately. For the following amino acids, the following abbreviations are used: diaminopropionic acid (Dap), diaminobutyric acid (Dab), selenocysteine ​​(Sec) and penicillamine (Pen). In the context of the present invention, penicillamine includes only D-penicillamine. Diaminopropionic acid (Dap) may also be abbreviated as Dpr, and diaminobutyric acid (Dab) may also be abbreviated as Dbu.

[0089] As mentioned above, part of the present invention is an antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof,

[0090]

[0091] It contains 1 to 6 -P-(W)sY inserted into the antibody disulfide bonds and linked by a linker -[(L1)-(L2)-(L3) m- a peptide of the formula P linked to a sulfide; wherein Z, L1, L2, L3, D, m, P, W, S, Y and n have the meanings given above.

[0092] In a specific embodiment, the antibody shuttle agent conjugate of the present invention is one in which the disulfide bonds originally present in the antibody are able to structurally retain the sulfide bonds in the structure under reducing conditions, and are able to form a new bridge between them by reacting the sulfide groups of the reduced disulfide bonds of the antibody with the linker L1, thereby incorporating the following diradicals between the sulfide groups of the antibody.

[0093]

[0094] The antibody shuttle conjugate of formula (I) or a pharmaceutically acceptable salt thereof can be in the form of a formulation comprising an antibody or a salt thereof and an excipient. For example, suitable excipients for bevacizumab (Avastin) are trehalose dihydrate, sodium phosphate, polysorbate 20 and water for injection preparations, and suitable excipients for pertuzumab (Perjeta) are glacial acetic acid, L-histidine, sucrose, polysorbate 20 and water for injection preparations; suitable excipients for cetuximab (Erbitux) are: sodium chloride, glycine, polysorbate 80, citric acid monohydrate, sodium hydroxide and water for injection preparations; and suitable excipients for trastuzumab (Herceptin) are, for example: L-histidine hydrochloride monohydrate, L-histidine, α, α-trehalose or polysorbate 20 dihydrate.

[0095] In a specific embodiment, the antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof is wherein L1 is linked to the -S- of the disulfide bond of the antibody via bond a and bond b, and bond c is linked to linker L2 via an amide bond between the C=O group immediately adjacent to bond c of linker L1 and the NH group of linker L2.

[0096] In another specific embodiment, alone or in combination with any of the above embodiments, the antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof is wherein the linker L2 is a diradical selected from the following:

[0097]

[0098] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof is wherein n is an integer from 1 to 4. In another specific embodiment, in combination with any of the above embodiments, the antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof is wherein n is 4, i.e., it has four peptides incorporated into the antibody structure. In another specific embodiment, alone or in combination with any embodiment of the invention, all peptides are identical.

[0099] In another specific embodiment, alone or in combination with any embodiment of the invention, the disulfide bonds of the antibody are interchain bonds. In another specific embodiment, alone or in combination with any embodiment of the invention, the disulfide bonds of the antibody are intrachain bonds. In another specific embodiment, alone or in combination with any embodiment of the invention, the disulfide bonds of the antibody are disulfide bonds formed by genetic engineering.

[0100] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is a diradical wherein P is a peptide selected from:

[0101] (a) a peptide comprising the amino acid sequence DapKAPETALD, which has an intrapeptide bond between Dap and D that is an amide bond, i.e., SEQ ID NO: 8:

[0102] (b) a peptide of 9 to 20 amino acid residues in length having intrapeptide bonds that are at least disulfide bonds and comprising the following amino acid sequence:

[0103] CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, i.e., SEQ ID NO: 9:

[0104] (c) a peptide of 9 to 11 amino acid residues in length having an intrapeptide bond that is at least a disulfide bond and consisting of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA having at least an intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, i.e.

[0105]

[0106] and

[0107] (d) a peptide having 16 amino acid residues and comprising the amino acid sequence CNCKAPETALCAAACH, which has intrapeptide disulfide bonds between the first and third cysteines, which are cysteine ​​1 and cysteine ​​11, and between the second and fourth cysteines, which are cysteine ​​3 and cysteine ​​15, i.e.,

[0108]

[0109] (e) A peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

[0110] A line between two amino acids of the sequence above or below represents an intrapeptide bond between two amino acid side chains. In a specific embodiment, a line between two amino acids of the sequence above or below represents an intrapeptide bond between two amino acid side chains.

[0111] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is a diradical wherein P is a peptide selected from: (a) a peptide having the amino acid sequence DapKAPETALD having 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 having at least an 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).

[0112] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is a diradical wherein P is the peptide DapKAPETALD having an intrapeptide bond between Dap and D that is an amide bond (SEQ ID NO: 7).

[0113] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is a diradical wherein P is a peptide having one intrapeptide bond. In another specific embodiment, the antibody shuttle agent conjugate is a diradical wherein P is a peptide having two intrapeptide bonds.

[0114] Antibodies are usually composed of two heavy chains (HC) and two light chains (LC) folded into constant and variable regions, although some antibodies, such as camel antibodies, contain only 2 heavy chains. It can exist alone in the form of a monomer (such as IgG), or in the form of a multimer of two units (such as IgA) to a multimer of five units (such as IgM). It has a disulfide intrachain bond. The antibody used for the purpose of the present invention can be a chimeric antibody, a humanized antibody or a fully human antibody. In the sense of the present invention, the antibody can be a natural antibody or a recombinant antibody. In a specific embodiment, the antibody is a therapeutic antibody. In another specific embodiment, the antibody is used for diagnosis. In another specific embodiment, the antibody is an antibody-drug conjugate (ADC).

[0115] The antibody can also be an antibody fragment, as long as it contains at least one disulfide bond, and as long as it maintains the function of the antibody from which it is derived. In particular, its therapeutic activity or diagnostic activity. For example, the antibody can be an antibody fragment, such as Fab, scFV, (Fab)2, double antibody, three antibody, four antibody, mini antibody or nano antibody. For example, by mutagenizing the nucleic acid sequence of the antibody and replacing one or more than one amino acid residue with cysteine ​​to encode a cysteine-modified antibody to incorporate cys, disulfide bonds can be incorporated into the antibody or its fragment (see CA2957354A1). Compared with full-length antibodies, antibody fragments have several advantages. Their smaller size allows better tissue penetration into solid tumors, and if antibodies are used as radioactive imaging agents, their shorter half-life is ideal. Suitable for applications where antibodies do not need to participate in the immune system, such as blocking signal molecules or receptors.

[0116] In a specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle conjugate is one in which the antibody comprises a heavy chain constant region of type IgA, IgD, IgE, IgG or IgM. IgA type antibodies can be divided into two isotypes: IgA1 or IgA2. IgG class antibodies can be divided into four isotypes: IgG1, IgG2, IgG3 and IgG4. The types of antibodies are included within the scope of the invention.

[0117] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is one wherein the antibody comprises a light chain constant domain, such as a light chain constant domain of the kappa or lambda type.

[0118] In another specific embodiment, alone or in combination with any embodiment of the present invention, the antibody shuttle conjugate is wherein 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. In particular, IgG1 is 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 toxicity). In a more specific embodiment, the antibody is a monoclonal chimeric antibody, a humanized antibody, or a fully human antibody. Antibody humanization allows the immunogenicity to be reduced by reducing the content of mouse monoclonal antibodies. This can be achieved, for example, by expressing isolated human variable domain genes in Escherichia coli. Common techniques developed for the production of fully human monoclonal antibodies can be used, such as phage display, in which a human antibody library is expressed on the surface of a phage, followed by selection and amplification in Escherichia coli, and transgenic mice expressing a human antibody library.

[0119] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is wherein the antibody is: (a) multispecific. Multispecific antibodies are a class of engineered antibodies and antibody-like proteins that combine multiple specific antigen binding elements in a single construct.

[0120] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is wherein the antibody is: a heterodimeric bispecific antibody, which is a traditional IgG molecule with one arm targeting one antigen and the other arm targeting a second antigen.

[0121] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle conjugate is a bispecific antibody fusion in which the antibody is a non-standard IgG molecule. The IgG is extended at the N-terminus of its respective heavy and light chains by additional variable domains of a second antibody.

[0122] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle conjugate is a trispecific antibody in which the antibody is a non-standard IgG molecule. The same techniques used to generate bispecific antibodies can also be combined to generate trispecific antibodies with different valencies.

[0123] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate is one in which the antibody is an scFv fused to an IgG via linkage of the scFv to the N-terminus or C-terminus of the heavy or light chain.

[0124] In a specific embodiment, the antibody shuttle conjugate of the present invention is an antibody wherein the antibody is selected from trastuzumab, bevacizumab, cetuximab, pertuzumab, aducanumab, bapineuzumab, nimotuzumab and naxituzumab. In a more specific embodiment, the antibody shuttle conjugate of the present invention is an antibody wherein the antibody is selected from trastuzumab, bevacizumab, cetuximab, pertuzumab.

[0125] In a specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate of the invention is wherein the antibody is trastuzumab. is an FDA-approved anti-HER2 antibody that is widely used in the clinical treatment of breast cancer. In another specific embodiment, alone or in combination with any embodiment of the present invention, the antibody shuttle conjugate of the present invention is trastuzumab

[0126] -DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-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.

[0127] In a specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate of the invention is wherein the antibody is cetuximab. FDA-approved anti-EGFR antibodies are widely used in the clinical treatment of colorectal cancer and head and neck cancer. In another specific embodiment, alone or in combination with any embodiment 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.

[0128] The term "DBM" corresponds to the linker L 1a The term "TTDS" corresponds to the linker L 2a These terms are used indiscriminately in two contexts.

[0129] DBM=L 1a With the formula:

[0130]

[0131] TTDS=L 2a With the formula:

[0132]

[0133] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate of the invention is wherein the antibody is bevacizumab. FDA-approved anti-VEGF-A antibody, which is widely used in the clinic to treat colon cancer, lung cancer, glioblastoma and renal cell carcinoma. In another specific embodiment, alone or in combination with any embodiment 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.

[0134] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle agent conjugate of the invention is wherein the antibody is Pertuzumab. FDA-approved anti-HER2 antibodies are widely used in the clinical treatment of breast cancer. In another specific embodiment, alone or in combination with any embodiment 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), which has an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid.

[0135] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle conjugate of the invention is wherein the antibody is aducanumab, an IgG1 antibody against an epitope of β-amyloid protein. In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle conjugate of the invention is wherein the antibody is bapilizumab, an IgG1 antibody against Aβ-amyloid protein. Both antibodies can be used to treat Alzheimer's disease.

[0136] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle conjugate of the invention is wherein the antibody is nimotuzumab, an IgG1 anti-EGFR antibody. In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody shuttle conjugate of the invention is wherein the antibody is nexituzumab, an IgG1 anti-EGFR antibody. Both antibodies can be used to treat brain metastases.

[0137] In specific embodiments, alone or in combination with any embodiment of the invention, a formulation comprising an antibody shuttle agent conjugate of the invention is one in which D is present, resulting in an antibody-drug shuttle agent conjugate, an antibody-radioligand shuttle agent conjugate, or an antibody-diagnostic agent shuttle agent conjugate.

[0138] In a specific embodiment, the biologically active substance is a pharmaceutically active ingredient. In another specific embodiment, the antibody shuttle agent conjugate of the present invention is wherein: the antibody is an anti-cancer therapeutic antibody, m=1, and the free radical of the active biological substance D is a free radical of an anti-cancer active pharmaceutical ingredient selected from the following: auristatin, duocarmycin, PBD dimer, maytansinoid, calicheamicin, anthracycline, camptothecin, alpha-amanitin, tubulysin, MMAE, T-DM1 and part of PROTAC.

[0139] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody is an anti-cancer therapeutic antibody, m=1, and D is a chemotherapeutic agent.

[0140] In another specific embodiment, alone or in combination with any embodiment of the invention, the antibody-drug shuttle conjugate is wherein the antibody is cetuximab and D is SN38. In another specific embodiment, the antibody-drug shuttle conjugate according to the invention is wherein the antibody is cetuximab, the drug is SN38, the peptide is MiniAp4, and the linker is TTDS-DBM.

[0141] In another specific embodiment, alone or in combination with any embodiment of the present invention, the formulation comprises an antibody that is an anti-cancer therapeutic antibody, m=1, and D is a radioligand. The formulation can be used for radioimmunotherapy. In another specific embodiment, alone or in combination with any embodiment of the present invention, D is biotin-yttrium 90 or biotin-iodine 131 or biotin-iodine 123. It can be linked to the antibody shuttle conjugate of the present invention via streptavidin, wherein when the diradical L3 is present in the antibody shuttle conjugate of the present invention, the streptavidin is linked to the amino acid side chain of the diradical L3.

[0142] In another specific embodiment, alone or in combination with any embodiment of the invention, D is a contrast agent for magnetic resonance imaging (MRI), such as gadolinium, in particular Gd-DTPA Gadolinium can be complexed with DOTA, and when the diradical L3 is present in the antibody shuttle agent conjugate of the present invention, DOTA is linked to the amino acid side chain of the diradical L3, or to an amino acid derivative through an available bond.

[0143] In another specific embodiment, the antibody shuttle agent conjugate of the present invention is wherein: the antibody is an anti-neurodegeneration therapeutic antibody, m=1, and the free radical of the active pharmaceutical ingredient is a free radical of an anti-neurodegeneration active pharmaceutical ingredient.

[0144] The antibody shuttle conjugate of the present invention can be prepared by a method comprising the following steps: a) reducing the disulfide bridge of the antibody; b) reacting the -SH group of the antibody with a dibromomaleimide-peptide (DBM-peptide) of formula (II) to re-bridge the disulfide bridge, i.e., to form a new bridge, DBM-P-(W). s -(Y), wherein the dibromomaleimide is attached to the peptide via the N-terminus of the peptide, wherein DBM, P, W, Y and s are as defined for the antibody shuttle conjugate of formula (I). Due to the reactivity of the cysteines that form the disulfide bridge, the DBM-peptide may bind to the antibody. This approach allows for a high degree of control over the number of peptides anchored per antibody molecule. In a specific embodiment, the antibody is trastuzumab.

[0145] The antibody shuttle agent conjugate of the present invention can be defined by its preparation method. Therefore, the antibody shuttle agent conjugate obtained by the method defined above is also considered to be part of the present invention.

[0146] In particular, the antibody shuttle agent conjugates as defined above can be obtained by reacting the sulfhydryl groups of the reduced disulfide bonds of the corresponding antibody or fragment thereof with the dibromomaleimide-peptide of formula (II) as defined above to rebridge the disulfide bonds with the underlying diradicals incorporated between the disulfide bonds of the antibody.

[0147]

[0148] In another specific embodiment, the antibody shuttle agent conjugate as defined above can be obtained by a method comprising the following steps: a) reducing the disulfide bridges of the antibody; b) re-bridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), c) optionally, performing hydrolysis; wherein q; L2, L3, D, P, m, W, s and Y are as defined in the antibody shuttle agent conjugate of formula (I).

[0149]

[0150] In a specific embodiment of the method, the disulfide bond is an interchain bond.All embodiments of the conjugate of formula (I) as product defined above are themselves also embodiments of the method for its preparation.

[0151] The shuttle peptide used in the conjugate of the present invention can be produced in whole or in part by chemical synthesis. The amino acids required for preparing the compound of formula (I) are commercially available. The compound of formula (I) can be easily prepared, for example, by liquid phase synthesis, or preferably by solid phase peptide synthesis, for which there are many disclosed steps (see M. Amblard et al., "Methods and protocols of modern solid-phase peptide synthesis". Molecular Biotechnology 2006, Vol. 33, p. 239-254). The compound of formula (I) can also be prepared by any combination of liquid phase synthesis and / or solid phase synthesis. For example, the main body of peptide P is synthesized by solid phase synthesis, and then the protecting group is removed in solution. The combination with the linker dibromomaleimide can be carried out in solid phase or solution. The construction of the linker can also be prepared by any combination of liquid 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 its similar derivatives.

[0152] The compound of formula (II) can be prepared by a method comprising the following steps: reacting a peptide derivatized 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 produce DBM-peptide. 1b The compound of formula (II) can be hydrolyzed by the corresponding linker L 1a The hydrolysis can be carried out under mild alkaline conditions.

[0153] In a specific embodiment, the DBM-peptide of formula (II) has the following formula:

[0154]

[0155] In another specific embodiment, the DBM-peptide of formula (II) has the following formula:

[0156]

[0157] In a specific embodiment, the antibody shuttle conjugate of the present invention is wherein the linker L1 is L 1b The conjugate can be prepared by hydrolyzing the corresponding linker L1 as linker under mild alkaline pH conditions. 1a The antibody shuttle agent conjugate is prepared.

[0158] The antibody shuttle conjugate of the present invention can be in the form of a pharmaceutically acceptable salt thereof. The term "pharmaceutically acceptable salt" as used herein includes any salt formed by a pharmaceutically acceptable non-toxic acid or non-toxic base, including an inorganic or organic acid or base. There is no restriction on the salt, except that if used for therapeutic purposes, it must be pharmaceutically acceptable. Since some compounds of formula (I) are basic compounds, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids and organic acids. These acids include, for example, 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, etc.

[0159] Examples of salt forms of antibodies according to the present invention are bevacizumab (Avastin) in salt form with trehalose dihydrate, sodium phosphate, polysorbate 20 salt; pertuzumab (Perjeta) in salt form with glacial acetic acid, L-histidine, sucrose or polysorbate 20; cetuximab (Erbitux) in salt form with sodium chloride, glycine, polysorbate 80, citric acid monohydrate or sodium hydroxide, trastuzumab (Herceptin) in salt form with L-histidine hydrochloride monohydrate, L-histidine, α, α-trehalose or polysorbate 20 dihydrate.

[0160] 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. In general, these salts are prepared, for example, by reacting these compounds in free acid form or free base form with a stoichiometric amount of a suitable pharmaceutically acceptable base or acid in water or an organic solvent or a mixture thereof.

[0161] Pharmaceutical compositions comprising a therapeutically effective amount of an antibody shuttle agent conjugate as defined above and a suitable amount of a pharmaceutically acceptable carrier or excipient are also part of the present invention.

[0162] 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 the compound to an organism. The term "pharmaceutically acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition or carrier. Each component must be pharmaceutically acceptable, i.e., compatible with the other components of the pharmaceutical composition. It must also be suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reaction, immunogenicity or other problems or complications, and commensurate with the benefit / risk ratio.

[0163] The expression "therapeutically effective amount" as used herein refers to an amount of a compound that, when administered, is sufficient to prevent the development of one or more symptoms of the disease being treated, or to alleviate to some extent one or more symptoms of the disease being treated. The specific dosage of the compound administered according to the present invention will of course be determined by the specific circumstances associated with the case, including the compound administered, the route of administration, the specific condition being treated, and similar considerations.

[0164] The expression "pharmaceutically acceptable excipient, diluent or carrier" refers to a pharmaceutically acceptable material, composition or carrier. Each ingredient must be pharmaceutically acceptable, i.e. compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with tissues or organs of humans and non-human animals without excessive toxicity, irritation, allergic reaction, immunogenicity or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0165] Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents or other liquid carriers, dispersing or suspending aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Unless to some extent any conventional excipient medium is incompatible with the substance or its derivatives, such as producing any undesirable biological effect or interacting in a deleterious manner with any other component or components of the pharmaceutical composition, its use is considered to be within the scope of the present invention.

[0166] In the pharmaceutical compositions of the present invention, the relative amounts of the active ingredient, pharmaceutically acceptable excipients and / or any other ingredients will vary depending on the identity, size and / or condition of the subject being treated and also on the route of administration of the composition.

[0167] The composition of the present invention can be administered in a parenteral form suitable for injection, for example, by intravenous bolus injection, intravenous infusion, implantation, oral administration, intrathecal administration or intranasal administration.

[0168] Antibody shuttle agent conjugates as defined above, for use as medicaments, are also part of the present invention. This includes antibody shuttle agent conjugates as well as antibody-drug shuttle agent conjugates according to the present invention.

[0169] The term "drug" as used herein is synonymous with widely accepted drugs or veterinary drugs (also referred to as pharmaceuticals, drugs, or simply drugs) used to cure, treat, or prevent disease in animals, including humans. Drugs are classified in various ways. A key distinction is between traditional small molecule drugs, which are typically derived from chemical synthesis, and biopharmaceuticals, which include recombinant proteins, vaccines, blood products for treatment (such as IVIG), gene therapy, monoclonal antibodies, and cell therapy (such as stem cell therapy).

[0170] The antibody shuttle agent conjugate as defined above, which is used to treat CNS disorders in mammals including humans, is also part of the present invention. This aspect can also be expressed as the use of the antibody shuttle agent conjugate as defined above for the preparation of a medicament for treating CNS disorders in mammals including humans. The present invention also relates to a method for treating a mammal including humans suffering from or susceptible to CNS disorders, the method comprising administering to the patient a therapeutically effective amount of the antibody shuttle agent conjugate as defined above, and a pharmaceutically acceptable excipient or carrier.

[0171] In a specific embodiment, the antibody shuttle agent conjugate is used for a CNS disorder, wherein the CNS disorder is cancer.

[0172] In another specific embodiment, the antibody shuttle agent conjugate of the present invention is used to treat primary brain tumors. In another specific embodiment, the antibody shuttle agent conjugate of the present invention is used to treat brain metastases (BM), which are major complications of several types of cancer, especially lung cancer, melanoma and breast cancer.

[0173] The antibody shuttle conjugates of the present invention, the antibody shuttle conjugates according to the present invention, and the antibody-drug shuttle conjugates can be used in the same manner as other known chemotherapeutic agents, i.e., in combination with other treatments simultaneously or sequentially, depending on the condition to be treated. They can be used alone or in combination with other suitable biologically active compounds. Therefore, the antibody shuttle conjugates of the present invention are used to treat cancer in mammals, including humans, in combination therapy with chemotherapeutic agents. Antibody-drug shuttle conjugates can be used, for example, in specific treatment regimens in combination therapy with other chemotherapeutic agents.

[0174] In another specific embodiment, the antibody shuttle conjugate of the present invention is used in combination with radioimmunotherapy. According to the present invention, two antibody shuttle conjugates can be used as antibody-radioligand shuttle conjugates. For example, a combination of radiotherapy and immunotherapy can be used to treat non-Hodgkin's lymphoma and other types of cancer, including brain tumors. The antibody-radioligand shuttle conjugate according to the present invention can bind to cancer cells and deliver high doses of radiation directly to the tumor.

[0175] Antibody shuttle agent conjugates as defined above, for use as diagnostic agents, are also part of the present invention. In a specific embodiment, the antibody shuttle agent conjugate according to the present invention is for use in a method for diagnosing a CNS disorder.

[0176] Throughout the specification and claims, the word "comprises / includes" and its variations are not intended to exclude other technical features, additives, ingredients or steps. In addition, the word "comprises / includes" includes the case of "consisting of..." Other objects, advantages and features of the present invention will become apparent to those skilled in the art after reading the specification or may be understood through the practice of the present invention. The following examples and drawings are provided in an illustrative manner and are not meant to limit the present invention. The reference numerals in the claims that are associated with the drawings and placed in brackets are only used to attempt to increase the understandability of the claims and should not be interpreted as limiting the scope of the claims. In addition, the present invention covers all possible combinations of the specific embodiments and preferred embodiments described herein.

[0177] Example

[0178] Protected amino acids, handles and resins were provided by Luxembourg Industries (Tel-Aviv, Israel), Neosystem (Strasbourg, France), CalbiochemNovabiochem AG (Laufelfingen, Switzerland), Bachem AG (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). Table 1 Other reagents and other solvents used are summarized. Table 1 Commercial suppliers and reagents used. DCM was passed through an Al2O3 column. DMF was stored over molecular sieves The mixture was stirred for 2 hours and nitrogen was bubbled through to remove volatile substances.

[0179] Protected amino acids were provided by Iris Biotech (Marktredwitz, Germany). ChemMatrix resin was purchased from PCAS BioMatrix (QC, Canada). Diisopropylethylamine (DIEA), N,N'-diisopropylcarbodiimide (DIC), and ninhydrin were provided by Fluka Chemika (Buchs, Switzerland). Solvents used 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.

[0180] Cell culture treated plates and bottles were purchased from Corning Costar. Culture medium was obtained from Lonza. XTT cell proliferation kit was purchased from Biological Industries (Cromwell, CT). Iodinated beads were obtained from Pierce. Desalting columns (MiniTrap and MidiTrap G-25) were purchased from GE-Healthcare.

[0181]

[0182] General methods for preparing conjugates of the invention

[0183] General notes about manual compositing: Solid phase peptide extension and other solid phase manipulations were performed manually in polypropylene syringes fitted with porous polyethylene disks. Solvents and soluble reagents were removed by aspiration. Washing between different synthesis steps was performed with dimethylformamide (DMF) (5 × 30 s) and dichloromethane (DCM) (5 × 30 s), using 10 mL of solvent / g of resin each time.

[0184] General notes about microwave-assisted synthesis: Microwave-assisted solid phase peptide synthesis was performed on a Liberty Blue automated microwave peptide synthesizer using H-Rink amide Protide resin (loading: 0.56 mmol / g). Linear peptides were synthesized on a 0.5 mmol scale using a 5-fold excess of Fmoc-amino acid (0.2 M) relative to the resin.

[0185] Identification Test: The tests used to identify and control the synthesis are as follows: A) Kaiser colorimetric assay for detection of primary amines bound to the solid phase (E. Kaiser et al., Anal. Biochem. 1970, vol. 34, pp. 595-598); B) p-nitrophenyl ester test of secondary amines bound to the solid phase (A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791).

[0186] Protocols used during manual synthesis of compounds : The compounds were synthesized on a 100 micromolar scale using the following method and protocol: The resin used for the manual synthesis was selected based on the group Y: If Y is OH, the terminal will be COOH and 2-chlorotrytil chloride resin will be selected from other available resins. If Y is NH2, the terminal will be CONH2 and Rink amide MBHA resin will be selected from other available resins.

[0187] Initial resin conditioning:The resin was conditioned by washing with MeOH (5×30 s), DMF (5×30 s), DCM (5×30 s), 1% TFA in DCM (1×30 s and 2×10 min), DCM (5×30 s), DMF (5×30 s), DCM (5×30 s), 5% DIEA in DCM (1×30 s, 2×10 min), DCM (5×30 s), DMF (5×30 s).

[0188] Removal of Fmoc group: The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was removed by treatment with piperidine 20 (vol / vol)% in DMF for 30 s, then twice for 10 min each. Two additional treatments (2 x 5 min) were performed with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) to ensure removal of the Fmoc group from the secondary amine (proline).

[0189] Coupling method described for 100 micromolar scale:

[0190] Coupling method 1: The protected amino acid (4 eq., 400 micromolar), TBTU (4 eq., 400 micromolar, 128 mg) dissolved in DMF (1 mL / g resin to 3 mL / g resin) were added to the resin in sequence, followed by DIEA (8 eq., 800 micromolar, 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 degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with 20 (vol / vol)% piperidine in DMF, using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected is proline, additional treatments (2×5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed to ensure removal of the Fmoc group.

[0191] Coupling method 2:The protected amino acid (4 eq., 400 μmol), PyBOP (4 eq., 400 μmol, 208 mg), HOAt (12 eq., 1.2 mmol, 163 mg) dissolved in DMF (1 mL / g resin to 3 mL / g resin) were added to the resin in sequence, followed by DIEA (12 eq., 1.2 mmol, 204 μL). The mixture was allowed to react for 1 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling reaction was performed twice under the same conditions. The degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with 20 (vol / vol)% piperidine in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, additional treatments (2 x 5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed to ensure removal of the Fmoc group.

[0192] Coupling method 3: The protected amino acid (4 eq., 400 μmol), PyBOP (4 eq., 400 μmol, 208 mg), HOBt (12 eq., 1.2 mmol, 162 mg) dissolved in DMF (1 mL / g resin to 3 mL / g resin) were added to the resin in sequence, followed by DIEA (12 eq., 1.2 mmol, 204 μL). The mixture was allowed to react for 1 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling reaction was performed twice under the same conditions. The degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with 20 (vol / vol)% piperidine in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, additional treatments (2 x 5 min) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed to ensure removal of the Fmoc group.

[0193] Coupling method 4, scale 100 μmol: The protected amino acid (3 eq., 300 μmol), DIC (3 eq., 300 μmol, 46 μL) and Oxyma (3 eq., 300 μmol, 43 mg) were dissolved in DCM / DMF (1:1). The mixture was allowed to react for 45 min 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 degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with 20 (vol / vol)% piperidine in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, additional treatments (2×5 min) were performed with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) to ensure removal of the Fmoc group.

[0194] Coupling method 5 , scale 100 μmol: The protected amino acid (3 eq., 300 μmol), DIC (3 eq., 300 μmol, 46 μL) and HOBt (3 eq., 300 μmol, 41 mg) were dissolved in DCM / DMF (1:1). The mixture was allowed to react for 45 min 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 degree of coupling was checked by Kaiser colorimetric assay. The Fmoc group was removed with 20 (vol / vol)% piperidine in DMF using a 30 s treatment and two 10 min treatments. If the amino acid to be deprotected was proline, additional treatments (2×5 min) were performed with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) to ensure removal of the Fmoc group.

[0195] Protocol used during microwave-assisted automated synthesis: The compounds were synthesized at a 500 micromolar scale using the following method and protocol: The resin used for microwave-assisted automated synthesis was selected based on the group Y: If Y is OH, the terminus will be COOH and Cl-TCP(Cl)ProTide resin will be selected from other available resins. If Y is NH2, the terminus will be CONH2 and Rink Amide ProTide resin will be selected from other available resins.

[0196] Initial resin conditioning: The resin was conditioned by washing with MeOH (5×30 s), DMF (5×30 s), DCM (5×30 s), 1% TFA in DCM (1×30 s and 2×10 min), DCM (5×30 s), DMF (5×30 s), DCM (5×30 s), 5% DIEA in DCM (1×30 s, 2×10 min), DCM (5×30 s), DMF (5×30 s).

[0197] Coupling and deprotection conditions for microwave-assisted automated peptide synthesis:

[0198] Coupling conditions:

[0199]

[0200] Deprotection conditions:

[0201]

[0202] method Heating time Total time Maximum temperature Standard deprotection 20-30s 1:05 90℃

[0203] Cyclization method of peptide sequence P:

[0204] Cyclization method 1: disulfide bond or diselenide bond: Cyclization is performed in solution after cleavage from the resin or on the resin after selective deprotection of Cys residues, Sec residues or Pen residues. The peptide is dissolved in a 100 μM aqueous solution of ammonium bicarbonate buffer at 10 mM and pH 8.0. The solution is vigorously stirred at room temperature for 24 h. Afterwards, the product is acidified to pH 2 to 3 with TFA, frozen and lyophilized.

[0205] Cyclization method 2: Amide bond: Cyclization was performed on the resin. The Fmoc group was removed with 20 (vol / vol)% piperidine in DMF, using a 30s treatment and two 10-minute treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 equivalents, 1000 micromoles, 56 mg) and DIEA (30 equivalents, 3000 micromoles, 240 μL). The deprotection of the OAl group and the Alloc group was first performed by adding tetrakis(triphenylphosphine)palladium(0) (0.1 equivalents, 10 μM, 12 mg), phenylsilane (10 equivalents, 1000 micromoles, 123 mg) in DCM (3×15 minutes). The resin was washed with sodium diethylcarbamate in 0.02M DCM (3×5 minutes). Then the coupling of the amino group of Dap and the carboxylate group of aspartic acid was achieved by adding PyBOP (4 equivalents, 400 micromoles, 208 mg), HOAt (12 equivalents, 1.2 mmoles, 163 mg), DMF (1 mL / g resin to 3 mL / g resin) and DIEA (12 equivalents, 1.2 mmoles, 204 μL). The coupling reaction was left for 1.5 h and repeated overnight.

[0206] Cyclization method 3: Amide bond:Cyclization is carried out on the resin. The Fmoc group is removed with a DMF solution of 20 (volume / volume)% piperidine, using 30s treatment and twice 10 minutes treatment. Use Boc2O (3 equivalents, 1000 micromoles, 56 mg) and DIEA (30 equivalents, 3000 micromoles, 240 μL) to protect the N-terminal amine with a Boc protecting group. The deprotection of the OAl group and the Alloc group is first carried out by adding tetrakis (triphenylphosphine) palladium (0) (0.1 equivalent, 10 μM, 12 mg), phenylsilane (10 equivalents, 1000 micromoles, 123 mg) in DCM (3×15 minutes). The resin is washed with sodium diethyldithiocarbamate in 0.02M DCM (3×5 minutes). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by two 30 min cycles of 4 equivalents of Oxyma (400 micromoles, 57 mg) and 4 equivalents of N,N'-diisopropylcarbodiimide (DIC) (400 micromoles, 61 μL).

[0207] Cyclization method 4: Amide bond: Cyclization was performed on the resin. The Fmoc group was removed with 20 (volume / volume)% piperidine in DMF, using 30s treatment and two 10-minute treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 equivalents, 1000 micromoles, 56 mg) and DIEA (30 equivalents, 3000 micromoles, 240 μL). The deprotection of the OAl group and the Alloc group was first performed by adding tetrakis(triphenylphosphine)palladium(0) (0.1 equivalents, 10 μM, 12 mg), phenylsilane (10 equivalents, 1000 micromoles, 123 mg) in DCM (3×15 minutes). The resin was washed with sodium diethyldithiocarbamate in 0.02M DCM (3×5 minutes). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by two 1-hour cycles of 4 equivalents of DIC (400 micromoles, 61 μL) and 4 equivalents of HOBt (400 micromoles, 54 mg).

[0208] General method for constructing joints

[0209] General method for disulfide formation: The disulfide bond can be completed by the reaction of two thiols. The thiol was dissolved in a 10 mM ammonium bicarbonate buffer solution at pH 8.0 at a concentration of 100 μM, and the solution was vigorously stirred at room temperature for 24 h. Afterwards, the solution was acidified to pH 2 to 3 with TFA, frozen and lyophilized.

[0210] General method for thioether formation: The thioether bond is achieved by reaction of the N-terminal bromoacetyl group with a cysteine ​​thiol as described in PL Barker et al., J. Med. Chem., 1992. vol 35, pp. 2040-2048.

[0211] General method for ether formation: The formation of ethers can be accomplished by reaction of a hydroxy group with a haloalkyl compound, preferably under basic conditions, as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter GM Wuts. 2014 John Wiley & Sons, Inc. pp. 26-29.

[0212] General method for ester formation: The formation of esters can be achieved by reaction of a hydroxyl group and a carboxylic acid using typical esterification conditions, such as Fischer esterification in the presence of acid catalyst, or by reaction of a hydroxyl group with a corresponding acid chloride as described in Greene's Protective Groups in Organic Synthesis, Fifth Edition. Peter G. M. Wuts. 2014 John Wiley & Sons, Inc. pp. 271-279.

[0213] 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.

[0214] Coupling of Fmoc-TTDS-OH: The coupling of Fmoc-TTDS-OH (2 equiv) was carried out by using 4 equiv of oxyma and 4 N,N'-diisopropylcarbodiimide (DIC) in DMF in 2 cycles of 30 min over 2 h. or 4 This was achieved with an equivalent of DIC and HOBt of 4 in DCM for 2 h. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed using 20 (v / v)% piperidine in DMF for 30 s followed by two 10 min treatments.

[0215] Coupling of 5-hexynoic acid: 5-Hexynoic acid (2 eq., 200 μmol, 23 mg) was coupled by using 4 eq. of Oxyma (400 μmol, 57 mg) and 4'-diisopropylcarbodiimide (DIC) (400 μmol, 61 μL) in DMF:DCM (1:1) for 2 cycles of 30 min. or The reaction was carried out by mixing 4 equivalents of DIC (400 μmol, 61 μL) and 4 equivalents of HOBt (400 mol, 54 mg) in DMF:DCM 1:1 for 4 h in DCM. orIt was achieved by 2 equivalents of PyBOP (400 micromoles, 208 mg) in DMF:DCM 1:1, 6 equivalents of HOAt (600 micromoles, 81.5 mg) and 6 equivalents of DIEA (600 micromoles, 102 μL) in DMF for 1.5 h. The solvent was removed by suction and the resin was washed with DMF (5×30s) and DCM (5×30s). The coupling was repeated under the same conditions. The degree of coupling was monitored using Kaiser colorimetric analysis.

[0216] Coupling of diglycolic anhydride: The coupling of diglycolic anhydride (10 equivalents, 1000 micromoles, 116 mg) was achieved by two 60 minute cycles of 10 equivalents of DIEA (1000 micromoles, 174 μL) in DMF. The solvent was removed by suction and the resin was washed with DMF (5×30s) and DCM (5×30s). The coupling was repeated under the same conditions. The degree of coupling was checked using the Kaiser colorimetric assay.

[0217] Methods for the alkyne-azide cycloaddition: Alkyne-azide cycloaddition (click reaction) couplings were performed in solution using the protocol described in SFM van Dongen et al.; Bioconjugate Chem. 2009, vol. 20, pp. 20-23.

[0218] General procedure for cleavage from resin: Final cleavage of the resin and side chain deprotection: This was performed by treating the resin with TFA (95%), H2O (2.5%) and TIS (2.5%) (2 h). Methyl tert-butyl ether was added to the resulting product and the mixture was centrifuged (3×8 min). The supernatant was discarded and the pellet was resuspended in a mixture of H2O, MeCN and TFA (1000:1000:1). The product was filtered out and frozen.

[0219] General methods for characterization of compounds:Compounds were analyzed by UPLC spectrometry (Acquity high-class system (PDA detector, sample manager FNT and quaternary solvent manager, Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 mL / min, using MeCN (0.036% TFA) and H2O (0.045% TFA) as solvents. In all cases, a 2-min linear gradient was used) and UPLC-MS spectrometry (Waters high class (PDA detector, sample manager FNT and quaternary solvent manager)) coupled to an electrospray ionization source ESI-MS Micromass ZQ and using MassLynx 4.1 software (Waters, Milford, MA). BEH C18 column (50×2.1 mm×1.7 μm, Waters). The flow rate was 0.6 ml / min, and MeCN (0.07% formic acid) and H2O (0.1% formic acid) were used as solvents. The samples were analyzed with positive ionization: ion spray voltage was 30 V, and capillary temperature was 1 kV) for characterization. Accurate mass was obtained by mass spectrometry: LTQ-FTUltra (Thermo Scientific), direct injection (Automated Nanoelectrospray). NanoMate (Advion BioSciences, Ithaca, NY, USA) aspirated samples from a 384-well plate (protein Lobind) with a disposable conductive pipette tip and injected the sample into the mass spectrometer through a nanoESI chip (20×20 array consisting of 400 nozzles). The spray voltage was 1.70 kV and the delivery pressure was 0.50 psi; ionization was NanoESI, positive ionization.

[0220] 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 mM to 4 mM in 90% H2O / 10% D2O and adjusting the pH to 2 to 3. Chemical shifts were referenced to the internal standard sodium 3-(trimethylsilyl)propane sulfonate (DSS). Suppression of the water signal was achieved by excitation shaping. Residue-specific assignments were obtained from 2D total correlation spectroscopy (TOCSY) and correlation spectroscopy (COSY) experiments, while 2D nuclear Overhauser effect spectroscopy (NOESY) allowed sequence-specific matching. 13C resonances were assigned from 2D 1H13C HSQC spectra. All experiments were performed at 298 K, except for the NOESY spectra which were acquired at 278 K. The amide proton temperature coefficients were determined from a series of one-dimensional spectra acquired between 278 K and 308 K. The TOCSY and NOESY mixing times were 70 ms and 250 ms, respectively.

[0221] Amino Acid Analysis: Amino acid analysis was performed to assess the amino acids present and the amount of each peptide obtained. To this end, ion exchange chromatography after acid hydrolysis was performed. The sample was hydrolyzed with 6M HCl at 110°C for 16h, then evaporated to dryness under reduced pressure and dissolved in 20mM HCl aqueous solution. 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 HCl (12M) and 20 μL of aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization reagent. The mixture was placed at 110°C overnight. Before executing the Waters AccQ-Tag protocol, the liquid was completely evaporated and 200 μL of 20mM HCl was added.

[0222] General methods for peptide purification and characterization:The crude product was purified by RP-HPLC on a semi-preparative scale and by UPLC spectroscopy (Acquity high-class system, PDA detector, sample manager FNT and quaternary solvent manager, Acquity BEH C18 (50×2 mm×1.7 μm) column, 0.61 ml / min, and MeCN (0.036% TFA) and H2O (0.045% TFA) were used as solvents. In all cases, a 2-minute linear gradient was used) and UPLC-MS spectroscopy (Waters high class (PDA detector, sample manager FNT and quaternary solvent manager)) was coupled to an electrospray ion source ESI-MS Micromass ZQ, and MassLynx 4.1 software (Waters, Milford, MA) was used. A BEH C18 column (50×2.1 mm×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 with positive ionization: ion spray voltage was 30 V and capillary temperature was 1 kV for characterization. Accurate mass was obtained by mass spectrometry: LTQ-FT Ultra (Thermo Scientific), direct injection (Automated Nanoelectrospray). NanoMate (Advion BioSciences, Ithaca, NY, USA) was used to draw samples from 384-well plates (protein Lobind) with disposable conductive pipette tips and injected into the mass spectrometer through nanoESIChip (20×20 array composed of 400 nozzles). The spray voltage was 1.70 kV and the delivery pressure was 0.50 psi; ionization was NanoESI, positive ionization. All peptides were obtained with a purity greater than 95%.

[0223] General approach to antibody modulation: Unless otherwise indicated for mixing, all mAb experiments were performed at room temperature in microcentrifuge tubes (1.5 mL, 2 mL, or 5 mL). All buffer solutions were prepared with MilliQ water. Borate buffered saline (BBS) represents 50 mM sodium borate, 50 mM NaCl, and 5 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.5. Phosphate buffered saline (PBS) represents 10 mM sodium phosphate, 137 mM sodium chloride, and 2.7 mM potassium chloride at pH 7.4. 10 mM (2.87 mg mL) of mAb was prepared in BBS immediately before use. -1 ) tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution.

[0224] Concentration was performed by using Amicon Ultra-15 low binding cellulose filters with a MWCO of 10 kDa. Centrifugation was performed in a Beckman Coulter Allegra 21K centrifuge, operating at 3500 rcf at 4°C.

[0225] The following abbreviations are used to describe antibody fragments based on their composition of heavy and light chains: heavy-heavy-light (HHL), heavy-heavy (HH), heavy-light (HL), heavy chain (H C ) and light chain (L C ).

[0226] General methods for characterizing antibodies: The LC-MS system is set up as follows. Using an Acquity UPLC system (Waters Corporation) equipped with a binary solvent manager and an automatic autosampler, 8 μL of sample is automatically injected into a BioSuite pPhenyl 1000 (Waters, 10 μm RPC 2.0×75 mm) chromatographic column at a flow rate of 100 microliters / minute. Intact protein (A = formic acid (FA) in 0.1% water, B = FA in 0.1% CH3CN) is eluted using a linear gradient from 5% to 80% B in 60 minutes. The chromatographic column outlet is directly introduced into the electrospray ionization (ESI) source of a Waters LCT-Premier XE mass spectrometer (TOF). The capillary voltage and cone voltage are set to 3000V and 100V, respectively. The desolvation and source temperatures are set to 350°C and 120°C, respectively. The conical gas flow and desolvation gas flow are set to 50L / h and 600L / h, respectively. The mass spectrometer was operated in positive polarity mode to obtain a full MS scan (400 to 4000 m / z).

[0227] Data were acquired using MassLynx software V4.1 SCN704 (Waters Inc.). The mass spectra corresponding to the chromatographic peaks were summed. Charged protein species in the resulting spectra were deconvoluted to their zero-charged mean mass using the integrated MaxEnt1 (maximum entropy) algorithm.

[0228] The output parameters are as follows: mass range 5000-70000, resolution 1Da / channel, uniform Gaussian model used, corresponding peak widths at half height.

[0229] Example 1: Preparation of (3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid (L1, DBM):

[0230]

[0231] DBM ((3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid) was prepared by the method described in Mol. Pharm. 12, 3986-3998 (2015). (1) 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). Concentration of the pure fractions afforded 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] - .

[0232] Example 2: NH2-TTDS-Dap-Lys- with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Preparation of Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (NH2-TTDS-SEQ ID NO: 1).

[0233]

[0234] For manual coupling of the first protected amino acid to the resin, coupling method 4 was applied using Fmoc-Asp(OA1)-OH (118.5 mg). Subsequent amino acids were coupled using coupling method 4 in the following order:

[0235] 46 μL of DIC and 43 mg of Oxyma were used in DMF / DCM (1:1). The mixture was allowed to react for 45 minutes with intermittent manual stirring. After each coupling, the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was removed by treatment with 20 (vol / vol)% piperidine in DMF for 30 s and then twice for 10 minutes each. Two additional treatments (2×5 minutes) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed to ensure the removal of the Fmoc group from the secondary amine (proline). The resin was cyclized according to cyclization method 2: the Fmoc group was removed with 20 (vol / vol)% piperidine in DMF, using a 30 s treatment and two 10 minute treatments. Use Boc2O (3 equivalents, 1000 micromoles, 56mg) and DIEA (30 equivalents, 3000 micromoles, 240μL) to protect the N-terminal amine with the Boc protecting group. First, the OAl group and the Alloc group are deprotected in DCM (3×15 minutes) by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 equivalent, 10μM, 12mg), phenylsilane (10 equivalents, 1000 micromoles, 123mg). The resin is washed with a DCM solution of 0.02M sodium diethylcarbamate (3×5 minutes). Then, the coupling of the amino group of Dap and the carboxylate group of aspartic acid is realized by adding PyBOP (4 equivalents, 400 micromoles, 208mg), HOAt (12 equivalents, 1.2 mmoles, 163mg), DMF (1mL / g resin to 3mL / g resin) and DIEA (12 equivalents, 1.2 mmoles, 204μL). The coupling was left for 1.5 h and repeated overnight.

[0236] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 eq., 200 mmol, 108.53 mg) was achieved by 4 eq. of DIC (400 mmol, 61 μL) and 4 eq. of HOBt (400 mmol, 54 mg) in DCM for 2 h. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group was then removed using piperidine in 20 (vol / vol)% DMF, using a 30 s treatment followed by two 10 min treatments each.

[0237] Example 3: DBM-TTDS-Dap-Lys- with an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2(DBM-TTDS-SEQ ID NO:1).

[0238] Starting from NH2-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (NH2-TTDS-SEQ ID NO: 1) prepared as in Example 2 and using DBM prepared as in Example 1, coupling method 2 was used to obtain the compound DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1).

[0239] DBM (4 eq., 400 μmol, 125 mg), PyBOP (4 eq., 400 μmol, 208 mg) and HOAt (12 eq., 1.2 mmol, 163 mg) in DMF (1 mL / g resin to 3 mL / g resin) were added to the resin in sequence, followed by 12 eq. of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling was repeated under the same conditions. The degree of coupling was checked using the Kaiser colorimetric assay. Molecular formula: C 59 H 94 Br2N 14 O 22 MW calculated value (Da): 1508.5034. MW found value (Da): 1508.4992. t R UPLC (min): 1.374. Purity: >95%. Yield: 5%.

[0240] Comparative Example 1:

[0241] NH2-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys- Preparation of Thr-Glu-Glu-Tyr-NH2 (NH2-TTDS-SEQ ID NO: 15)

[0242] For 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 using coupling method 4 in the following order:

[0243]

[0244]

[0245] 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-fluorenylmethoxycarbonyl (Fmoc) protecting group was removed by treatment with 20 (vol / vol%) piperidine in DMF for 30 s and then twice for 10 minutes each. Two additional treatments (2×5 minutes) with DBU, toluene, piperidine, DMF (5%, 5%, 20%, 70%) were performed to ensure the 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 (vol / vol)% piperidine in DMF, using a 30 s treatment and two 10 minute treatments. The N-terminal amine was protected with a Boc protecting group using Boc2O (3 eq., 1000 micromoles, 56 mg) and DIEA (30 eq., 3000 micromoles, 240 μL). The OAl and Alloc groups were first deprotected in DCM (3×15 min) by the addition of tetrakis(triphenylphosphine)palladium(0) (0.1 eq., 10 μM, 12 mg), phenylsilane (10 eq., 1000 micromoles, 123 mg). The resin was washed with a 0.02 M solution of sodium diethylcarbamate in DCM (3×5 min). The coupling of the amino group of Dap and the carboxylate group of aspartic acid was then achieved by the addition of PyBOP (4 eq., 400 micromoles, 208 mg), HOAt (12 eq., 1.2 mmoles, 163 mg), DMF (1 mL / g resin to 3 mL / g resin) and DIEA (12 eq., 1.2 mmoles, 204 μL). The coupling was left for 1.5 h and repeated overnight.

[0246] Coupling of Fmoc-TTDS-OH: Coupling of Fmoc-TTDS-OH (2 eq., 200 μmol, 108.53 mg) was achieved by 4 eq. of DIC (400 μmol, 61 μL) and 4 eq. of HOBt (400 μmol, 54 mg) in DCM for 2 h, followed by removal of the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group using 20 (v / v)% piperidine in DMF, using a 30 s treatment followed by two 10 min treatments each.

[0247] Comparative Example 2:

[0248] DBM-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys- Preparation of Thr-Glu-Glu-Tyr (DBM-TTDS-SEQ ID NO: 15)

[0249] Starting from NH2-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr (NH2-TTDS-SEQ ID NO: 15) prepared in Comparative Example 1, and using DBM prepared as in Example 1, coupling method 2 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).

[0250] DBM (4 eq., 400 μmol, 125 mg), PyBOP (4 eq., 400 μmol, 208 mg) and HOAt (12 eq., 1.2 mmol, 163 mg) in DMF (1 mL / g resin to 3 mL / g resin) were added to the resin in sequence, followed by 12 eq. of DIEA (1.2 mmol, 204 μL). The mixture was allowed to react for 1.5 h with intermittent manual stirring. The solvent was removed by suction and the resin was washed with DMF (5×30 s) and DCM (5×30 s). The coupling was repeated under the same conditions. The degree of coupling was checked using the Kaiser colorimetric assay. Molecular formula: C 124 H 176 Br2N 32 O 39 MW calculated value (Da): 2895.1139. MW found value (Da): 2895.1296. t R UPLC (min): 1.415. Purity: >95%. Yield: 10%.

[0251] Example 4: Trastuzumab-DBM-having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Preparation of TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1).

[0252] Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in Example 3 and trastuzumab following the general method for monoclonal antibody modulation, the following scheme was used for coupling to obtain the compound of formula Tz-DBM-TTDS-SEQ ID NO: 1.

[0253] Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL of sterile water, and the buffer was completely exchanged to BBS pH 8.5 using a PD10 g25 column (GE Healthcare) following the general method for monoclonal antibody regulation. The concentration was determined by UV / Vis absorbance (for trastuzumab, ε280 = 215380 M was used). -1 cm -1 ), the proteins were stored in snap-frozen aliquots at -20°C. For experiments, the aliquots were thawed and used immediately.

[0254] The coupling protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) Briefly, trastuzumab (111 μM, 4.9 mL, 544 nmoles) 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 eq.) was added, and the reaction was incubated at 37 °C for 2 h with gentle stirring. TCEP was removed by SEC using a PD10 G25 column with BBS as buffer according to the manufacturer's instructions. Subsequently, DBM peptide (Example 3) in dry DMF (10 mM, 247 μL, 4.35 μmol, 8 eq.) 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 and PBS. The final conjugate was characterized by LC-MS to confirm the integrity of the antibody after peptide conjugation.

[0255] Comparative Example 3: Trastuzumab-DBM-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys- Preparation of Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Tyr-NH2 (Tz-DBM-TTDS-SEQ ID NO: 15).

[0256] Starting from DBM-TTDS-Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Ty r-NH2 (DBM-TTDS-SEQ ID NO: 15) prepared as in Comparative Example 2 and trastuzumab following the general method of monoclonal antibody regulation, coupling was performed using the following protocol to obtain a compound of formula Tz-DBM-TTDS-SEQ ID NO: 15.

[0257] Trastuzumab was obtained in its clinical form (Roche, lyophilized), resuspended in 7.2 mL of sterile water, and the buffer was completely exchanged to BBS pH 8.5 using a PD10 g25 column (GE Healthcare) following the general method for monoclonal antibody regulation. The concentration was determined by UV / Vis absorbance (for trastuzumab, ε280 = 215380 M was used).-1 cm -1 ), the proteins were stored in snap-frozen aliquots at -20°C. For experiments, the aliquots were thawed and used immediately.

[0258] The coupling protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) Briefly, trastuzumab (111 μM, 4.9 mL, 544 nanomoles) 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 micromoles, 6 equivalents) was added, and the reaction was incubated at 37 °C for 2 h with gentle stirring. TCEP was removed by SEC using a PD10 G25 column with BBS as buffer according to the manufacturer's instructions. Subsequently, DBM peptide (Comparative Example 2) in dry DMF (10 mM, 247 μL, 4.35 micromoles, 8 equivalents) 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 and PBS. The final conjugate was characterized by LC-MS to confirm the integrity of the antibody after peptide conjugation.

[0259] Example 5: Cetuximab-DBM-having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Preparation of TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Cx-DBM-TTDS-SEQ ID NO: 1).

[0260] Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO: 1) prepared as in Example 3 and cetuximab following the general method for monoclonal antibody modulation, the following scheme was used for coupling to obtain a compound of formula Cx-DBM-TTDS-SEQ ID NO: 1.

[0261] Cetuximab was obtained in its clinical form (SelleckChem, lyophilized), resuspended in sterile water, and the buffer was completely exchanged into BBS, pH 8.5, using a PD10 g25 column (GE Healthcare) following the general method for monoclonal antibody regulation. The concentration was determined by UV / Vis absorbance (for cetuximab, ε280 = 215380 M was used). -1 cm -1 ), the proteins were stored in snap-frozen aliquots at -20°C. For experiments, the aliquots were thawed and used immediately.

[0262] The coupling protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) Briefly, cetuximab (1 mg, 660 nanomoles) 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 micromoles, 6 equivalents) was added, and the reaction was incubated at 37°C for 2 h with gentle stirring. DTT was removed by SEC using a PD10 G25 column with BBS as buffer according to the manufacturer's instructions. Subsequently, the DBM peptide (Example 3) was added to the reduced cetuximab in dry DMF (10 mM, 5.3 μL, 3.52 micromoles, 8 equivalents), and the reaction was left at room temperature for 30 minutes. Excess reagents were then removed by SEC using a PD10 G25 column and PBS. The final conjugate was characterized by LC-MS to confirm the integrity of the antibody after peptide coupling. The masses of Cetuximab and Cetuximab-DBM-TTDS-SEQ ID NO: 1 were characterized by deglycosylation with PNGase F as 148182 and 153654, respectively.

[0263] Example 6: Bevacizumab-DBM-having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Preparation of TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Bv-DBM-TTDS-SEQ ID NO: 1).

[0264] Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO:1) prepared as in Example 3 and bevacizumab following the general method of monoclonal antibody regulation, the following scheme was used for coupling to obtain the compound of formula Bv-DBM-TTDS-SEQ ID NO:1.

[0265] Bevacizumab was obtained in its clinical form (HSJD, lyophilized), resuspended in sterile water, and the buffer was completely exchanged into BBS, pH 8.5, using a PD10 g25 column (GE Healthcare) following the general method for monoclonal antibody regulation. The concentration was determined by UV / Vis absorbance (for bevacizumab, ε280 = 215380 M was used). -1 cm -1 ), the proteins were stored in snap-frozen aliquots at -20°C. For experiments, the aliquots were thawed and used immediately.

[0266] The coupling protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) Briefly, bevacizumab (1 mg, 660 nanomoles) 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 micromoles, 6 equivalents) was added, and the reaction was incubated at 37°C for 2 h with gentle stirring. DTT was removed by SEC using a PD10 G25 column with BBS as buffer according to the manufacturer's instructions. Subsequently, the DBM peptide (Example 3) in dry DMF (10 mM, 5.3 μL, 3.52 micromoles, 8 equivalents) was added to the reduced bevacizumab, and the reaction was left at room temperature for 30 minutes. Excess reagents were then removed by SEC using a PD10 G25 column and PBS. The final conjugate was characterized by LC-MS to confirm the integrity of the antibody after peptide coupling. The masses of bevacizumab and bevacizumab-DBM-TTDS-SEQ ID NO: 1 were characterized by deglycosylation with PNGase F as 146322 and 151794, respectively.

[0267] Example 7: Pertuzumab-DBM-having an amide bond between the Dap side chain amino group and the Asp side chain carboxylic acid Preparation of TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-DBM-TTDS-SEQ ID NO: 1).

[0268] Starting from DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (DBM-TTDS-SEQ ID NO:1) prepared as in Example 3 and Pertuzumab following the general method for monoclonal antibody modulation, the following scheme was used for coupling to obtain a compound of formula Pt-DBM-TTDS-SEQ ID NO:1.

[0269] Pertuzumab was obtained in its clinical form (SelleckChem, lyophilized), resuspended in sterile water, and the buffer was completely exchanged into BBS, pH 8.5, using a PD10 g25 column (GE Healthcare) following the general method for monoclonal antibody regulation. The concentration was determined by UV / Vis absorbance (for Pertuzumab, ε280 = 215380 M was used). -1 cm -1 ), the proteins were stored in snap-frozen aliquots at -20°C. For experiments, the aliquots were thawed and used immediately.

[0270] The coupling protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). (4) Briefly, pertuzumab (1 mg, 660 nanomoles) was diluted to a final concentration of 22.9 μM with BBS (pH 8.5). A fresh solution of DTT (10 mM, 4 μL, 2.64 micromoles, 6 equivalents) was added, and the reaction was incubated at 37°C for 2 h with gentle stirring. DTT was removed by SEC using a PD10 G25 column with BBS as buffer according to the manufacturer's instructions. Subsequently, DBM-peptide (Example 3) in dry DMF (10 mM, 5.3 μL, 3.52 μmol, 8 equivalents) was added to the reduced pertuzumab, and the reaction was left at room temperature for 30 min. Excess reagents were then removed by SEC using a PD10 G25 column and PBS. The final conjugate was characterized by LC-MS to confirm the integrity of the antibody after peptide coupling. The masses of Pertuzumab and Pertuzumab-DBM-TTDS-SEQ ID NO: 1 were characterized by deglycosylation with PNGase F to be 145214 and 150686, respectively.

[0271] Embodiment 8: Stability of shuttle agents in mouse serum.

[0272] A major advantage of the shuttle agents of the present invention is that, unlike the vast majority of peptides composed only of L-amino acids (which are rapidly metabolized by a range of enzymes present in blood serum, thus limiting their therapeutic effect), these peptides are made of D-amino acids and are therefore not recognized by metabolizing enzymes present in serum, thereby significantly increasing their half-life in serum.

[0273] Regarding the stability study of the BBB-shuttle peptides present in Example 4 and Comparative Example 3 in mouse serum, these peptides were incubated at a concentration of 150 μM in HBSS buffer at 37° C. in the presence of 90% mouse serum. At a series of times, 50 μL aliquots were collected, to which methanol was added to precipitate serum proteins. The samples were centrifuged, filtered, and analyzed by HPLC to determine the degree of degradation. Figure 7 Stability studies of the BBB-shuttle agent peptides presented in Example 4 and Comparative Example 3 in mouse serum are shown.

[0274] Example 9: Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) Binding to breast cancer cells.

[0275] In vitro binding to HER2-positive BT-474 and SKBR-3 breast cancer cells was determined by flow cytometry. Confluent cells were detached from the flask with trypsin and neutralized with DMEM supplemented with FBS. Cells in suspension were washed in ice-cold PBS, counted and separated into individual 1.5 mL tubes (10 cells per tube).6 cells).

[0276] The binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) was carried out in ice-cold PBS at increasing concentrations at 4°C for 30 minutes. The cells were then washed and incubated with anti-human Dylight 650 secondary antibody (Abcam plc) at 4°C for 30 minutes in ice-cold PBS. The cells were washed with ice-cold PBS and analyzed by flow cytometry (10,000 gated events under each condition). Figure 8 The binding of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) to HER-2 overexpressing cells is shown.

[0277] Example 10: Binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to breast cancer cells.

[0278] 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 and neutralized with DMEM supplemented with FBS. The cells in suspension were washed in ice-cold PBS, counted and separated into individual 1.5 mL tubes (10 cells per tube). 6 cells).

[0279] Binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) was performed in ice-cold PBS at increasing concentrations at 4°C for 30 minutes. The cells were then washed and incubated with anti-human Dylight 488 secondary antibody (Abcam plc) in ice-cold PBS for 30 minutes at 4°C. The cells were washed with ice-cold PBS and analyzed by flow cytometry (2000 gated events per condition). Fig. 9 Binding of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5) to EGFR-positive MDA-MB-231 breast cancer cells is shown.

[0280] Example 11: Binding of Pt, Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to breast cancer cells.

[0281] The in vitro binding ability of Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to HER2-positive BT-474 breast cancer cells was determined by flow cytometry. The fused cells were separated from the culture flask with trypsin and neutralized with DMEM supplemented with FBS. The suspended cells were washed in ice-cold PBS, counted, and separated into separate 1.5 mL tubes (10 cells per tube). 6 cells).

[0282] Binding experiments were performed for Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) with increasing concentrations in ice-cold PBS for 30 minutes at 4°C. The cells were then washed and incubated with anti-human Dylight 488 secondary antibody (Abcam plc) in ice-cold PBS for 30 minutes at 4°C. After washing the cells with ice-cold PBS, they were analyzed by flow cytometry (2000 gated events per condition). Fig.10 The binding of Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) to HER-2 overexpressing cells is shown.

[0283] Example 12: Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM- Cell cycle arrest of TTDS-SEQ ID NO: 15 (Comparative Example 3).

[0284] 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 1 mg / mL RNaseA (EURX Ltd. Gdansk, Poland), and stained with propidium iodide (2.5 μg / ml). Cell cycle was analyzed using a BD LSR II flow cytometer (BD Biosciences).

[0285] 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 cells, BT-474 cells 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. The cells were stained with propidium iodide and the cell cycle was analyzed by flow cytometry. The results are shown in Figure 2. Fig.11 shown.

[0286] 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

[0287] Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) were radiolabeled using PierceTM iodinated beads (Life Technologies). Briefly, two beads were used for each protein, washed with 500 μL of reaction buffer (50 mM NaPi, pH 6.5) and dried on filter paper. In a glass vial, the calculated amount of carrier-free Na 125 I (1 mCi / mg protein) was added to the beads in 200 μL of reaction buffer. The reaction was incubated for 5 min. The protein was then added and reacted for 15 min with occasional mixing. The reaction was stopped by removing the solution from the reaction vessel and adding it to a PD MiniTrap G-25 column (GE Healthcare) previously equilibrated with PBS. The iodinated protein was dialyzed ( Mini dialysis device, 20 KDa, 0.5 mL) was used with PBS overnight to further remove unincorporated 125 I. The radioactivity of 10 μL fractions was measured for 2 minutes using a Packard Cobra II Gamma counter and the protein concentration was determined using the BCA assay (ThermoScientific). The samples were diluted with Ringer Hepes to a final concentration of 100 nM.

[0288] Example 14: Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5); Bv, Bv-DBM-TTDS-SEQ ID AlexaFluor488-NHS labeling and quantitative analysis of SEQ ID NO:1 (Example 6), Pt, and Pt-DBM-TTDS-SEQ ID NO:1 (Example 7) quantity

[0289] Briefly, two 0.2 μL aliquots of AlexaFluor 488 (10 mg mL -1 100 μL of selected mAb (0.25 mg, 82 nmol, NaPi pH 8) was added every 15 min in DMSO and samples were mixed in the dark for one hour. Excess dye was removed by SEC using PDmini G25 columns with PBS as buffer according to the manufacturer's instructions.

[0290] Example 15: Permeability assay in an in vitro human BBB cell model

[0291] These experiments were performed using a model developed in the laboratory of Professor R. Cecchelli. (5) Briefly, endothelial cells derived from pluripotent stem cells and bovine pericytes were thawed in gelatin-coated culture 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 in 12-well Transwell chambers (8,000 cells / well) and pericytes were seeded in 12-well plates previously coated with Matrigel and gelatin, respectively (50,000 cells / well). Both cell lines used sECM culture medium, which was changed every 2 to 3 days. Seven to eight days after seeding, the assay was performed by placing the chamber containing the endothelial cells into a new well without pericytes.

[0292] For the assay, 500 μL of unlabeled or 125 I-labeled mAb was added to the donor compartment in ECM medium or Ringer's HEPES solution, and 1500 μL of ECM medium or Ringer's HEPES solution was introduced into the recipient compartment. Lucifer yellow (25 μM) was added as a barrier integrity (Papp < ​​15·10 -6 cm / s). For unlabeled compounds, the plates were incubated for 16 h, but 500 μL was removed from the receptor compartment after 2 h for analysis and replaced with fresh medium. 125 I-labeled monoclonal antibodies, the plates were incubated at 37°C for 2 h, and the solutions in both compartments were recovered and analyzed. For AlexaFluor 488-labeled monoclonal antibodies, the plates were incubated at 37°C for 2 h, and the solutions in both compartments were recovered 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:

[0293]

[0294] Among them, P app In cm / s, Q A (t) is the amount of compound in the receptor well at time t, V D is the volume in the donor pore, t is the time (s), A is the area of ​​the membrane (cm 2 ), Q D (t0) is the amount of compound in the donor compartment at the beginning of the experiment.

[0295] Proteins from the receptor compartment for MS analysis were purified by immunoprecipitation using protein A magnetic beads following the manufacturer's instructions. Briefly, 25 μL of magnetic beads were placed in a 1.5 mL microcentrifuge tube, diluted with PBST and gently mixed. The tube was placed in a magnetic stand to facilitate the removal of the supernatant. 500 μL of PBS solution was added to the tube to wash the beads. After mixing, the solution was removed after collecting the beads with the magnetic stand. This operation was repeated 3 times. 1 mL of the receptor solution was added and mixed with the beads o / n at 4°C. The supernatant was then discarded and the magnetic beads were washed (3×500 μL PBST and 3×500 μL PBS). The mAb was eluted with 50 μL 0.1 M glycine, pH 2, and the solution was neutralized with 8 μL 3 M Tris, pH 8.5. The samples were analyzed by LCT-MS. Fig.12 The permeability results of Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) in the human in vitro BBB cell model are shown. Fig.13 , Fig.14 , Fig.15 The permeability results of Cx, Cx-DBM-TTDS-SEQ ID NO: 1 (Example 5), Bv, Bv-DBM-TTDS-SEQ ID NO: 1 (Example 6), Pt and Pt-DBM-TTDS-SEQ ID NO: 1 (Example 7) in the same model are shown respectively. Fig.16 A comparison of the permeability of these conjugates is shown.

[0296] Example 16: Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) Biodistribution study in mice.

[0297] The biodistribution study of mice was conducted by ChemPartner Animal Experiment Center according to the protocol approved by ChemPartner Institutional Animal Care and Use Committee (IACUC), following the guidelines for laboratory animal care and assessment (AAALAC). Tz, Tz-DBM-TTDS-SEQ ID NO: 1 (Example 4) and Tz-DBM-TTDS-SEQ ID NO: 15 (Comparative Example 3) (10 mg / kg) were injected into CD-1 male mice aged 6 to 8 weeks by tail vein injection. Blood was collected 8 hours after injection to generate serum, and the brain was collected at the terminal. The amount of antibody in brain tissue and serum was determined by ELISA (goat anti-human IgG F (c) antibody, Sigma, number # 609-101-017, anti-human IgG (Fab specific)-peroxidase, Sigma, number # A0293). Fig.17 Biodistribution studies in mice are shown.

[0298] Example 17: Coupling of DBM peptide to IgG1 mAb at pH 8.5

[0299] The coupling protocol was adapted from Org. Biomol. Chem. 15, 2947-2952 (2017). Briefly, the selected monoclonal antibody (mAb) (1 mg, 660 nanomoles) was diluted with BBS (pH 8.5) to a final concentration of 22.9 μM. Freshly prepared DTT solution (10 mM, 4 μL, 2.64 micromoles, 6 equivalents) was added and incubated at 37 ° C for 2 hours with gentle stirring. DTT was removed by SEC using a PDmini G25 column using BBS as a buffer according to the manufacturer's instructions. Subsequently, DBM peptide in dry DMF (10 mM, 5.3 μL, 3.52 micromoles, 8 equivalents) was added to the reduced mAb, and the reaction was left at room temperature for 30 minutes. Excess reagents were then removed by SEC using a PDmini G25 column containing PBS. The final conjugate was characterized by LC-MS. The DBM peptides used were:

[0300] DBM-TTDS-MiniAp4 ([M+H+] theoretical value: 1509.269Da; [M+H+] experimental value: 1508.517Da

[0301]

[0302] DBM-TTDS-SN38-MiniAp4 (M+H+] theoretical value: 2125.900Da; [M+H+] experimental value: 2126.710Da

[0303]

[0304] DBM-TDS-Ang2( [M+H+] theoretical value: 2898.765Da; [M+2H+ / 2] experimental value: 1449.571Da)

[0305]

[0306] DBM-TTDS-SN38-Ang2 ([M+H+] theoretical value: 3516.380Da; [M+2H+ / 2] experimental value: 1758.172Da)

[0307]

[0308] Example 18: Labeling of conjugates with AlexaFluor 488-NHS

[0309] use All antibodies were labeled with 488 (λex: 485 / λem: 535 nm) for transport analysis, where antibodies were analyzed at 500 nm and quantified using a fluorimeter.

[0310] Briefly, two aliquots of 0.2 μL of AlexaFluor 488 (10 mg mL in DMSO) were added every 15 min. -1 ) was added to 100 μL of the selected mAb (0.25 mg, 82 nmol, NaPi pH 8) and the samples were mixed for one hour in the dark. Excess dye was removed by SEC using PDmini G25 columns with PBS as buffer according to the manufacturer's instructions.

[0311] Example 19: Permeability determination of in vitro human BBB cell model

[0312] These experiments were performed using a model developed in the laboratory of Professor R. Cecchelli. (5) Briefly, endothelial cells derived from pluripotent stem cells and bovine pericytes were thawed in gelatin-coated culture dishes (Corning). Pericytes were cultured in DMEM pH 6.8, while endothelial cells were cultured in supplemented endothelial cell growth medium (sECM) (Sciencecells). After 48 h, endothelial cells were seeded in 12-well Transwell inserts (8,000 cells / well) and pericytes were seeded in 12-well plates pre-coated with matrigel and gelatin, respectively (50,000 cells / well). sECM medium was used for both cell lines and was changed every 2 to 3 days. The assay was performed 7 to 8 days after seeding by placing the insert containing the endothelial cells into a new well without pericytes.

[0313] To perform the assay, 500 μL of fluorescently labeled mAb (1 μM) in Ringer's HEPES solution was added to the donor chamber, and 1500 μL of Ringer's HEPES solution was added to the receptor chamber. Lucifer yellow (25 μM) was used to assess barrier integrity (Papp < ​​15 10 -6 cm / s). The monoclonal antibody was placed at 37°C for 2 h, and the solution was recovered from both compartments and analyzed by fluorescence (λex: 485 nm; λem: 535 nm). Samples were evaluated in triplicate. A gamma counter was used to quantify the amount of protein, and the following formula was used to quantify the apparent permeability:

[0314]

[0315] Where P app Obtained in cm / s, Q A (t) is the amount of compound in the receptor pore at time t, V Dis the volume in the donor pore, t is the time in seconds, A is the membrane area in cm, Q D (t0) is the amount of compound in the donor compartment at the beginning of the experiment.

[0316] The results are as follows Fig.18 The results show that the permeability of the modified antibody (Cx-DBM_MiniAp4) according to the present invention is higher than that of the antibody (Cx-mal-MiniAp4) modified with maleimide MiniAp4 in Example 23 of WO2015 / 001015A1.

[0317] on the other hand, Fig.19 The ratio of Papp of Cx modified with peptide shuttles (MiniAp4 or Ang2) conjugated to SN38 and Cx modified with naked shuttles (MiniAp4 and Ang2) measured at 1 μM in a human in vitro BBB cell model is shown. The results show that the MiniAp4 conjugate increases the transport of SN38 more effectively than the Ang2 conjugate.

[0318] Terms

[0319] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0320] Item 1: an antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof,

[0321]

[0322] It contains 1 to 6 disulfide bonds inserted into the antibody in the form of -P-(W)sY and connected via a linker -[(L1)-(L2)-(L3) m -] a peptide of formula P linked to a sulfide;

[0323] in:

[0324] Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof;

[0325] The disulfide bond is any disulfide bond originally present in the antibody and capable of structurally retaining a sulfide bond in the structure under reducing conditions, the disulfide bond being selected from the group consisting of the naturally occurring interchain disulfide bonds of the antibody, the naturally occurring intrachain disulfide bonds of the antibody, and the disulfide bonds introduced into the antibody by genetic engineering;

[0326] L1 is selected from L 1a and L 1b The connector;

[0327]

[0328] q is an integer from 1 to 6;

[0329] L1 is linked to -S- of the antibody disulfide bond via bonds a and b, and bond c is linked to linker L2 via an amide bond, ester bond, or thioester bond between the C=O group immediately adjacent to bond c of linker L1 and the NH group, O group, or S group on the left side of the drawn LA of linker L2 below;

[0330] L2 is a diradical containing 2 to 8 radicals selected from LA, LB, LC, and having the formula -LA-(LB) u -LC-.

[0331] LA is a diradical selected from the following: -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-;

[0332] LB is a diradical independently selected from the following: -NH-(CH2) r’ -C(=O)-; -C(=O)-(CH2) r’ -C(=O)-; -S-(CH2) r’ -C(=O)-; -O-(CH2) r’ -C(=O)-; -NH-(CH2) r’ -; -C(=O)-(CH2) r’ -;-S-(CH2) r’ -;-O-(CH2) r’ -;-NH-CH-((CH2) 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 combinations thereof;

[0333] LC is a diradical selected from the following: -NH-(CH2) r’ -C(=O)-; -NH-CH-((CH2) r’ -NH2)-C(=O)-;-C(=O)-(CH2) r’ -C(=O)-; -S-(CH2) r’ -C(=O)-;-S-CH2-CH(NH2)-C(=O)-;-O-(CH2) r’ -C(=O)-; -(CH2) r’ -C(=O)-;

[0334] u is an integer from 0 to 6;

[0335] r' is an integer from 1 to 5;

[0336] When u=0, LA is linked to the diradical LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LA formula and the functional group on the left side of the LC formula;

[0337] When u=1, LA is linked to the diradical LB via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LA formula and the functional group on the left side of the LB formula; and LB is linked to the diradical LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LB formula and the functional group on the left side of the LC formula;

[0338] When u is greater than 1, LB is the same or different, and they are connected via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester; one end of LB is connected to LA via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, and the bond is formed between the functional group on the right side of the LA formula and the functional group on the left side of the LB formula; and the other end of LB is connected to LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, and the bond is formed between the functional group on the right side of the LB formula and the functional group on the left side of the LC formula;

[0339] L3 is a diradical selected from the following: an amino acid selected from Lys, Orn, Dap, Dab; ​​Glu, and Asp; selected from -C(=O)-(CH2) r-C(=O)-; -C(=O)-(CH2) t -NH-; -C(=O)-(CH2) t -S-; -C(=O)-(CH2) t Amino acid derivatives of Lys, Orn, Dap and Dab derived from the amino acid side chain by connecting the diradical of -O- to the amino group of the amino acid side chain, wherein the amino group is connected to the C=O terminal group on the left side of the diradical; selected from the group selected from -NH-(CH2)t r -C(=O)-; -NH-(CH2) t -NH-; -NH-(CH2) t -S-; -NH-(CH2) t Amino acid derivatives of Glu and Asp derived from the diradical of -O- linked to the C=O group of the amino acid side chain, wherein the diradical is linked to the C=O group via the NH group on the left side of the diradical; and any previous amino acid or amino acid derivative of CH2CH2NCH2CO2H)4(DOTA) or streptavidin is also linked via a feasible bond;

[0340] t is an integer from 1 to 5;

[0341] m is an integer selected from 0 or 1;

[0342] D is a substance connected to the linker L3, which is selected from a biologically active substance, a substance used in a diagnostic method; and a radioligand used in radiotherapy;

[0343] P is a single peptide diradical which is the same or different and is selected from: (a) a peptide comprising the amino acid sequence X1KAPETALX2, which has an intrapeptide bond between X1 and X2 which is an amide bond; wherein X1 is selected from Dap (2,3-diaminopropionic acid) and Dab (2,4-diaminobutyric acid); X2 is selected from D (aspartic acid) and E (glutamic acid); i.e.

[0344]

[0345] (b) a peptide of 12 to 20 amino acid residues in length having at least an intrapeptide bond that is a disulfide bond or a diselenide bond, and comprising the following amino acid sequence: X3KAPETALX4AAA; having at least an intrapeptide disulfide bond or an intrapeptide diselenide bond between X3 and X4, wherein X3 and X4 are identical and are selected from C (cysteine), Sec (selenocysteine) and Pen (penicillamine); i.e.

[0346]

[0347] (c) a peptide of 9 to 11 amino acid residues in length having at least an intrapeptide bond that is a disulfide bond or a diselenide bond, and consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6; X5KAPETALX6A; and X5KAPETALX6AA having at least an intrapeptide disulfide bond or an intrapeptide diselenide bond between X5 and X6; wherein X5 and X6 are identical and are selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine), i.e.

[0348]

[0349] (d) having 16 amino acid residues and comprising the amino acid sequence X7NX8KAPETALX9AAAX 10 H peptide, between X7 and X9 and between X8 and X 10 There is an intra-peptide disulfide bond or an intra-peptide diselenide bond between them; wherein X7-X 10 are independently selected from C (cysteine), Sec (selenocysteine) and Pen (penicillamine); provided that X7 and X9 are the same, and X8 and X 10 the same; that is

[0350]

[0351] and (e) a peptide comprising the amino acid sequence X1KAPETALX2, wherein X1 is selected from Dap and Dab; ​​and X2 is a linear peptide selected from D (aspartic acid) and E (glutamic acid) (SEQ ID NO: 7);

[0352] W is selected from -NH-(CH2) r -C(=O)- and -NH-CH((CH2) r NH2)-C(=O)- diradical;

[0353] r is an integer independently selected from 1 to 5;

[0354] s is an integer independently selected from 0 to 1;

[0355] Y is a free radical selected from -NH2, -OH, -OR3 and -NHR3;

[0356] When m=0, L3 and D are absent, and P is directly linked to 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 of the peptide sequence P;

[0357] When m=1, D is present and connected to a functional group of an amino acid side chain of linker L3, or to an amino acid derivative of linker L3, by derivatization thereof, wherein the connection is made by an amide, ester, disulfide bond or thioester bond; L3 is connected to the LC of L2 by an amide bond formed between the C=O terminal group on the left side of the LC formula and the amine group of linker L3; and P is directly connected to L3 by an amide bond formed between the C=O terminal group on the right side of the LC formula and the amine group of the first amino acid of the peptide sequence P; and

[0358] When s=0, P is directly linked to Y via an amide bond, a carboxylic acid bond or an 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 free radical Y, the free radical Y being -NH2, -OH, -OR3 or -NHR3; and

[0359] When s=1, P is linked to the free radical W through an amide bond formed between the free radical W and the C=O at the C-terminus of the last amino acid of the sequence P, the bond being formed between the functional group on the left side of the drawn W formula and the functional group (C=O) at the C-terminus of the last amino acid of the sequence P on the right side of the drawn sequence; and W is linked to Y as follows: -C(=O)-NH-(CH2) r- C(=O)-Y, or -C(=O)-NH-CH((CH2) r NH2)-C(=O)-Y;

[0360] n is an integer independently selected from 1 to 6;

[0361] Represents a connection point; and

[0362] S in the formula (I) represents a sulfide.

[0363] Item 2: The antibody shuttle agent conjugate according to Item 1, which has four copies of the peptide P, and all the peptides are identical.

[0364] Clause 3: The antibody shuttle agent conjugate according to any one of clauses 1 to 2, wherein the disulfide bond is an interchain bond.

[0365] Clause 4: The antibody shuttle agent conjugate according to any one of clauses 1 to 3, wherein P is a diradical of a peptide selected from:

[0366] (a) A peptide comprising the amino acid sequence DapKAPETALD, which has an intrapeptide bond between Dap and D, which is an amide bond, i.e., SEQ ID NO: 8:

[0367] (b) a peptide of 9 to 20 amino acid residues in length having intrapeptide bonds that are at least disulfide bonds and comprising the following amino acid sequence:

[0368] CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, i.e., SEQ ID NO: 9:

[0369] (c) a peptide of 9 to 11 amino acid residues in length having an intrapeptide bond that is at least a disulfide bond and consisting of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA having at least an intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, i.e.

[0370]

[0371] and

[0372] (d) a peptide having 16 amino acid residues and comprising the amino acid sequence CNCKAPETALCAAACH, which has intrapeptide disulfide bonds between the first and third cysteine ​​residues, which are cysteine ​​1 and cysteine ​​11, and between the second and fourth cysteine ​​residues, which are cysteine ​​3 and cysteine ​​15, i.e.,

[0373]

[0374] (e) A peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

[0375] Item 5: The antibody shuttle agent conjugate according to Item 4, wherein P is a diradical of a peptide selected from:

[0376] (a) a peptide having an amino acid sequence of DapKAPETALD, which has an intrapeptide bond between Dap and D that is an amide bond (SEQ ID NO: 7);

[0377] (b) a peptide having the amino acid sequence CKAPETALC, which has at least an intrapeptide disulfide bond between the cysteines at positions 1 and 9 (SEQ ID NO: 10);

[0378] (c) A peptide having the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

[0379] Clause 6: The antibody shuttle agent conjugate according to any one of clauses 1 to 5, wherein the antibody is selected from trastuzumab, bevacizumab, cetuximab, pertuzumab, aducanumab, bapizumab, nimotuzumab and necituzumab.

[0380] Item 7: The antibody shuttle conjugate according to Item 1, which is selected from trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-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; Cetuximab

[0381] -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; Bevacizumab

[0382] -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; and Pertuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-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, wherein DBM is the linker L 1a And TTDS is connector L 2a .

[0383] Clause 8: The antibody shuttle agent conjugate according to Clause 1, wherein:

[0384] a) the antibody is an anti-cancer therapeutic antibody, m=1, and the free radical of the active biological substance D is a free radical of an anti-cancer active pharmaceutical ingredient selected from the group consisting of auristatins, duocarmycins, PBD dimers, maytansines, calicheamicins, anthracyclines, camptothecins, α-amanitin, tubulysin, MMAE, T-DM1 and PROTAC moieties; or alternatively,

[0385] b) The antibody is an anti-neurodegeneration therapeutic antibody, m=1 and D is a free radical of an anti-neurodegeneration active pharmaceutical ingredient.

[0386] Clause 9: A method for preparing an antibody shuttle agent conjugate as described in any one of clauses 1 to 8, comprising:

[0387] a) reducing the disulfide bridges of the antibody;

[0388] b) by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II) to rebridge the disulfide bridges,

[0389]

[0390] c) optionally, performing hydrolysis;

[0391] wherein q; L2, L3, D, P, m, W, s and Y are as defined in the antibody shuttle agent conjugate of formula (I).

[0392] Clause 10: A pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle agent conjugate as defined in any one of Clauses 1 to 8 and a suitable amount of a pharmaceutically acceptable carrier or excipient.

[0393] Clause 11: An antibody shuttle agent conjugate as defined in any one of clauses 1 to 8 for use as a medicament.

[0394] Clause 12: An antibody shuttle agent conjugate as described in any one of Clauses 1 to 8 for use in treating a CNS disorder in a mammal including a human.

[0395] Item 13: The antibody shuttle agent conjugate for use according to Item 12, wherein the CNS disorder is cancer.

[0396] Item 14: The antibody shuttle agent conjugate for use according to any one of items 12 to 13, wherein the antibody shuttle agent conjugate is used in combination therapy with conventional chemotherapeutic agents or radiotherapy.

[0397] Clause 15: An antibody shuttle agent conjugate as defined in any one of clauses 1 to 8 for use as a diagnostic agent or radioligand for radiotherapy.

[0398] Reference List

[0399] Patent Literature

[0400] WO2015 / 001015A1

[0401] Non-patent literature

[0402] -Anthony Regina et al., in "ANG4043, a novel brain-penetrant Peptide-mAbconjugate, is efficacious against HER2-positive Intracranial Tumors in Mice", mct.aacrjournals.org

[0403] -Abstract Macarena Sánchez 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)

[0404] -M. Amblard et al., "Methods and protocols of modern solid-phase peptide synthesis" Molecular Biotechnology 2006, Vol. 33, p. 239-254)

[0405] -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)

[0406] -A. Madder et al., Eur. J. Org. Chem. 1999, pp. 2787-2791

[0407] -P. L. Barker et al., J. Med. Chem., 1992. vol 35, pp. 2040-2048.

[0408] -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

[0409] -M. Kazemi et al., Journal of Sulfur Chemistry, 2015, vol. 36:6, pp. 613-623.

[0410] - M.T. Nguyen et al., J. Org. Chem. 1998, 63, vol. 20, pp. 6878 - 6885.

[0411] - F. David et al., Org. Process Res. Dev. 2010, 14, 4, pp. 999 - 1007.

[0412] - https: / / www.cliffsnotes.com / study-guides / chemistry / organic-chemistry-ii / aldehydes-and-ketones / reactions-of-aldehydes-and-ketones.

[0413] - Eur J Pharm Biopharm, 2017, 115, 149 - 158 or triphosgene as described in J. Med. Chem. 2008, 51, 21, 6916 - 6926.

[0414] - Dongen et al.; Bioconjugate Chem. 2009, vol. 20, pp. 20 - 23.

[0415] - CA2957354A1

Claims

1. An antibody shuttle agent conjugate of formula (I) or a pharmaceutically acceptable salt thereof, It contains 1 to 6 disulfide bonds inserted into the antibody in the form of -P-(W)sY and connected via a linker -[(L1)-(L2)-(L3) m - a peptide of formula P linked to a sulfide; in: Z represents the structure of a monoclonal antibody or a monoclonal antibody fragment thereof; The disulfide bond is any disulfide bond originally present in the antibody and capable of structurally retaining a sulfide bond in the structure under reducing conditions; the disulfide bond is selected from the group consisting of the naturally occurring interchain disulfide bond of the antibody, the naturally occurring intrachain disulfide bond of the antibody, and the disulfide bond introduced into the antibody by genetic engineering; L1 is selected from L 1a and L 1b The connector; q is an integer from 1 to 6; L1 is linked to the -S- of the disulfide bond of the antibody via bonds a and b, and bond c is linked to the linker L2 via an amide bond, an ester bond, or a thioester bond between the C=O group immediately adjacent to bond c of linker L1 and the NH group, O group, or S group on the left side of the drawn LA of the following linker L2; L2 is a diradical containing 2 to 8 radicals selected from LA, LB, LC, and having the formula -LA-(LB) u -LC- LA is a diradical selected from the following: -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 diradical independently selected from the following: -NH-(CH2) r’ -C(=O)-; -C(=O)-(CH2) r’ -C(=O)-; -S-(CH2) r’ -C(=O)-; -O-(CH2) r’ -C(=O)-; -NH-(CH2) r’ -; -C(=O)-(CH2) r’ -;-S-(CH2) r’ -;-O-(CH2) r’ -;-NH-CH-((CH2) 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 combinations thereof; LC is a diradical selected from the following: -NH-(CH2) r’ -C(=O)-; -NH-CH-((CH2) r’ -NH2)-C(=O)-;-C(=O)-(CH2) r’ -C(=O)-; -S-(CH2) r’ -C(=O)-;-S-CH2-CH(NH2)-C(=O)-;-O-(CH2) r’ -C(=O)-; -(CH2) r’ -C(=O)-; u is an integer from 0 to 6; r' is an integer from 1 to 5; When u=0, LA is linked to the diradical LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LA formula and the functional group on the left side of the LC formula; When u=1, LA is linked to the diradical LB via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LA formula and the functional group on the left side of the LB formula; and LB is linked to the diradical LC via a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, the bond being formed between the functional group on the right side of the LB formula and the functional group on the left side of the LC formula; When u is greater than 1, LB is the same or different, and they are connected by a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester; one end of LB is connected to LA by a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, and the bond is formed between the functional group on the right side of the LA formula and the functional group on the left side of the LB formula; and the other end of LB is connected to LC by a chemically feasible bond selected from amine, amide, ether, thioether, disulfide, ester and thioester, and the bond is formed between the functional group on the right side of the LB formula and the functional group on the left side of the LC formula; L3 is a diradical selected from the following: an amino acid selected from Lys, Orn, Dap, Dab; ​​Glu, and Asp; selected from -C(=O)-(CH2) r -C(=O)-; -C(=O)-(CH2) t -NH-; -C(=O)-(CH2) t -S-; -C(=O)-(CH2) t Amino acid derivatives of Lys, Orn, Dap and Dab derived from a diradical of -O- linked to the amino group of the amino acid side chain, wherein the C=O terminal group on the left side of the diradical is linked to the amino group; selected from the group consisting of -NH-(CH2)t r -C(=O)-; -NH-(CH2) t -NH-; -NH-(CH2) t -S-; -NH-(CH2) t Amino acid derivatives of Glu and Asp derived from the diradical of -O- linked to the C=O group of the amino acid side chain, wherein the diradical is linked to the C=O group via the NH group on the left side of the diradical; and any previous amino acid or amino acid derivative of CH2CH2NCH2CO2H)4(DOTA) or streptavidin is also linked via a feasible bond; t is an integer from 1 to 5; m is an integer selected from 0 or 1; D is a substance connected to the linker L3, which is selected from biologically active substances, substances used in diagnostic methods; and radioligands used in radiotherapy; P is a single peptide diradical which may be identical or different and is selected from: (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 Dap (2,3-diaminopropionic acid) and Dab (2,4-diaminobutyric acid); and X2 is selected from D (aspartic acid) and E (glutamic acid); i.e. (b) a peptide of 12 to 20 amino acid residues in length having at least an intrapeptide bond that is a disulfide bond or a diselenide bond and comprising an amino acid sequence of X3KAPETALX4AAA; having at least an intrapeptide disulfide bond or an intrapeptide diselenide bond between X3 and X4, wherein X3 and X4 are identical and are selected from C (cysteine), Sec (selenocysteine) and Pen (penicillamine); i.e. (c) a peptide of 9 to 11 amino acid residues in length having at least an intrapeptide bond that is a disulfide bond or a diselenide bond, and consisting of an amino acid sequence selected from the group consisting of X5KAPETALX6; X5KAPETALX6A; and X5KAPETALX6AA having at least an intrapeptide disulfide bond or an intrapeptide diselenide bond between X5 and X6; wherein X5 and X6 are identical and are selected from the group consisting of C (cysteine), Sec (selenocysteine) and Pen (penicillamine), i.e. (d) having 16 amino acid residues and comprising the amino acid sequence X7NX8KAPETALX9AAAX 10 H peptide, between X7 and X9 and between X8 and X 10 There is an intra-peptide disulfide bond or an intra-peptide diselenide bond between them; wherein X7 to X 10 are independently selected from C (cysteine), Sec (selenocysteine) and Pen (penicillamine); provided that X7 and X9 are the same, and X8 and X 10 the same; that is and (e) a peptide comprising the amino acid sequence X1KAPETALX2, wherein X1 is selected from Dap and Dab; ​​and X2 is selected from a linear peptide of D (aspartic acid) and E (glutamic acid) (SEQ ID NO: 7); W is selected from -NH-(CH2) r -C(=O)- and -NH-CH((CH2) r NH2)-C(=O)- diradical; r is an integer independently selected from 1 to 5; s is an integer independently selected from 0 to 1; Y is a free radical selected from -NH2, -OH, -OR3 and -NHR3; When m=0, L3 and D are absent, and P is directly linked to 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 of the peptide sequence P; When m=1, D is present and connected to a functional group of an amino acid side chain of linker L3, or to an amino acid derivative of linker L3, by derivatization thereof, wherein the connection is made by an amide, ester, disulfide bond or thioester bond; L3 is connected to the LC of L2 by an amide bond formed between the C=O terminal group on the left side of the LC formula and the amine group of linker L3; and P is directly connected to L3 by an amide bond formed between the C=O terminal group on the right side of the LC formula and the amine group of the first amino acid of the peptide sequence P; and When s=0, P is directly linked to Y via an amide bond, a carboxylic acid bond or an ester bond, the bond being formed between the C=O at the C-terminus of the last amino acid of the sequence P and the free radical Y, and the free radical Y is -NH2, -OH, -OR3 or -NHR3; and When s=1, P is linked to the free radical W via an amide bond formed with the C=O at the C-terminus of the last amino acid of the sequence P, the bond being formed between the functional group on the left side of the drawn W formula and the functional group (C=O) at the C-terminus of the last amino acid of the sequence P on the right side of the drawn sequence; and W is linked 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; Represents a connection point; and S in the formula (I) represents a sulfide.

2. The antibody shuttle conjugate according to claim 1, wherein the disulfide bonds originally present in the antibody can structurally retain the sulfide bonds in the structure under reducing conditions, and can form a new bridge between the sulfide groups of the reduced disulfide bonds of the antibody and the linker L1 through the reaction between them, thereby incorporating the following diradicals between the sulfide groups of the antibody 3. The antibody shuttle agent conjugate according to any one of claims 1 to 2, which has four copies of the peptide P, and all the peptides are identical.

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

5. The antibody shuttle agent conjugate according to any one of claims 1 to 4, wherein P is a diradical of a peptide selected from: (a) A peptide comprising the amino acid sequence DapKAPETALD, which has an intrapeptide bond between Dap and D that is an amide bond, i.e., SEQ ID NO: 8: (b) a peptide of 9 to 20 amino acid residues in length having intrapeptide bonds that are at least disulfide bonds and comprising the following amino acid sequence: CKAPETALCAAA having at least an intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, i.e., SEQ ID NO: 9: (c) a peptide of 9 to 11 amino acid residues in length having an intrapeptide bond that is at least a disulfide bond and consisting of an amino acid sequence selected from the group consisting of CKAPETALC; CKAPETALCA; and CKAPETALCAA having at least an intrapeptide disulfide bond between cysteine ​​1 and cysteine ​​9, i.e. (d) a peptide having 16 amino acid residues and comprising the amino acid sequence CNCKAPETALCAAACH, which has intrapeptide disulfide bonds between the first and third cysteine ​​residues, which are cysteine ​​1 and cysteine ​​11, and between the second and fourth cysteine ​​residues, which are cysteine ​​3 and cysteine ​​15, i.e., (e) A peptide comprising the amino acid sequence DapKAPETALD (SEQ ID NO: 14).

6. The antibody shuttle agent conjugate according to claim 5, wherein P is a diradical of a peptide selected from the group consisting of: (a) a peptide having an amino acid sequence of DapKAPETALD, which has 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, which has at least an 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).

7. The antibody shuttle agent conjugate according to any one of claims 1 to 5, wherein the antibody is selected from trastuzumab, bevacizumab, cetuximab, pertuzumab, aducanumab, bapizumab, nimotuzumab and necituzumab.

8. The antibody shuttle agent conjugate of claim 1, which is selected from: trastuzumab-DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Tz-DBM-TTDS-SEQ ID NO: 1), which has an amide bond between the amino group of the Dap side chain and the carboxylic acid of the Asp side chain; 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; 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; and Pertuzumab -DBM-TTDS-Dap-Lys-Ala-Pro-Glu-Thr-Ala-Leu-Asp-NH2 (Pt-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, wherein DBM is the linker L 1a And TTDS is connector L 2a 9. The antibody shuttle agent conjugate according to claim 1, wherein: a) the antibody is an anti-cancer therapeutic antibody, m=1, and D is a free radical of an anti-cancer active pharmaceutical ingredient selected from the group consisting of auristatins, duocarmycins, PBD dimers, maytansines, calicheamicins, anthracyclines, camptothecins, α-amanitin, tubulysin, MMAE, T-DM1, and PROTAC moieties; or alternatively, b) The antibody is an anti-neurodegeneration therapeutic antibody, m=1 and D is a free radical of an anti-neurodegeneration active pharmaceutical ingredient.

10. The antibody shuttle agent conjugate according to claim 1, wherein the antibody is cetuximab, m=1 and D is SN38.

11. A method for preparing an antibody shuttle agent conjugate as defined in any one of claims 1 to 10, comprising: a) reducing the disulfide bridges of the antibody; b) re-bridging the disulfide bridges by reacting the -SH groups of the antibody with a dibromomaleimide-peptide of formula (II), c) optionally, performing hydrolysis; Where q; L2, L3, D, P, m, W, s and Y are as defined in the antibody shuttle agent conjugate of formula (I).

12. A pharmaceutical composition comprising a therapeutically effective amount of the antibody shuttle agent conjugate as defined in any one of claims 1 to 10 and an appropriate amount of a pharmaceutically acceptable carrier or excipient.

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

14. An antibody shuttle agent conjugate as claimed in any one of claims 1 to 10 for use in treating CNS disorders in mammals including humans.

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

16. The antibody shuttle agent conjugate for use according to any one of claims 14 to 15, wherein the antibody shuttle agent conjugate is used in combination therapy with conventional chemotherapeutic agents or radiotherapy.

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

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