Anti-HIV antibody drug conjugate

Abstract

It has been found that a CD mimic compound provides a synergistic anti-HIV effect when used in combination with a neutralizing antibody which specifically recognizes a V3 loop exposed in the middle of an HIV invasion mechanism, but the effect thereof cannot be said to be sufficient, particularly in vivo. The present invention provides an anti-HIV antibody drug conjugate in which a CD4 mimic compound and an anti-HIV antibody are connected via a linker.

Description

Anti-HIV antibody-drug conjugates

[0001] The present invention relates to an anti-HIV antibody-drug conjugate, more specifically, to a conjugate of an anti-HIV antibody and a CD4 mimic compound having anti-HIV activity. The present invention also relates to an HIV infection inhibitor containing the anti-HIV antibody-drug conjugate as an active ingredient, and a pharmaceutical composition containing the same.

[0002] Human immunodeficiency virus (HIV) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). Currently, combination antiretroviral therapy (cART), which includes a combination of several anti-HIV drugs, is used in clinical treatment to suppress viral replication and control disease progression. However, due to the emergence of drug-resistant strains of HIV, as well as significant side effects and high costs, novel anti-HIV drugs with different mechanisms of action are needed.

[0003] There are two types of HIV: HIV-1 and HIV-2, and HIV-1 is further classified into subtypes A to K. HIV-1 is common in the Western Hemisphere, Europe, Asia, and central, southern, and eastern Africa, while HIV-2 is common in western Africa. Compared to HIV-1, which is more common, HIV-2 is less infectious and is only prevalent in limited areas, so anti-HIV drugs and AIDS vaccine development are primarily targeting HIV-1.

[0004] HIV-1 is a type of retrovirus. Mature viruses are spherical, 100-110 nm in diameter, and contain a bullet-shaped core structure containing two copies of the single-stranded RNA genome, reverse transcriptase, integrase, and other proteins. The envelope structure is composed of a lipid bilayer and a protein coat. The surface of the virus particle contains trimers of the coat proteins gp120 and gp41, which specifically bind to CD4, CXCR4, and CCR5 on human CD4+ T cells (helper T cells) and macrophages, playing an important role in HIV entry into host cells.

[0005] The first step in HIV-1 entry into host cells is the interaction of the HIV-1 envelope protein gp120 with the host cell surface protein CD4 (first receptor). This interaction induces a major structural change in gp120, exposing a region called the V3 loop. Next, interaction of this V3 loop with the second receptor (coreceptor, CCR5 or CXCR4) exposes gp41 on the surface, which penetrates the host cell membrane and enters the host cell via membrane fusion.

[0006] Our group has previously developed CD4 mimic compounds as small molecule inhibitors of HIV entry that compete with the HIV envelope protein gp120 for binding to the primary host cell receptor CD4 (Patent Documents 1 and 2). Similar to CD40, binding of CD4 mimic compounds to gp120 induces a conformational change in gp120, exposing the HIV-1 coreceptor binding site that can be recognized by anti-HIV neutralizing antibodies (Non-Patent Document 1). Therefore, we have reported that the combined use of CD4 mimic compounds and anti-HIV antibodies is effective against HIV infection. Furthermore, the use of CD4 mimic compounds is advantageous from a safety perspective, since the coreceptor binding site can be exposed without binding to the host cell receptor (Non-Patent Document 2).

[0007] Patent No. 6710376 Patent No. 7297220

[0008] Kobayakawa, T. et al., J. Med. Chem., 2021, 64, 1481Ohashi, N. et al., ChemMedChem., 2016, 11, 940

[0009] As mentioned above, CD mimic compounds have been shown to have synergistic anti-HIV effects when combined with neutralizing antibodies that specifically recognize the V3 loop, which is exposed during HIV entry. However, although this combination was effective in vitro, its effectiveness in vivo was not sufficient.

[0010] The present inventors synthesized antibody-drug conjugates (ADCs) composed of a CD4 mimic compound and an anti-HIV neutralizing antibody in various ways using various linkers, and evaluated their anti-HIV activity. As a result, they found that the use of the ADCs as a conjugate can more effectively inhibit viral entry than the use of a CD4 mimic compound and an anti-HIV neutralizing antibody in combination separately, thereby completing the present invention.

[0011] That is, the present invention provides the following: 1. An anti-HIV antibody-drug conjugate in which a CD4 mimic compound and an anti-HIV antibody are linked via a linker. 2. The CD4 mimic compound is represented by the following general formula (I): [wherein X is a halogen atom selected from Cl, Br, and F, A is an alkylene group having 1 to 5 carbon atoms, and B is a group represented by the following formulas (II) to (IV): (In the formula, R 1 and R 2 are each independently an alkylene group having 1 to 5 carbon atoms which may contain a carbonyl group), (In the formula, R 3 is an alkylene group having 1 to 5 carbon atoms which may contain a carbonyl group), and

[0023] 1. The anti-HIV antibody-drug conjugate according to claim 1, wherein the compound is a compound represented by the formula:

[0024] 2. The anti-HIV antibody-drug conjugate according to claim 1, wherein the anti-HIV antibody is a neutralizing antibody specific for the V3 loop on the surface of HIV-1.

[0025] 3. The anti-HIV antibody-drug conjugate according to claim 1, wherein the anti-HIV antibody is a neutralizing antibody specific for the V3 loop on the surface of HIV-1.

[0026] 4. The anti-HIV antibody-drug conjugate according to claim 1, wherein the linker is a peptide linker or a PEG linker.

[0027] 5. An HIV infection inhibitor comprising, as an active ingredient, the anti-HIV antibody-drug conjugate according to any one of claims 1 to 4.

[0028] 6. A pharmaceutical composition for treating or preventing HIV infection, comprising the HIV infection inhibitor according to claim 5.

[0029] This specification incorporates the disclosures of Japanese Patent Application No. 2024-088735, from which the present application claims priority.

[0012] The present invention can significantly improve anti-HIV activity compared to when a CD4 mimic compound is simply combined with an anti-HIV neutralizing antibody. The present invention is particularly advantageous when considering in vivo therapy. While antibody-based therapy is expensive, the present invention can achieve high anti-HIV activity with a small amount of antibody, which may also lead to cost reduction.

[0013] 1A and 1B are schematic diagrams showing the structure of an antibody-drug conjugate of the present invention in which an antibody and a CD4 mimic compound are non-selectively linked. 2A and 2B are schematic diagrams showing the structure of an antibody-drug conjugate of the present invention in which an antibody and a CD4 mimic compound are linked using the CCAP method. 3A and 3B are schematic diagrams showing the structure of an antibody-drug conjugate of the present invention in which an antibody and a CD4 mimic compound are linked using the tCAP method.

[0014] The present invention is described in detail below: The present invention provides an anti-HIV antibody-drug conjugate in which a CD4 mimic compound and an anti-HIV antibody are linked via a linker.

[0015] As used herein, a CD4 mimic compound refers to a small molecule that mimics a portion of the CD4 molecule and binds to a pocket in the HIV-1 coat protein gp120 called the Phe43 cavity, thereby causing a conformational change in gp120. Examples of such compounds include the small molecule compound NBD-556 disclosed in Qian, Z. et al., Virology 339, 213-225 (2005) and derivative compounds derived from this as a lead compound, the compounds disclosed in Ding, S. et al., J. Virol. 2019 Nov 26; 93(24): e01325-19, and the compounds disclosed in Japanese Patent Nos. 6710376 and 7297220.

[0016]

[0017] More specifically, examples of CD4 mimic compounds include low molecular weight compounds having a halobenzene and oxamide skeleton. Compounds having a ring structure in addition to the halobenzene and oxamide skeleton can also be suitably used.

[0018] In a preferred embodiment, the CD4 mimic compound is represented by the following general formula (I) disclosed in Japanese Patent No. 6710376: [wherein X is a halogen atom selected from Cl, Br, and F, A is an alkylene group having 1 to 5 carbon atoms, and B is a group represented by the following formulas (II) to (IV): (In the formula, R 1 and R 2 are each independently an alkylene group having 1 to 5 carbon atoms which may contain a carbonyl group), (In the formula, R 3 is an alkylene group having 1 to 5 carbon atoms which may contain a carbonyl group), and and a derivative thereof.

[0019] In one aspect of the above preferred embodiment, the CD4 mimic compound is YIR-821, a compound having the following structure: wherein X is hydrogen. Derivatives disclosed in Japanese Patent No. 7297220, in which a polyethylene glycol chain or an alkyl chain is introduced at the X position, can also be suitably used.

[0020]

[0021] The CD4 mimic compound constituting the conjugate of the present invention can be synthesized using the description herein and techniques commonly used in the art, for example, by a synthetic route starting from the parent compound YIR-821, but the synthesis method is not particularly limited. YIR-821 can be synthesized, for example, based on the description in WO 2016 / 190331.

[0022] Furthermore, the antibody constituting the complex of the present invention is not particularly limited, but since the CD4 mimic compound can cause a structural change in gp120 upon binding to HIV, thereby exposing the V3 loop, it is preferable to use a neutralizing antibody, particularly a neutralizing monoclonal antibody specific for the V3 loop of HIV, or a functional fragment thereof that retains the binding specificity for the V3 loop.

[0023] Various research and development efforts are underway in the field of anti-HIV monoclonal antibodies, and a representative monoclonal antibody against the HIV V3 loop is KD-247 (generic name: suvizumab), which is currently undergoing clinical trials. Details of anti-HIV monoclonal antibodies are described, for example, in Journal of Virology, June 2006, pp. 5552-5562; Journal of Virology, June 2006, pp. 5563-5570; Kaketsukenshoho Reimei, 23: 42-54 (2014); Japanese Patent Nos. 2989862 and 5526386. The amino acid sequences of the VH and VL regions of KD-247 are also disclosed in the above Journal of Virology, June 2006, pp. 5552-5562. The method for obtaining and the effects of another monoclonal antibody, 916B2, used in the examples, along with other monoclonal antibodies, are described, for example, in Virology 475 (2015) 187-203. Those skilled in the art can obtain the KD-247 antibody and other anti-HIV antibodies based on this information.

[0024] In the conjugate of the present invention, a peptide linker, PEG linker, alkyl linker, etc. can be appropriately used as a linker connecting the CD4 mimic compound and the antibody. The linker is not limited to, but commercially available products such as mini-PEG (Peptide Institute, Inc.) can also be used. One unit of mini-PEG is approximately 10 Å, and it can be used alone or in combination.

[0025] The CD4 mimic compound and antibody can be linked via a linker using any method known in the art, including nonselective or site-specific binding. However, site-specific binding, which allows selective drug introduction, is preferred for stable delivery of the same complex. Suitable site-specific binding methods include the CCAP method, which uses an Fc affinity peptide (Kishimoto, S. et al., Bioconjug. Chem., 2019, 30, 698-702), and the tCAP method, which uses the reagent tCAP (N3) available from Peptide Institute, Inc. The tCAP method has the advantage of minimizing the risk of immunogenicity because the affinity peptide does not remain in the final complex.

[0026] In the present invention, the CD mimic compound may be bound to any site on the antibody except for the antigen-binding region, and the binding site is not particularly limited. However, it is more preferable to use a site-specific binding method so that the CD mimic compound binds only to the Fc region, which is the antigen-recognition region of the antibody, and not to the Fab region. Furthermore, when developing and supplying the compound as a pharmaceutical, it is necessary to prepare a complex bound to the same site, regardless of lot or scale.

[0027] Furthermore, the antibody-drug ratio (number of drug molecules per antibody molecule) in the conjugates of the present invention is not particularly limited, but is typically in the range of 0.5 to 10, preferably 1 to 5, and more preferably approximately 1 to 2. A typical antibody is composed of two heavy chains and two light chains and thus has two antigen-binding sites. Therefore, a particularly high synergistic effect can be expected when one or two molecules of a CD4 mimic compound are bound to one antibody molecule. If the antibody-drug ratio is too high, the drug may also be introduced into the Fab region of the antibody, potentially reducing the antibody's ability to recognize antigens. Therefore, from this perspective, it is preferable to use a site-specific conjugation method such as the CCAP method or tCAP method described above.

[0028] The complex of the present invention can competitively inhibit the binding of CD4 to the Phe43-cavity of HIV gp120 due to the function of the CD4 mimic compound. Furthermore, without being bound by any theory, the conformational change in gp120 induced by the complex of the present invention is not identical to the change caused by interaction with CD4; it exposes the V3 loop but does not allow subsequent binding to a coreceptor, thereby inhibiting HIV entry into host cells. Therefore, the present invention provides an HIV infection inhibitor comprising the above-described complex of the present invention as an active ingredient.

[0029] The conjugate of the present invention can be administered as an HIV infection inhibitor as it is, but can also be administered as a pharmaceutical composition by appropriately adding, in addition to this active ingredient, carriers, excipients, preservatives, oxidation stabilizers, etc. that are commonly used in pharmaceutical compositions. Thus, the present invention also provides a pharmaceutical composition for treating or preventing HIV infection, which comprises the above-mentioned HIV infection inhibitor.

[0030] The HIV infection inhibitor and pharmaceutical composition of the present invention can be administered orally or parenterally, for example, orally; or by intravenous, intramuscular, transdermal, subcutaneous, intradermal, or intraperitoneal injection or infusion, but this is not particularly limited. The administration route may be preferably intravenous, intramuscular, or subcutaneous injection. Those skilled in the art can appropriately determine a suitable administration route for administering the HIV infection inhibitor and pharmaceutical composition of the present invention.

[0031] The dose of the HIV infection inhibitor of the present invention to be administered to a human depends on the age, body weight, symptoms, etc. of the patient to be administered, and is not particularly limited, but can be, for example, in the range of 100 μg / kg to 100 mg / kg body weight per day, preferably 500 μg / kg to 50 mg / kg body weight, and more preferably 1 mg / kg to 30 mg / kg body weight per day.

[0032] Furthermore, the HIV infection inhibitor of the present invention can be used alone, but is also intended to be used in combination with other anti-HIV drugs that have inhibitory effects via different mechanisms. Examples of other anti-HIV drugs include, but are not limited to, reverse transcriptase inhibitors, protease inhibitors, and integrase inhibitors. Examples of reverse transcriptase inhibitors include, but are not limited to, zidovudine, lamivudine, abacavir, tenofovir, emtricitabine, and efavirenz; protease inhibitors include atazanavir, darunavir, and ritonavir; and integrase inhibitors include raltegravir. The anti-HIV infection inhibitor and other anti-HIV drugs can be contained in the same or different pharmaceutical compositions. The HIV infection inhibitor of the present invention and other anti-HIV drugs can be administered simultaneously, sequentially, or in completely different ways. Furthermore, the routes of administration of the HIV infection inhibitor of the present invention and other anti-HIV drugs can be the same or different.

[0033] In the following examples, two anti-HIV neutralizing antibodies and the anti-HER2 antibody trastuzumab were used as a negative control to prepare antibody-drug conjugates. Both nonspecific modification using active esters and site-specific conjugation were used as conjugation methods. The linker length was adjusted by the number of linker unit repeats.

[0034] As a result, a significant improvement in anti-HIV activity was observed for the conjugate of the present invention compared to the activity of the antibody and CD4 mimic compound used alone, and the conjugate showed anti-HIV activity 7 to 10 times higher than that of the neutralizing antibody used alone.

[0035] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0036] First, a representative example of the synthesis method of the conjugate of the present invention is shown below. Reactions using air- or moisture-sensitive reagents were carried out in dry glassware under a nitrogen or argon atmosphere using commercially available solvents and reagents unless otherwise noted.

[0037] For analysis of the obtained compounds, thin layer chromatography (TLC) was carried out on Merck 60F254 precoated silica gel and visualized by fluorescence quenching with ultraviolet light or staining with molybdenum phosphate, para-anisaldehyde, or ninhydrin.

[0038] Flash column chromatography was performed using silica gel 60N (Kanto Chemical) or Isolera One (Biotage) equipped with a SNAP Ultra Silica Cartridge.

[0039] Low- and high-resolution mass spectra (LRMS and HRMS) were recorded using a Bruker Daltonics microTOF focus (electrospray ionization mass spectrometry) spectrometer in either positive or negative ion detection mode. For antibody analysis, a Bruker Daltonics ultrafleXtreme (matrix-assisted laser desorption / ionization mass spectrometry) spectrometer was used in positive ion detection mode. Preparative GPC was performed in THF at a flow rate of 8 mL / min using a Phenogel 10 μm column (300 × 212 mm, Phenomenex, USA) mounted on a SHIMADZU LC-20AP, CBM-20A (Shimadzu Corporation). Eluted products were detected by RID using a SHIMADZU RID-20A (Shimadzu Corporation, Japan).

[0040] Reverse-phase high-performance liquid chromatography (HPLC) was performed on Cosmosil 5C 18 -ARII preparative column (20 x 250 mm, Nacalai Tesque) (flow rate 10.0 mL / min) and Cosmosil 5C 18 An ARII analytical column (4.6 × 250 mm, Nacalai Tesque) was used (flow rate: 1.0 mL / min), and the effluent was detected by UV light at 220 nm. A solvent system containing 0.1% (v / v) TFA / HO (solvent A) and 0.1% (v / v) TFA / MeCN (solvent B) was used for reversed-phase HPLC elution.

[0041] Hydrophobic interaction chromatography (HIC-HPLC) was performed using TSKgel HIC-ADC (4.6 mm × 100 mm, Tosoh) and TSKgel HIC-Butyl-NPR (4.6 mm × 100 mm, Tosoh).

[0042] Condition 1: Flow rate 0.5 mL / min, detection at 280 nm. A solvent system containing 25 mmol / L phosphate buffer (pH 7.0) containing 1.5 mol / L ammonium sulfate (solvent A) and 25 mmol / L phosphate buffer (pH 7.0) containing 20% ​​2-propanol (solvent B) was used for HIC-HPLC.

[0043] Condition 2: Flow rate 0.5 mL / min, detection at 280 nm. A solvent system containing 25 mmol / L phosphate buffer (pH 7.0) containing 2.0 mol / L ammonium sulfate (solvent A) and 25 mmol / L phosphate buffer (pH 7.0) containing 20% ​​2-propanol (solvent B) was used for HIC-HPLC. Antibody purification was performed using an Amicon centrifuge filter (10 kDa MWCO, 0.5 mL volume) from Merck. For ease of explanation, compounds are numbered in the order of their appearance in the examples.

[0044] Example 1 Synthesis of the Conjugate of the Present Invention 1 (Non-selective Binding Method) (1) Synthesis of Linker First, a linker for linking the CD4 mimic compound to the antibody was synthesized. For convenience, the side chain portion of the CD4 mimic compound was linked to a PEG linker.

[0045]

[0046] To a solution of compound 1 (660 mg, 1.22 mmol) in CHCl (15 mL), HATU (464 mg, 1.22 mmol), Boc-PEG 11The α-amine (compound 2) (787 mg, 1.22 mmol) and DIPEA (0.42 mL, 2.44 mmol) were added at 0 °C. After stirring the reaction mixture overnight at room temperature, the mixture was quenched with saturated aqueous NaHCO3 and extracted with CHCl2. The organic layer was then dried over MgSO4 and concentrated under reduced pressure. The reaction mixture was purified by silica gel flash column chromatography (Buchi, Pure C-815) using CHCl3 / MeOH (100:0 to 85:15) and further purified by GPC to give compound 3 (787 mg, 55%) as a yellow oil.

[0047] 1 H-NMR (500 MHz, CDCl3) δ: 1.43-1.45 (m, 15H), 1.65-1.73 (m, 1H), 1.81-1.91 (m, 3H), 2.08 (s, 3H), 2.51 (s, 3H), 2.57 (s, 3H), 2.95 (s, 3H), 3.29-3.30 (m, 2H), 3.36-3.40 (m, 2H), 3.51-3.54 (m, 4H), 3.61-3.64 (m, 45H), 3.72 (s, 3H), 3.91-3.98 (m, 1H), 4.51-4.54 (m, 1H), 5.10-5.11 (m, 1H), 6.40-6.60 (m, 2H), 6.94-6.97 (m, 2H); 13C-NMR (125 MHz, CDCl3) δ 12.6, 18.1, 19.4, 25.1, 25.6 (3C), 28.7 (2C), 29.3, 31.4, 31.6, 33.4, 39.5, 40.5, 40.6, 43.4, 52.6, 53.6, 70.2, 70.4 (10C), 70.6 (10C), 79.3, 98.6, 117.6, 124.7, 129.0, 132.6, 138.6, 156.1, 158.9, 172.6, 172.7, 172.8; HRMS (ESI), m / z C 53 H 94 NO 20 S + [M+H] + Calculated value 1167.6317, measured value 1167.6317.

[0048]

[0049] To a solution of compound 3 (668 mg, 0.572 mmol) in THF (5.7 mL) was added 1N aqueous LiOH solution (1.43 mL) at room temperature. After stirring the reaction mixture at room temperature for 1.5 hours, the mixture was filtered, quenched with 1N aqueous hydrochloric acid solution, and extracted with CHCl. ​​The organic layer was dried over MgSO and concentrated under reduced pressure to give compound 4.

[0050] (2) Linking of CD4 mimic compound to linker Next, the linker prepared above was linked to the backbone portion of the CD4 mimic compound.

[0051]

[0052] To a solution of compound 5 (137 mg, 0.351 mmol) in THF (7 mL) was added LiAlH (44.0 mg, 1.16 mmol) at 0 °C. The mixture was stirred at room temperature for 1.5 hours, followed by the addition of saturated aqueous sodium potassium tartrate at 0 °C and the addition of 1 M aqueous NaOH to adjust the pH to 13-14. The mixture was extracted with CHCl, the organic layer was dried over MgSO, and concentrated under reduced pressure. The reaction mixture was dissolved in DMF (1.75 mL), and COMU (89.9 mg, 0.21 mmol), compound 4 (241 mg, 0.21 mmol), and DIPEA (0.071 mL, 0.42 mmol) were added at 0 °C. The reaction mixture was stirred overnight at room temperature, then quenched with saturated aqueous NaHCO, extracted with CHCl, and the organic layer was dried over MgSO. The reaction was purified using CHCl3 / MeOH (100:0 to 75:25) and GPC to give compound 7 (192 mg, 60%) as a colorless oil.

[0053] 1H-NMR (500 MHz, CDCl3) δ: 1.46-1.49 (m, 15H), 1.66-1.98 (m, 8H), 2.12-2.15 (m, 5H), 2.51 (, 3H), 2.57-2.67 (m, 5H), 2.79-2.99 (s, 19H), 3.32 (s, 2H), 3.41 (s, 2H), 3.54-3.67 (m, 46H), 4.02-4.14 (m, 1H), 7.33-7.36 (m, 2H), 7.60-7.62 (m, 2H); 13 C-NMR (125 MHz, CDCl3) δ 12.4, 19.2, 21.5, 24.5, 24.6(2C), 24.7, 28.4(3C), 28.6(2C), 31.2, 31.4, 33.4, 39.4(2C), 39.5, 40.3, 41.7, 41.8, 41.9, 43.0, 48.2, 48.5, 48.9, 50.0, 67.5, 69.5, 70.3(11C), 70.4(11C), 79.2, 85.8, 114.7, 117.0, 121.0 (2C), 129.2, 129.3 (2C), 130.3, 130.5, 134.9, 135.0, 156.1, 156.9, 159.7, 159.8, 159.9, 172.6, 172.7, 172.9; HRMS (ESI), m / z C 72 H 120 ClN 10 O 21 S + [M+H] + The calculated value is 1527.8034 and the measured value is 1527.8033.

[0054]

[0055] Compound 7 (2 mg, 1.31 μmol) was dissolved in CHCl (13 mL) and added with TFA (1.36 μL) / TIPS (1.62 μL) / HO (0.14 μL) at 0 °C. The mixture was stirred at room temperature for 5 h, concentrated under reduced pressure, and then purified by HPLC to give the trifluoroacetate salt of compound 8 as a colorless oil (1.8 mg, 91%); Rt = 20.5 min (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile; A:B = 95:5 to 20:80 over 30 min; flow rate = 1.0 mL / min, 220 nm). HRMS (ESI), m / z C 54 H 96 ClN 10 O6 + [M+H] + Calculated value 1175.6689, measured value 1175.6689.

[0056]

[0057] To a solution of compound 8 (19.0 mg, 16.0 μmol) in CHCl (19 mL) was added disuccinimidyl glutarate (DSG) (105 mg, 0.323 mmol) and TEA (7.85 μL, 56.4 μmol) at room temperature. The mixture was stirred at room temperature for 4 hours, concentrated under reduced pressure, and then purified by HPLC to give the trifluoroacetate salt of compound 9 as a colorless oil (5.6 mg, 25%); Rt = 22.5 min (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile; A:B = 95:5 to 20:80 over 30 minutes; flow rate = 1.0 mL / min at 220 nm).

[0058] 1H-NMR (500 MHz, CDCl3)δ: 1.20-1.32 (m, 2H), 1.42-1.53 ​​(m, 4H), 1.71-1.80 (m, 4H), 1.86-1.97 (m, 4H), 2.07-2.14 (m, 4H), 2.34-2.38 (6H), 2.62-2.72 (6H), 2.84-2.88 (m, 4H), 3.11-3.22 (m, 2H), 3.41-3.47 (m, 4H), 3.56-3.58 (m, 4H), 3.63-3.66 (m, 45H), 4.13-4.14 (m, 1H), 7.34-7.36 (m, 2H), 7.61-7.63 (m, 2H); 13 C-NMR (125 MHz, CDCl3) δ 20.2, 20.7, 21.2, 21.4, 21.5, 21.8, 25.6 (2C), 29.9, 30.0, 30.1, 31.3, 31.4, 34.4, 36.5, 39.2, 70.1 (11C), 70.3 (11C), 121.0, 121.1, 125.3 (2C), 128.2 (2C), 129.0, 129.3, 130.4, 132.0, 135.0, 137.9, 156.9, 157.4, 157.5, 159.6, 159.7, 166.7, 168.4, 169.3 (2C), 172.0, 172.7, 173.4, 173.9; HRMS (ESI), m / z C 63 H 105 ClN 11 O 21 + [M+H] + The calculated value is 1386.7170 and the measured value is 1386.7170.

[0059] (3) Preparation of Conjugates. Trastuzumab or KD-247 (2.00 mg mL, PBS, pH 7.4) was diluted with 1, 5, 10, 30, or 50 equivalents of compound 9 (10 mM in DMSO) obtained in (2) and 20% DMA to 800 μL of trastuzumab or KD-247 (2.00 mg mL, PBS, pH 7.4). The mixture was incubated at 20°C for 3 hours to prepare antibody-drug conjugates (Figure 1). After 3 hours, the mixture was dialyzed six times (PBS, 5-fold dilution) using a dialysis filter (10 kDa, 0.5 mL) to remove unreacted compound 9 and DMA. The concentration of each sample was calculated from the absorbance measurement results.

[0060] As a result, conjugates with the antibody-drug ratios shown in Table 1 were obtained. The antibody-drug ratios in the conjugates obtained in this example ranged from 0.3 to 16, depending on the amount of CD4 mimic compound (compound 9) added.

[0061]

[0062] Example 2 [Synthesis of the Complex of the Present Invention 2 (CCAP Method)] (1) Preparation of CD4 Mimic Compound

[0063] To a solution of compound 5 (49.7 mg, 127.8 μmol) in THF (1.3 mL) was added LiAlH (16.0 mg, 421.7 μmol) at 0 °C. After stirring the mixture at room temperature for 1.5 h, saturated aqueous potassium sodium tartrate was added at 0 °C and the mixture was basified with 1 M aqueous NaOH (pH = 13–14). After extraction with CHCl, the organic phase was dried over MgSO and concentrated under reduced pressure. The crude mixture was dissolved in DMF (1 mL), and HATU (10.4 mg, 71.1 μmol), Fmoc-Arg(Pbf)-OH (49.1 mg), and DIPEA (22 μL, 129.2 μmol) were added at 0 °C. The reaction mixture was stirred overnight at room temperature, quenched by the addition of saturated aqueous NaHCO, extracted with CHCl, and the organic phase was dried over MgSO. After crude purification by column chromatography, the crude product was dissolved in piperidine (540 μL) / CHCl (30 μL). The mixture was stirred at room temperature for 1 hour, quenched with water, and extracted with CHCl. ​​The organic phase was dried over MgSO. The synthesized compound 11 was crudely purified by column chromatography and used in the next reaction.

[0064] (2) Linking the CD4 mimic compound to a linker

[0065] Peptide resins 12 (n = 0), 13 (n = 3), and 14 (n = 6) were synthesized using 0, 3, and 6 mini-PEGs (Peptide Institute, Inc.) and NovaSyn® TGR resin (0.25 mmol / g, 0.1 mmol scale), respectively. 9-Fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis (SPPS) was performed manually and automatically (PurePrepChorus, Gyros Protein Technologies, AZ, USA). Furthermore, peptide resins 15, 16, and 17 were synthesized using 20 equivalents of compound 11 (1).

[0066] After peptide elongation was complete, the resin was washed with DMF (3 × 5 mL) and DCM (3 × 5 mL). Next, peptide resins 15, 16, and 17 were treated with 5 mL of a TFA / EDT / TIPS / HO (89:7.5:1:2.5) solution for 2 h. 5 mL of DMSO was added to the HPLC-prepared peptides, followed by 20 equivalents of 30% HO (49.8 μL, 488 μmol) and 2 equivalents of 2 M NH3-MeOH (24.4 μL, 48.8 μmol). The reaction mixture was stirred at room temperature for 12 h. DMSO and volatile organics were removed under reduced pressure, and 1 mL of DMF and 20 equivalents of disuccinimido glutarate (DSG) linker were added to yield peptides 18, 19, and 20. 2 mL of HO (0.05% TFA) was added to this solution, and the product was purified by preparative reverse-phase HPLC. The elution conditions were as follows: mobile phase A = 0.1% TFA in water; mobile phase B = 0.1% TFA in acetonitrile; A:B = 100:0 to 55:45 over 40 min; flow rate = 10.0 mL min-1; detection was by absorbance at 220 nm. The solution was lyophilized to give peptides 18, 19, and 20 as colorless solids, as described below.

[0067] Peptide 18 (n=0):t R = 22.5 min (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile; A:B = 95:5 to 20:80 over 30 min; flow rate = 1.0 mL / min, 220 nm), HRMS (ESI) [M+2H] 2+ m / z calculated 1331.5, observed 1331.5.

[0068] Peptide 19 (n=3): t R = 22.5 min (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile; A:B = 95:5 to 20:80 over 30 min; flow rate = 1.0 mL / min, 220 nm), HRMS (ESI) [M+2H] 2+ m / z calculated 1549.1, observed 1548.8.

[0069] Peptide 20 (n=6): t R = 22.5 min (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile; A:B = 95:5 to 20:80 over 30 min; flow rate = 1.0 mL / min, 220 nm), HRMS (ESI) [M+2H] 2+ m / z calculated 1766.8, observed 1766.5.

[0070] (3) Conjugate Preparation: Antibody conjugation was performed using peptides 18, 19, and 20 synthesized in (2) (Figure 2). Peptides 18, 19, and 20 were dissolved in DMF to a concentration of 5 mM. The antibodies used were the anti-HER2 antibody trastuzumab and the neutralizing antibodies KD-247 and 916B2. 200 μL (2.5 nmoL) of 2.00 mg / mL antibody was transferred to a 50 mM acetate buffer solution at pH 5.5 using a dialysis filter. Five equivalents of a 5 mM DMF solution of peptides 18, 19, and 20 were added to the transferred antibody solution. After allowing to stand at room temperature for 2 hours, the solution was transferred to a peptide dissociation buffer (50 mM citrate buffer solution at pH 2.5) using a dialysis filter. Unreacted peptide was removed, and the solution was immediately replaced with 50 mM PBS. The antibody-drug ratio and molecular weight of the resulting antibody-drug conjugates were measured using high-performance liquid chromatography (HIC) and MALDI-TOF-MS. Furthermore, the concentration of each sample was calculated from the results of absorbance measurement.

[0071] As a result, conjugates were obtained with the antibody-drug ratios shown in Table 2. The antibody-drug ratios in the conjugates obtained in this example were in the range of approximately 0.5 to 2, indicating that the drug was selectively introduced.

[0072]

[0073] Example 3 Synthesis of the Conjugate of the Present Invention 3 (tCAP Method) (1) Linking of CD4 Mimic Compound and Linker

[0074] Peptide resins 21 (n = 6), 22 (n = 9), and 23 (n = 12) were synthesized using 6, 9, and 12 mini-PEGs (Peptide Institute, Inc.), respectively, and NovaSyn® TGR resin (0.25 mmol / g, 0.1 mmol scale). 9-Fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis (SPPS) was performed manually and automatically (PurePrepChorus, Gyros Protein Technologies, AZ, USA). Furthermore, peptide resins 24, 25, and 26 were synthesized using 20 equivalents of compound 11, synthesized in Example 2(1).

[0075] After peptide elongation, the resin was washed with DMF (3 × 5 mL) and DCM (3 × 5 mL). Next, peptide resins 24, 25, and 26 were treated with 5 mL of a 95:2.5:2.5 TFA / TIPS / HO solution for 2 hours. After separating each peptide by HPLC, 2 mL of DMF was added, followed by 20 equivalents of dibenzocyclooctyne-N-hydroxysuccinimidyl (DBCO-NHS) and stirring at room temperature for 12 hours. The DMF was removed under reduced pressure, and 2 mL of acetonitrile (0.05% TFA) and 2 mL of HO (0.05% TFA) were added. The resulting product was purified by preparative reverse-phase HPLC. The elution conditions were as follows: mobile phase A = 0.1% TFA in water; mobile phase B = 0.1% TFA in acetonitrile; A:B gradient from 80:20 to 55:45 over 30 min; flow rate = 10.0 mL / min; detection was by absorbance at 220 nm. The solution was lyophilized to give peptides 27, 28, and 29 as colorless solids, as described below.

[0076] Peptide 27 (n=6): t R = 23.5 min (A = 0.1% TFA water; B = 0.1% TFA acetonitrile; A:B = 95:5 to 40:60 over 30 min gradient; flow rate = 1.0 mL / min, 220 nm) HRMS (ESI) [M+2H] 2+ m / z calculated 974.5, observed 974.6.

[0077] Peptide 28 (n=9): t R = 23.5 min (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile; A:B = 95:5 to 40:60 over 30 min gradient; flow rate = 1.0 mL / min, 220 nm), HRMS (ESI) [M+2H] 2+ m / z calculated 1192.6, observed 1192.3.

[0078] Peptide 29 (n=12): t R = 23.5 min (A = 0.1% TFA in water; B = 0.1% TFA in acetonitrile; A:B = 95:5 to 40:60 over 30 min gradient; flow rate = 1.0 mL / min, 220 nm), HRMS (ESI) [M+3H] 3+ m / z calculated 940.5, observed 940.2.

[0079] (2) Preparation of Conjugates. Antibody conjugation was performed using peptides 27, 28, and 29 synthesized in (1) (Figure 3). Peptides 27, 28, and 29 were dissolved in DMF to a concentration of 10 mM. The antibodies used were the anti-HER2 antibody trastuzumab and the neutralizing antibody KD-247. 100 μL (1.25 nmoL) of 2.00 mg / mL antibody was substituted with 50 mM carbonate-bicarbonate buffer at pH 9.0 using a dialysis filter. 21.2 μL (4 equivalents) of an aqueous solution of tCAP(N3) reagent was added to the substituted antibody solution. After allowing to stand at room temperature for 30 minutes, the solution was substituted with peptide dissociation buffer (50 mM citrate buffer, pH 2.5) to remove unreacted peptide.

[0080] The reaction mixture was again replaced with a 100 mM carbonate-bicarbonate buffer solution at pH 9.0 using a dialysis filter. 21.2 μL (4 equivalents) of tCAP(N3) reagent solution was added to the replaced antibody solution. After 30 minutes at room temperature, the solution was replaced with a peptide dissociation buffer solution (50 mM citrate buffer, pH 2.5) to remove unreacted peptide. The solution was then replaced with 50 mM PBS at pH 7.0 using a dialysis filter. Five equivalents of a 10 mM DMF solution of peptides 27, 28, and 29 were added to the replaced solution. After 3 hours at room temperature, the peptides were removed using a dialysis filter. The antibody-drug conjugates were analyzed for antibody-drug ratio and molecular weight using HIC-HPLC and MALDI-TOF-MS. Furthermore, the concentration of each sample was calculated from the absorbance measurement results.

[0081] As a result, conjugates were obtained with the antibody-drug ratios shown in Table 3. The antibody-drug ratio in the conjugates obtained in this example was around 2, indicating that the drug was selectively introduced.

[0082]

[0083] Example 4 [Evaluation of Anti-HIV Activity 1] The anti-HIV activity (infection-inhibiting activity) of the conjugate obtained in Example 2 was evaluated in comparison with the activity when the antibody and the CD4 mimic compound YIR-821 were simply used in combination. Activity evaluation was performed by a reporter assay using HIV-1 CH058 (provided by the NIH HIV Reagent Program, J. Virol. 2012 Mar; 86(5): 2715-2728. doi: 10.1128 / JVI.06157-11 PMID: 22190722) obtained by transfecting 293T cells with a plasmid containing the full-length HIV-1 virus polynucleotide sequence, and TZM-bl cells (obtained from the NIH AIDS Reagent Program (https: / / www.aidsreagent.org / Index.cfm)).

[0084] TZM-bl cells are indicator cells that express luciferase upon HIV infection. They contain a β-galactosidase gene linked to the HIV-1 LTR sequence. Upon infection with HIV-1, the HIV-1 tat gene expresses the transcriptional activator Tat, which then acts on the LTR promoter region to express β-galactosidase. Addition of a substrate results in enzymatic cleavage, producing galactose and luciferin. The luciferase oxidizes the luciferin, producing chemiluminescence that can be detected with a luminometer, allowing the number of HIV-1-infected cells to be measured.

[0085] Specifically, 50 μl of HIV-1 CH058 adjusted to 4000 TCID50 / ml (TCID; tissue culture infectious dose) and 100 μl of a dilution series of inhibitors (antibodies, CD4 mimic compounds, or antibody-drug conjugates) (all in D-MEM 10% FBS) were added to a 96-well flat-bottom microculture plate and incubated at 37°C for 1 hour. 5 100 μl of TZM-bl cells adjusted to cells / ml were added and cultured at 37°C for 2 days. After culture, Britelite plus (PerkinElmer) was added to each well and the luminescence due to the luciferase enzyme reaction was measured using a luminometer. For evaluation, wells to which only virus and cells were added were considered to be 100% infected, and wells to which only cells and no virus or sample were added were considered to be 0% infected. The IC of the sample was calculated as follows: 50 (50% inhibitory concentration) was determined.

[0086] As a result, as shown in Table 4, the anti-HIV antibodies KD-247 and 916B2 each had anti-HIV activity alone and showed higher infection-inhibiting activity when combined with a CD4 mimic compound. However, antibody-drug conjugates of anti-HIV antibodies and CD4 mimic compounds showed significantly higher infection-inhibiting activity, demonstrating the synergistic effect of the antibodies and CD4 mimic compounds.

[0087] In particular, the conjugate prepared with six mini-PEGs as a linker for the KD-247 antibody showed approximately 7-fold higher infection-inhibitory activity than the KD247 antibody alone, and approximately 5.5-fold higher infection-inhibitory activity than the KD247 antibody in combination with a CD4 mimic compound (YIR-821). On the other hand, the conjugate prepared with three mini-PEGs as a linker for the 916B2 antibody showed approximately 10-fold higher infection-inhibitory activity than the 916B2 antibody alone, and approximately 2-fold higher infection-inhibitory activity than the 916B2 antibody in combination with a CD4 mimic compound (YIR-821).

[0088] Furthermore, when anti-HIV antibodies were not used (CD4 mimic compound alone, in combination with trastuzumab, and a complex of CD4 mimic compound and trastuzumab), the CD4 mimic compound demonstrated infection-inhibiting activity.

[0089]

[0090] Example 5 [Anti-HIV Activity Evaluation 2] The anti-HIV activity of the conjugate obtained in Example 3 was evaluated in comparison with the activity when the antibody was used alone, in the same manner as in Example 4. As a result, when the KD-247 antibody and the CD4 mimic compound were conjugated by the tCAP method, the conjugates obtained using 6, 9, and 12 mini-PEGs as linkers showed almost the same IC 50 Value and IC 90 The values ​​are shown, demonstrating that both have significantly higher infection-inhibiting activity than the KD-247 antibody alone (YMU 39) and the combined use of the KD-247 antibody and the CD4 mimic compound (YIR-821) (lower panel).

[0091]

[0092] The present invention makes it possible to provide an HIV infection inhibitor that has significantly higher anti-HIV activity than previously studied CD4 mimic compounds. In particular, the present invention makes it possible to provide an HIV infection inhibitor that is clinically very advantageous, as well as a pharmaceutical composition for treating or preventing HIV infection. An HIV infection inhibitor containing the anti-HIV antibody-drug conjugate of the present invention as an active ingredient can reduce the amount of antibody used compared to using an antibody alone. Because antibodies are expensive, the effects of the present invention may also lead to cost savings and lower drug prices. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. An anti-HIV antibody-drug conjugate in which a CD4 mimic compound and an anti-HIV antibody are linked via a linker, wherein the CD4 mimic compound is represented by the following general formula (I): [wherein X is a halogen atom selected from Cl, Br, and F, A is an alkylene group having 1 to 5 carbon atoms, and B is a group represented by the following formulas (II) to (IV): (In the formula, R 1 and R 2 are each independently an alkylene group having 1 to 5 carbon atoms which may contain a carbonyl group), (In the formula, R 3 is an alkylene group having 1 to 5 carbon atoms which may contain a carbonyl group), and and n is a group selected from the group consisting of:

2. The anti-HIV antibody-drug conjugate according to claim 1, wherein the anti-HIV antibody is a neutralizing antibody specific for the V3 loop on the surface of HIV-1.

3. The anti-HIV antibody-drug conjugate according to claim 1, wherein the linker is a peptide linker or a PEG linker.

4. An HIV infection inhibitor comprising the anti-HIV antibody-drug conjugate according to any one of claims 1 to 3 as an active ingredient.

5. A pharmaceutical composition for treating or preventing HIV infection, comprising the HIV infection inhibitor according to claim 4.

Images