Targeted dendrimer complex

Abstract

Provided herein is a dendrimer-targeting agent conjugate comprising a dendrimer, a HER2 targeting agent, and a therapeutic agent, wherein the dendrimer comprises a core unit and a lysine or lysine analogue unit, the HER2 targeting agent is a peptide moiety having a molecular weight of up to about 80 kDa and comprising an antigen-binding site, and is covalently linked to the dendrimer by a spacer group, and the therapeutic agent is covalently linked to the dendrimer surface unit. Also provided herein are compositions comprising the conjugate and methods of treatment, particularly cancer treatment, using the conjugate.

Description

[Technical Field]

[0001] This disclosure relates to therapies that are generally useful for targeting cancers that overexpress HER2. More specifically, this disclosure relates to targeted delivery of therapeutic moieties using dendrimer-targeting moieties that include a HER2 antibody fragment or mimetic and a therapeutic moiety such as a hypercytotoxic agent residue. [Background technology]

[0002] According to the WHO, cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. The most common forms of cancer are lung cancer, breast cancer, colorectal cancer, prostate cancer, skin cancer, and stomach cancer.

[0003] Much effort is being made to develop effective cancer treatments. However, finding novel therapies that have sufficient efficacy at the target, appropriate pharmacokinetic properties to deliver to the site of action, sufficient duration of action, and a good safety profile is extremely difficult, time-consuming, and costly.

[0004] The development of monoclonal antibody therapies such as trastuzumab (Herceptin®) and rituximab (Mabthera®) represents a notably successful approach. For example, Herceptin®, approved in 1998, is used to treat HER2-overexpressing breast and gastric cancers. This antibody works by binding to the HER2 / ERBB2 receptor, which is overexpressed in some cancer morphologies, and blocking the signal that stimulates cancer cell growth. However, Herceptin® also has drawbacks. Known side effects include myocardial damage and heart failure. Furthermore, Herceptin® is not sufficiently effective in some patients whose cancer is HER2-positive, and several resistance mechanisms have been proposed (Pohlmann, Mayer and Mernaugh, 2009).

[0005] Another contrasting therapeutic approach involves the use of cytotoxic agents that are active against rapidly dividing cells. Cisplatin, a long-established example, is used to treat various cancers such as ovarian cancer, bladder cancer, testicular cancer, and squamous cell carcinoma. Another group of cytotoxic agents are hypercytotoxic agents such as auristatins, including monomethyl auristatin E (MMAE), which are extremely potent but can simultaneously cause serious toxicity and adverse side effects in some patients. As a result, such hypercytotoxic agents cannot be safely administered to patients as chemotherapeutic agents on their own. Research has been conducted to identify ways to utilize such hypercytotoxic agents, leading to the development of antibody-drug conjugates such as brentuximab vedotin (Adcetris®), which contains MMAE conjugated to brentuximab (CD30 antibody) via a linear linker and has been approved as a treatment for Hodgkin lymphoma and anaplastic large cell lymphoma.

[0006] Many drug candidates fail in clinical trials due to a low therapeutic index (i.e., the ratio of therapeutic effect to toxicity). Some of the underlying reasons for this failure include lack of efficacy, lack of specificity, low drug absorption / bioavailability, uncontrollable biodistribution, rapid metabolism and clearance, and instability during storage. In practice, a balance must be struck between preventing the achievement of therapeutic cytotoxicity necessary to kill cancer cells and reducing harmful toxicity and side effects to a level tolerable by patients.

[0007] Various approaches are being investigated to test and improve the therapeutic effects of compounds with suboptimal properties. For example, sustained-release formulations can be developed for compounds that are rapidly eliminated from the body. One such example in the field of chemotherapy is the liposomal formulation of the active ingredient irinotecan, product Onivyde®, which significantly reduces plasma excretion compared to conventional formulations (Messerer et al., 2004). Other technologies include the use of depot injection formulations (e.g., Lupron Depot®). Dendrimer technology is also being studied to improve the properties of pharmaceutically active agents and avoid formulation problems; for example, International Publication No. 2012 / 167309 describes the provision of drug-dendrimer conjugates, particularly those containing the poorly soluble pharmaceutically active agent docetaxel.

[0008] Despite these technological and other advancements, there is still a need for further development of safe and effective treatments for diseases such as cancer.

[0009] None of the considerations of documents, actions, materials, devices, articles, etc. described herein constitute any or all of the prior art basis or common general knowledge in the art relevant to this disclosure. [Overview of the project]

[0010] In the first embodiment, a dendrimer-targeting agent conjugate is provided, the conjugate is a) i) Core unit (C), and ii) A dendrimer comprising a constituent unit (BU), wherein each constituent unit is a lysine residue or an analog thereof, A dendrimer is formed in which a core unit is covalently attached to at least two constituent units via amide linkages, and each amide linkage is formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit; b) A HER2 targeting agent having a maximum molecular weight of approximately 80 kDa and containing an antigen-binding site, which is covalently linked to a dendrimer by a spacer group; c) A therapeutic agent covalently linked to the surface constituent units of a dendrimer; d) A hydrophilic polymer group covalently bonded to the surface constituent unit of the dendrimer.

[0011] In some embodiments, the peptide moiety is selected from heavy chain antibodies, Fab, Fv, scFv, or single-domain antibodies. In some embodiments, the peptide moiety is heavy chain variable (V H ) containing domains or heavy chain variable (V H ) consists of domains. In some embodiments, the peptide portion is light chain variable (V L ) Consists of domains or light chain variable (V L It consists of domains.

[0012] In some embodiments, the targeting agent has a molecular weight of about 5 kDa to about 30 kDa. In some embodiments, the targeting agent has a molecular weight of about 5 kDa to about 18 kDa. In some embodiments, the targeting agent has a molecular weight of about 5 kDa to about 15 kDa. In some embodiments, the targeting agent has a molecular weight of about 10 kDa to about 16 kDa. In some embodiments, the targeting agent has a molecular weight of about 14 kDa to about 18 kDa.

[0013] In some embodiments, the targeting agent consists of fewer than 120 amino acid residues.

[0014] In some embodiments, the targeting agent comprises or consists of any of the amino acid sequences defined herein.

[0015] In some embodiments, one targeting agent is covalently linked to the dendrimer. In some embodiments, two or more targeting agents are covalently linked to the dendrimer.

[0016] In some embodiments, the targeting agent precursor comprises a non-natural amino acid residue, and the non-natural amino acid residue has a side chain comprising a reactive functional group.

[0017] In some embodiments, the non-natural amino acid residue is

Chemical Formula

[0018] In some embodiments, the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting moiety.

[0019] In some embodiments, the spacer group precursor comprises a reactive functional group that is an alkyne group. In some embodiments, the alkyne group is a dibenzocyclooctyne group.

[0020] In some embodiments, the spacer group is covalently attached to a surface building unit of a dendrimer. In some embodiments, the spacer group is covalently attached to a core unit.

[0021] In some embodiments, the covalent linkage between the targeting agent and the spacer group is formed by the reaction between a complementary reactive functional group present on the targeting agent precursor and the spacer group precursor. In some embodiments, the spacer group consists of an alkene group.

[0022] In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent. In some embodiments, the therapeutic agent is a supercytotoxic agent. In some embodiments, the therapeutic agent is auristatin or a maytansinoid. In some embodiments, the therapeutic agent is monomethyl auristatin E. In some embodiments, the therapeutic agent is monomethyl auristatin F. In some embodiments, the therapeutic agent is SN-38. In some embodiments, the therapeutic agent is cabazitaxel.

[0023] In some embodiments, the therapeutic agent is covalently linked to the surface constituent units of the dendrimer via a linker. In some embodiments, the therapeutic agent is covalently linked to the surface constituent units of the dendrimer via a cleavable linker. In some embodiments, the cleavable linker contains a Val-Cit-PAB group.

[0024] In some embodiments, the composite includes hydrophilic polymer groups covalently bonded to the surface constituent units of the dendrimer. In some embodiments, the hydrophilic polymer groups are PEG groups, which are covalently bonded to the surface constituent units of the dendrimer. In some embodiments, the PEG groups have an average molecular weight in the range of about 500 to about 2500 g / mol.

[0025] In some embodiments, the spacer group includes a PEG group.

[0026] In some embodiments, the core unit has the following structure: [ka] Includes.

[0027] In some embodiments, the core unit has the following structure: [ka] Includes.

[0028] In some embodiments, the core unit is BHALys.

[0029] In some embodiments, the dendrimer has 1 to 5 generations of structural units. In some embodiments, the dendrimer has 3 to 5 generations of structural units. In some embodiments, the dendrimer has 3 generations of structural units. In some embodiments, the dendrimer has 4 generations of structural units. In some embodiments, the dendrimer has 5 generations of structural units.

[0030] In some embodiments, each of the structural units is

Chemical Formula

[0031] In some embodiments, the conjugate is for administration in combination with an additional active agent.

[0032] In some embodiments, the conjugate is internalized into HER2-expressing cells.

[0033] In some embodiments, administration of the conjugate results in reduced side effects compared to administration of an equivalent dose of free therapeutic agent.

[0034] In some embodiments, administration of the conjugate results in at least a 50% reduction in the maximum plasma concentration of the released therapeutic agent compared to administration of an equivalent dose of free therapeutic agent.

[0035] In a second aspect, there is provided a composition comprising a plurality of conjugates as defined herein.

[0036] In a third aspect, i) a conjugate as described herein; ii) a pharmaceutically acceptable excipient; provided is a pharmaceutical composition comprising the foregoing.

[0037] In some embodiments, the composition is formulated for parenteral delivery.

[0038] In some embodiments, the complex or composition is intended for use in the treatment of cancer.

[0039] A fourth aspect provides a method for treating cancer, comprising administering a therapeutically effective amount of the complex or composition described herein to a subject in need thereof.

[0040] A fifth aspect provides the use of the complex or composition described herein in the manufacture of a drug for the treatment of cancer.

[0041] In some embodiments, the cancer is ovarian cancer, breast cancer, gastric cancer, uterine cancer, or another cancer characterized by abnormal expression of the ERBB2 gene.

[0042] In the sixth aspect, a method for killing HER2-expressing cells, A method is provided which involves contacting a complex defined herein with HER2-expressing cells, thereby internalizing the complex into the cells, and thereby causing the therapeutic agent to kill the HER2-expressing cells.

[0043] Further aspects, embodiments, and examples are described herein, and it will be understood that these may include one or more of the embodiments or features described above. [Brief explanation of the drawing]

[0044] [Figure 1] This figure shows the SDS-Page analysis results for compounds 74, 75, and 76 stained with Coomassie blue. [Figure 2] This figure shows the SDS page and fluorescence imaging analysis of compounds 77 and 78. [Figure 3] The figures show the SDS page and fluorescence imaging analysis of compound 41 (lane A: crude mixture; lane B: purified complex), 42 (lane C: crude mixture; lane D: purified complex), 43 (lane E: crude mixture; lane F: purified complex), 44 (lane G: crude mixture; lane H: purified complex), and 45 (purified complex). [Figure 4] This figure shows the mean fluorescence intensity values ​​over 24 hours for compound 36 (control) and compound 41 (target) in MDA-MB-231, MDA-MB-231 / HER2, and SKOV-3 cells. At least 10,000 cells were counted for each measurement. Values ​​are mean ± standard deviation (SD; n=3). [Figure 5-1] This figure shows the flow cytometry results when dendrimers were incubated in HER2-positive cells (MDA-MB-231 / HER2) and HER2-negative cells (MDA-MB-231) at 3.33 nM for 24 hours at 37°C. At least 10,000 cells were counted per measurement. Values ​​are mean ± standard deviation (SD; n=3). [Figure 5-2] (Same as above.) [Figure 6] a) Confocal microscopy images of MDA-MB-231 cells treated with compound 36 (control) or b) compound 41 (target) at a concentration of 3.33 nM for 24 hours. Green, blue, and red fluorescence represent the cell membrane stained with AF-488-WGA, the nucleus stained with DAPI, and the dendrimer labeled with Cy5, respectively. Scale bar = 50 μm. [Figure 7] a) Confocal microscopy images of MDA-MB-231 / HER2 cells treated with compound 36 (control) or b) compound 41 (target) at a concentration of 3.33 nM for 24 hours. Green, blue, and red fluorescence represent the cell membrane stained with AF-488-WGA, the nucleus stained with DAPI, and the dendrimer labeled with Cy5, respectively. Scale bar = 50 μm. [Figure 8] a) Confocal microscopy images of SKOV-3 cells treated with compound 36 (control) or b) compound 41 (target) at a concentration of 3.33 nM for 24 hours. Green, blue, and red fluorescence represent the cell membrane stained with AF-488-WGA, the nucleus stained with DAPI, and the dendrimer labeled with Cy5, respectively. Scale bar = 50 μm. [Figure 9]This figure shows the distribution data of tumors and blood after 48 hours of sacrifice of dendrimers of 3H-labeled compounds 40 and 45. All data were normalized by tissue volume. All data represent mean ± SEM (n=5); (NS = not significant; * = p value < 0.05). [Figure 10] This figure shows the representative ex-vivo tumor distribution of compounds 36 and 41 after euthanasia at 48 hours. The data show typical fields of view for (a) non-target dendrimer (compound 36) and (b) target dendrimer (compound 41). [Figure 11] This image shows that the target dendrimer (compound 41) was taken up in the central and peripheral regions of the tumor, while the control compound 36 was not taken up. [Figure 12] This figure shows a plot of the mean tumor volume over time in mice inoculated with SKOV3 cells, after treatment with vehicle, control compound 36, target complex 41, and Kadcyla® or Herceptin®. [Figure 13] This figure shows the time course of survival in mice inoculated with SKOV3 cells after treatment with vehicle, control compound 36, target complex 41, and Kadcyla® or Herceptin®. [Figure 14] This figure shows the average percentage change in body weight over time after administering a vehicle, control compound 36, target complex 41, Kadcyla®, or Herceptin® to mice inoculated with SKOV3 cells. [Figure 15] This figure shows the internalization rates of fourth-generation dendrimers and single (compound 90, MFI single) or multiple (compound 91, MFI multiple) conjugate anti-HER2 nanobodies. [Figure 16]These are confocal microscope images showing SKOV-3 cells after incubation with compound 91 (multiple 2D3-dendrimer complexes) at 37°C for a) 1 hour, b) 3 hours, c) 6 hours, or d) 24 hours. Compound 91 is stained with Cy5 (magenta), the cell membrane with AF488-WGA (green), and the nucleus with Hoechst 33342 (blue). Scale bar = 30 μM [Figure 17] These are confocal microscope images showing SKOV-3 cells after incubation with compound 90 (a single 2D3-dendrimer complex) at 37°C for a) 1 hour, b) 3 hours, c) 6 hours, or d) 24 hours. Compound 91 is stained with Cy5 (magenta), the cell membrane with AF488-WGA (green), and the nucleus with Hoechst 33342 (blue). Scale bar = 30 μM [Figure 18] This is a radioactive TLC image of the compound of this disclosure showing that 89Zr is bound to the dendrimer. [Figure 19-1] This graph shows the percentage of zirconium injected per gram in (a) the kidney, (b) the liver, and (c) tumors over a 9-day period for compounds 89, 90, and 92. [Figure 19-2] (Same as above.) [Figure 20] This figure shows representative maximum intensity projections of PET images of radioisotope complexes in animals from 4 hours to 9 days after birth. The data is expressed in becquerels / voxels (cm³), and thresholds have been set to highlight tumor uptake. [Figure 21] This table shows the tumor:organ ratio of ex vivo signaling for injected zirconium (dose / gram) for the compounds of this disclosure on days 2 and 9. [Figure 22] This table shows the percentage of injected zirconium (dose / gram) in ex vivo tumors and organs for the compounds of this disclosure on days 2 and 9. [Figure 23] This figure shows the flow cytometry analysis and average fluorescence intensity values ​​over 24 hours for compound 70 (control) and compound 78 (target) in MDA-MB-231 / HER2 cells. [Figure 24] This figure shows the flow cytometry analysis and average fluorescence intensity values ​​over 24 hours for MDA-MB-231 cells with compound 70 (control) and compound 78 (target). [Figure 25] This figure shows the flow cytometry analysis and mean fluorescence intensity values ​​over 24 hours for compound 70 (control) and compound 78 (target) in SKOV-3 cells. Sequence listing symbols: SEQ ID NO: 1: 2D3 nanobody. SEQ ID NO: 2D3 nanobody with N-terminal tag, TEV, and C-terminal azide. SEQ ID NO: 3: 2D3 nanobody with C-terminal tag, TEV, and azide. [Modes for carrying out the invention]

[0045] General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art (e.g., chemistry, biochemistry, medicinal chemistry, polymer chemistry, etc.).

[0046] In this specification, the word “comprise,” or variations such as “comprises” or “comprising,” is understood to mean encompassing the element, integer or step, or group of elements, integer or step, described herein, and not to mean excluding any other element, integer or step, or group of elements, integer or step. When used herein, the term “and / or,” for example “X and / or Y,” is understood to mean either “X and Y” or “X or Y,” and is deemed to explicitly support both meanings or either meaning.

[0047] When used herein, unless otherwise stated, the term “about” means ±10%, more preferably ±5%, of the specified value.

[0048] As used herein, the terms "a," "an," and "the" include both singular and plural aspects unless the context clearly indicates otherwise.

[0049] In this specification, the term “subject” refers to any organism susceptible to diseases or conditions associated with HER2 (human epidermal growth factor receptor 2). In one embodiment, the subject may be an animal. In one example, the subject is a mammal. In one embodiment, the subject is a human. In one embodiment, the subject is a non-human animal. In one embodiment, the subject has cancer. In one embodiment, the subject has HER2-positive cancer.

[0050] In this specification, the term “treating” includes alleviating symptoms associated with a particular disorder or condition. For example, as used herein, the term “treating cancer” includes alleviating symptoms associated with cancer. In one embodiment, the term “treating cancer” refers to reducing the size of a cancerous tumor. In one embodiment, the term “treating cancer” refers to reducing the invasiveness of cancer. In one embodiment, the term “treating cancer” refers to extending progression-free survival. In this specification, the term “progression-free survival” refers to the length of time during and after treatment for cancer in which a patient has the disease, i.e., cancer, but does not experience a recurrence of the disease or an increase in the symptoms of the disease.

[0051] In this specification, the term “prevention” includes preventing a particular disorder or condition. For example, in this specification, the term “prevent cancer” means preventing the onset or persistence of symptoms associated with cancer. In one embodiment, the term “prevent cancer” means slowing or stopping the progression of cancer. In one embodiment, the term “prevent cancer” means slowing or preventing metastasis.

[0052] As used herein, “therapeutic effective dose” refers to a complex containing a therapeutic agent, administered in an amount sufficient to alleviate or, to some extent, prevent one or more of the symptoms of the disorder or condition being treated. The result may be a reduction and / or mitigation of the signs, symptoms or causes of the disease or condition, or any other desirable change in the biological system. In one embodiment, the term “therapeutic effective dose” refers to an amount of the complex administered sufficient to result in a reduction in the size of a cancerous tumor. In one embodiment, the term “therapeutic effective dose” refers to an amount of the complex administered sufficient to result in an extension of progression-free survival. As used herein, “effective dose” refers to an amount of the complex that is effective to achieve the desired pharmacological effect or therapeutic improvement without excessive side effects, or to achieve the desired pharmacological effect or therapeutic improvement with a reduced side effect profile. The therapeutic effective dose may be determined by routine experiments, such as, for example, dose-escalation clinical trials, but is not limited to these. “Therapeutic effective dose” includes, for example, a prophylactic effective dose. In one embodiment, a prophylactic effective dose is an amount sufficient to prevent metastasis. It is understood that the “effective dose” or “therapeutic effective dose” may vary from person to person due to variations in the compound’s metabolism, age, weight, the subject’s overall health, the condition being treated, the severity of the condition being treated, and the prescribing physician’s judgment. The appropriate “effective dose” for any individual can be determined by those skilled in the art using routine experiments.

[0053] As used herein, the term “attached” refers to a connection between chemical components by covalent bonds. The term “covalent bond” is used interchangeably with the term “covalent attachment.”

[0054] Dendrimer In the first embodiment, a dendrimer-targeting agent conjugate is provided, the conjugate is a) i) Core unit (C), and ii) A dendrimer comprising a constituent unit (BU), wherein each constituent unit is a lysine residue or an analog thereof, A dendrimer is formed in which a core unit is covalently attached to at least two constituent units via amide linkages, and each amide linkage is formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit; b) A HER2 targeting agent having a maximum molecular weight of approximately 80 kDa and containing an antigen-binding site, which is covalently linked to a dendrimer by a spacer group; c) A therapeutic agent covalently linked to the surface constituent units of a dendrimer; d) A hydrophilic polymer group covalently bonded to the surface constituent unit of the dendrimer.

[0055] Dendrimer complexes have been demonstrated to be highly effective anticancer agents in in vitro and in vivo studies, and experimental studies have shown improved efficacy compared to Herceptin® and Kadcycla® (HER2-emtansine antibody-drug conjugates). In contrast to Herceptin® itself, this complex is rapidly internalized in HER2-overexpressing cancer cells. The rapid internalization property is expected to help deliver the therapeutic agent administered via the complex to the desired site of action at a high rate. The ability to specifically deliver the therapeutic portion to target cells, through rapid internalization, helps reduce unwanted side effects (i.e., toxicity) associated with the exposure of other non-cancerous cells to the cytotoxic agent. Such an approach is particularly advantageous when delivering cytotoxic therapeutic portions (e.g., hypercytotoxic therapeutic portions), which can have toxic effects if they circulate freely in plasma.

[0056] In this specification, the term “dendrimer” refers to a molecule comprising a core and dendrons attached to that core. Each dendron consists of generations of branched constituent units, with the number of branches increasing with each generation of constituent units. A “complex” may include pharmaceutically acceptable salts or solvates as defined herein.

[0057] As used herein, the term “constituent unit” refers to a branched molecule which is a lysine residue or analogue having three functional groups. One functional group (e.g., derived from a carboxylic acid group) is for attachment to the core or previous generation constituent unit, and at least two functional groups (e.g., derived from an amine) are for attachment to the next generation constituent unit or for forming the surface of the dendrimer molecule.

[0058] Those skilled in the art will understand that complexes can be produced in various forms, such as salts (for example, when ionizable groups present in the complex are present), and different solvates. This disclosure will be understood to relate to all such forms of dendrimer-target partial complexes.

[0059] Suitable salts of the complex include those formed with organic or inorganic acids or bases. In this specification, the expression “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt. Exemplary acid addition salts include, but are not limited to, salts of sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, vitalates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, saccharates, formates, benzoates, glutamates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, and pamoates (i.e., salts of 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Examples of base addition salts, but not limited to these, include ammonium salts, alkali metal salts, e.g., potassium and sodium salts, alkaline earth metal salts, e.g., calcium and magnesium salts, salts with organic bases, e.g., dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono, di, tri lower alkylamines, e.g., ethyl, tert-butyl, diethyl, diisopropyl-, triethyl-, tributyl-, or dimethyl-propylamine, or mono-, di-, tri-hydroxy lower alkylamines, e.g., mono-, di-, triethanolamine. A pharmaceutically acceptable salt may contain other molecules, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic part that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.Furthermore, non-pharmaceutically acceptable salts may be useful as intermediates in the preparation of pharmaceutically acceptable salts, or as useful during storage or transport, and will therefore be understood to be within the scope of this disclosure.

[0060] Those skilled in organic and medicinal chemistry will understand that many organic compounds can form complexes with solvents that react with, precipitate, or crystallize them. Such complexes are known as “solvates.” For example, a complex with water is known as a “hydrate.” In this specification, the terms “pharmaceutically acceptable solvate” or “solvate” refer to the association of one or more solvent molecules with a compound of the disclosure. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0061] Core unit The dendrimer's core unit (C) is covalently attached to the constituent units via amide linkages, each amide linkage formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit. Therefore, the core unit may be formed from, for example, a core unit precursor containing an amino group. Any suitable amino-containing molecule may be used as the core unit precursor.

[0062] Typically, the core unit may contain functional groups to provide additional functionality to the nitrogen atoms used for covalent attachment of the constituent units, for example, to attach the targeted moiety via a spacer group. In other words, the HER2 targeter is typically covalently linked to the dendrimer's core unit by a spacer group. In some embodiments, the core unit can be derived from a precursor having three reactive nitrogen atoms, two of which may be used to attach the constituent units and one of which may be used to attach the spacer group.

[0063] In some embodiments, the core unit has the following structure: [ka] It holds.

[0064] In some embodiments, the core unit is a lysine molecule having three attachment points, structure: [ka] It holds.

[0065] In some embodiments, the core unit can be derived from a precursor having two reactive nitrogen atoms, which can be used to attach the constituent units. For example, in some embodiments, the core unit may be derived from ethylenediamine, 1,4-diaminobutane, or 1,6-diaminohexane. In some embodiments, the core unit has the following structure: [ka] The core unit has, that is, the acid portion is capped with benzhydrylamine to form a lysine residue (BHA-Lys) that forms the corresponding amide, for example, a core unit precursor having two reactive (amino)nitrogens: [ka] It can be formed from.

[0066] When using core unit precursors having only two reactive nitrogen atoms, such as BHA-Lys, the two amino groups are typically functionalized in the constituent unit, and the spacer group (and thus the targeting agent) is typically attached via the surface constituent unit. In the dendrimer-targeting agent complex of the present invention, multiple terminal groups can be presented on the surface of the dendrimer-targeting agent complex in a controlled manner. In particular, when a lysine constituent unit is used, the arrangement of the targeting agent and / or hydrophilic polymer group and / or therapeutic agent on the alpha or epsilon nitrogen atom of the constituent unit can be predetermined as described below. In some preferred embodiments, the hydrophilic polymer group, therapeutic agent, and targeting agent are provided to the surface of the dendrimer via attachment via the constituent unit. In other words, in those embodiments, the core unit does not provide any attachment sites for the targeting agent other than via the constituent unit. In such embodiments, it will be understood that any functional groups present in the core unit that are not used for covalent attachment to the constituent units are either unreacted (i.e., unreacted under the conditions to which the complex is exposed) or capped with suitable capping groups to prevent further reaction. An example of such a core unit is the BHA-Lys group described above.

[0067] Components A constituent unit (BU) is a lysine residue or an analogue thereof, and may be formed from a suitable constituent unit precursor, e.g., a lysine or lysine analogue containing a suitable protecting group. The lysine analogue has two amino nitrogen atoms for binding to a later-generation constituent unit and an acyl group for binding to a previous-generation constituent unit or core unit. Examples of suitable constituent units include: [ka] These include, The acyl group of each component unit provides covalent attachment sites for attachment to the core or previous generation component units, and each nitrogen atom provides covalent attachment sites for attachment to subsequent generation component units or terminal groups such as linked therapeutic agents.

[0068] In some preferred embodiments, the constituent units are, [ka] and; The acyl group of each component unit provides a covalent attachment site for attachment to a core unit or a previous generation component unit, and each nitrogen atom provides a covalent attachment site for attachment to a subsequent generation component unit or a terminal group such as a linked therapeutic agent.

[0069] In some preferred embodiments, the constituent units are, [ka] and; The acyl group of each component unit provides covalent attachment sites for attachment to the core or previous generation component units, and each nitrogen atom provides covalent attachment sites for attachment to subsequent generation component units or terminal groups such as linked therapeutic agents.

[0070] The outermost generation of building blocks (BU) outer As mentioned above, the outermost generation of constituent units (BU) may be formed by lysine or lysine analog constituent units used in other generations of constituent units (BU). outer ) is the outermost generation of constituent units from the dendrimer's core unit, i.e., the outermost generation of constituent units (BU) outer Subsequent generation component units will not be attached to it.

[0071] It will be understood that the dendrons of a dendrimer can be synthesized to the required number of generations, for example, by appropriately attaching constituent units (BUs). In some embodiments, the constituent units (BUs) of each generation may be formed from the same constituent units, for example, all generations of the constituent units may be lysine constituent units. In some other embodiments, the constituent units of one or more generations may be formed from different constituent units than the constituent units of the other generations.

[0072] The dendrimer portion typically has 1 to 5 generations of constituent units. In some embodiments, the dendrimer is a 1-generation constituent unit dendrimer. In some embodiments, the dendrimer is a 2-generation constituent unit dendrimer. In some embodiments, the dendrimer is a 3-generation constituent unit dendrimer. In some embodiments, the dendrimer is a 4-generation constituent unit dendrimer. In some embodiments, the dendrimer is a 5-generation constituent unit dendrimer. For example, a 3-generation constituent unit dendrimer is a dendrimer having a structure that includes three constituent units covalently linked to each other, for example, when the constituent units are lysine, and this structure: [ka] It may include.

[0073] In some embodiments, the dendrimer has constituent units of a complete generation. For example, a third-generation dendrimer has three constituent units of a complete generation. In the case of a core having two reactive amine groups, such a third-generation dendrimer would contain 14 constituent units (i.e., core unit + 2BU + 4BU + 8BU). However, given the nature of the synthetic process for producing dendrimers, it will be understood that one or more reactions performed to produce a dendrimer may not be fully completed. Therefore, in some embodiments, the dendrimer may contain constituent units of an incomplete generation. For example, a population of dendrimers can be obtained in which the dendrimers have a distribution of the number of constituent units per dendrimer. In some embodiments, a population of dendrimers can be obtained in which each dendrimer has an average number of constituent units of at least 8, or at least 9, or at least 10, or at least 11, or at least 12, or at least 13. In some embodiments, a population of dendrimers can be obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 10 or more constituent units. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers are obtained as a group of dendrimers having 12 or more constituent units.

[0074] In some embodiments, each generation of the constituent units in each dendron (X) is given by Equation [BU]2 (b-1) It may also be expressed as, where b is the number of generations. A dendron (X) with three complete constituent units of three generations is represented as [BU]1-[BU]2-[BU]4.

[0075] Targeting agent The dendrimer-targeting agent conjugates described herein include a HER2 targeting agent, i.e., the targeting agent is capable of binding to human epidermal growth factor receptor 2 (HER2, also known as ERBB2; Gene ID No. 2064, NCBI). The HER2 targeting agents described herein are useful for targeting the disclosed dendrimer conjugates to tumor and cancer cells.

[0076] The targeting agent contains a peptide moiety with a molecular weight of up to approximately 80 kDa and includes an antigen-binding site that specifically binds to the molecule (i.e., HER2) or has affinity for the target molecule (i.e., HER2). This interaction can arise from any type of binding or association, such as covalent bonds, ionic bonds, hydrogen bonds, or van der Waals forces.

[0077] In one embodiment, the HER2 targeting agent described herein has a molecular weight of about 3 kDa to about 80 kDa, or about 3 kDa to about 60 kDa, or about 3 kDa to about 50 kDa, or about 3 kDa to about 40 kDa, or about 3 kDa to about 30 kDa, or about 3 kDa to about 20 kDa, or about 3 kDa to about 15 kDa, or about 3 kDa to about 13 kDa, or about 5 kDa to about 15 kDa, or about 5 kDa to about 12 kDa, or about 5 kDa to about 10 kDa.

[0078] In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 80 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 60 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 50 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 40 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 30 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 20 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 15 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 3 kDa to about 13 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 5 kDa to about 15 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 5 kDa to about 12 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of from about 5 kDa to about 10 kDa.

[0079] As used herein, "kDa" or "kilodalton" refers to a unit of molecular weight consisting of 1000 daltons.

[0080] In some embodiments, the targeting agent is an antibody fragment. As used herein, the term "antibody fragment" is intended to mean a portion or fragment of an antibody capable of specifically binding an antigen, for example, F as defined herein V , V H , V L or a variable region. It will be understood that the term encompasses fragments directly derived from antibodies, as well as proteins produced using recombinant means. In one embodiment, the antibody fragment is selected from Fab, Fv, scFv, heavy chain antibody, domain antibody, heavy chain antibody, diabody or triabody.

[0081] As used herein, the term "Fv" means any protein in which VL and VH associate to form a complex having an antigen-binding domain, i.e., any protein that can specifically bind to an antigen, whether it is composed of multiple polypeptides or a single polypeptide (scFV). H and V L This may be a single polypeptide chain or different polypeptide chains. In one embodiment, Fv of the Disclosure (as well as any protein of the Disclosure) may have multiple antigen-binding sites that may or may not bind the same antigen. This term will be understood to encompass fragments directly derived from antibodies, as well as proteins produced using recombinant means. In some examples, V H It is not linked to the heavy chain constant domain CH1, and / or V L The light chain constant domain (CL), for example, is not linked to the domain antibody. Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, etc. Fab fragments consist of monovalent antigen-binding fragments of immunoglobulin and can be produced by digesting the whole antibody with the enzyme papain to produce a fragment consisting of the intact light chain and part of the heavy chain, or by using recombinant means. Fab fragments are generally V H and C H 1 and V L and C L It contains or is composed of these. The "Fab' fragment" of the antibody is obtained by reducing the intact light chain and V after treating the entire antibody with pepsin. H This can be obtained by generating a molecule consisting of a portion of the heavy chain containing a single constant domain. Two Fab' fragments are obtained from antibodies treated in this way. Fab' fragments can also be produced by recombinant means. A "single-chain Fv" or "scFv" is a recombinant molecule containing a variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable and flexible polypeptide linker.

[0082] In some embodiments, the antibody fragment is selected from heavy chain antibodies, Fab, Fv, scFv, or single-domain antibodies.

[0083] As used herein, a "single-domain antibody (sdAb)," also called a "domain antibody (dAb)" or "nanobody," is a single variable region heavy chain V H or light chain V L It consists of. In one embodiment, the variable region is derived from the camelid family. In one embodiment, the variable region is derived from the shark. In one embodiment, V H V, which originates from the camelid family. H That is the case.

[0084] In some embodiments, the targeting agent is a single-domain antibody. In some embodiments, the targeting agent is a VH single-domain antibody. In some embodiments, the targeting agent is a VL single-domain antibody.

[0085] In one embodiment, the single-domain antibody comprises a single-domain amino acid sequence described, for example, European Publication No. 2215125(A1), U.S. Patent No. 20110028695, Hussack et al. (2018), or Arezumand et al. (2017). In one embodiment, the single-domain antibody comprises a single-domain amino acid sequence described in U.S. Patent No. 20110028695. In some embodiments, the single-domain antibody is a nanobody described by Vaneycken et al. (Vaneycken et al. (2011)). In some embodiments, the single-domain antibody is 2Rs15d (Vaneycken et al. (2011)). In some embodiments, the single-domain antibody is 2Rb17c (Vaneycken et al. (2011)). In some embodiments, the single-domain antibody is 1R59b (Vaneycken et al. (2011)). In some embodiments, the single-domain antibody is 2R5a (Vaneycken et al. (2011)). In some embodiments, the single-domain antibody is 1R136d (Vaneycken et al. (2011)). In some embodiments, the single-domain antibody is 1R143c (Vaneycken et al. (2011)). In some embodiments, the single-domain antibody is C3 (Wu et al. (2018)). In some embodiments, the single-domain antibody is 5F7GGC (Pruszynski et al. (2013)). In one embodiment, the single-domain antibody is 5F7 containing an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology with that shown in U.S. Patent No. 20110028695.

[0086] In one embodiment, the single-domain antibody comprises the single-domain amino acid sequence disclosed in Example 7. In one embodiment, the single-domain antibody is 2D3 comprising the amino acid sequence shown in Sequence ID No. 1986 of U.S. Patent No. 20110028695.

[0087] In one embodiment, the single-domain antibody comprises the amino acid sequence EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS.

[0088] In some embodiments, the targeting agent does not compete for HER2 binding with trastuzumab and / or pertuzumab. In some embodiments, the targeting agent competes for HER2 binding with trastuzumab and / or pertuzumab. In one embodiment, the targeting agent is 2D3, and the targeting agent competes for HER2 binding with trastuzumab and / or pertuzumab. In some embodiments, the targeting agent binds to the extracellular domain of HER2. In some embodiments, the targeting agent binds to the extracellular domains I, II, III, and / or IV of HER2. In one embodiment, the targeting agent binds to the extracellular domain III of HER2. In some embodiments, the targeting agent binds to the dimerized arm of ErbB2. In examples where the targeting agent binds to the dimerized arm of ErbB2, the targeting agent may compete for HER2 binding with pertuzumab. In some embodiments, the targeting agent binds to HER2 with a binding affinity of at least 1 nM, at least 2 nM, at least 5 nM, or at least 10 nM, as determined by SPR testing.

[0089] In some embodiments, the HER2 targeting agent is a single-domain antibody having a molecular weight of approximately 4 kDa to approximately 80 kDa, or approximately 5 kDa to approximately 80 kDa, or approximately 5 kDa to approximately 60 kDa, or approximately 5 kDa to approximately 50 kDa, or approximately 5 kDa to approximately 40 kDa, or approximately 5 kDa to approximately 30 kDa, or approximately 5 kDa to approximately 20 kDa, or approximately 5 kDa to approximately 15 kDa, or approximately 5 kDa to approximately 12 kDa, or approximately 10 kDa to approximately 16 kDa.

[0090] In some embodiments, the HER2 targeter contains less than 500, less than 400, less than 300, less than 200, less than 150, less than 140, less than 130, less than 120, less than 110, or less than 100 amino acid residues. In some embodiments, the HER2 targeter contains more than 50, more than 75, more than 100, or more than 120 amino acid residues. In some embodiments, the HER2 targeter contains less than 120 amino acid residues. In some embodiments, the HER2 targeter contains more than 100 to 120 amino acid residues.

[0091] In some embodiments, the HER2 targeting agent is a mimetic of an antibody or antibody fragment. Hereinafter, the terms “mimetic” or “mimetics” refer to compounds that can bind an antigen but are not structurally related to an antibody, such as an antibody or antibody fragment. This term is understood to be exclusive to the antibodies or antibody fragments described herein. This term is understood to encompass synthetic (in vitro produced) mimetics and mimetics produced using recombinant means. This term is understood to encompass protein mimetics.

[0092] In one embodiment, the mimetic is selected from afibody, aptamer, affin, affimer, afitin, antikalin, avimer, alphabody, monobody, DARPin, Fyomer, fibronectin type III-derived protein scaffold, phytocystatin-derived protein scaffold, and paratope mimetic peptides. Similar to antibodies, the mimetic can also be used as a targeting site.

[0093] In one embodiment, the mimetic is derived from one of the following protein scaffolds: the z domain of protein A, γ-B crystallin, ubiquitin, cystatin, sac7d, triple helix, coiled coil, lipocalin, cyclotide, the A domain of membrane receptors, ankyrin repeat motif, the sh3 domain of Fym, the Kunits domian of protease inhibitors, the type III domain of fibronectin, the IgG-like form of Clostridium perfringens, or one of the thermostable carbohydrate-binding module family 32 (CBM32).

[0094] In one embodiment, the mimetic range is approximately 3kDa to 20kDa, or approximately 4kDa to 18kDa, or approximately 6kDa to 16kDa, or approximately 6kDa to 14kDa, or approximately 6kDa to 12kDa, or approximately 6kDa to 10kDa, or approximately 6kDa to 8kDa. In one embodiment, the mimetic range is approximately 3kDa to 20kDa. In one embodiment, the mimetic range is approximately 3kDa to 20kDa. In one embodiment, the mimetic range is approximately 4kDa to 18kDa. In one embodiment, the mimetic range is approximately 6kDa to 16kDa. In one embodiment, the mimetic range is approximately 6kDa to 14kDa. In one embodiment, the mimetic range is approximately 6kDa to 12kDa. In one embodiment, the mimetic value is approximately 6 kDa to approximately 10 kDa. In another embodiment, the mimetic value is approximately 6 kDa to approximately 8 kDa.

[0095] In some embodiments, the targeting agent is an affibody. Hereinafter, the term “affibody” refers to any of the classes of very small (approximately 6 kDa) polypeptide antibody mimetics based on a domain of three α-helix bundles, approximately 58 amino acids in length, known as the “Z domain.” Typically, the scaffold of an affibody is based on a modified B domain of protein A. Affibodies are characterized by extremely high stability (tolerant to high temperatures of 90°C and possessing target affinity ranging from nanomolar to picomolar). See, for example, Nord et al., (1995), Protein Eng., 8:601-608. Examples of known affibodies include, for example, affibodies against HER2 (e.g., Antibody-HER2 Antibody®, AFFIBODY AB, Bromma, Sweden; U.S. Patent No. 7,993,650).

[0096] In some embodiments, the targeting agent is an affibody having a molecular weight in the range of about 3 kDa to about 10 kDa, or about 3 kDa to about 8 kDa, or about 4 kDa to about 8 kDa, or about 4 kDa to about 7 kDa, or about 5 kDa to about 7 kDa, or about 6 kDa.

[0097] In some embodiments, the targeting agent is an affibody having fewer than 80 amino acid residues, or fewer than 70 amino acid residues, or fewer than 65 amino acid residues, or fewer than 60 amino acid residues. In some embodiments, the targeting agent consists of 40 to 80 amino acid residues, or 50 to 70 amino acid residues, or 55 to 65 amino acid residues, or 56 to 60 amino acid residues, or about 58 amino acid residues.

[0098] For the purposes of this disclosure, the term “antibody” includes four-chain proteins, such as two light chains and two heavy chains, including recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primate-like antibodies, deimmunized antibodies, and half-antibodies, bispecific antibodies) that can specifically bind to one or a few closely related antigens by Fv. Antibodies generally include a constant domain, which can be arranged into a constant region and a constant fragment, a crystallizable fragment (Fc). Exemplary forms of antibodies include a tetrachain structure as the basic unit. Full-length antibodies include two heavy chains (about 50–70 kDa) and two light chains (about 23 kDa each) linked by covalent bonds. Light chains generally include a variable region and a constant domain, which in mammals are either κ-light chains or λ-light chains. Heavy chains generally include a variable region and one or two constant domains, which are linked to additional constant domains by hinge regions. Mammalian heavy chains are one of the following types: α, δ, ε, γ, or μ. Each light chain is covalently linked to one of the heavy chains. For example, two heavy chains, and heavy and light chains, are held together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary depending on the type of antibody. Each chain has an N-terminal variable region (VH or VL, approximately 110 amino acids long each) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL: approximately 110 amino acids long) is aligned with and disulfide-bonded to the first constant domain of the heavy chain (CH: approximately 330-440 amino acids long). The variable region of the light chain is aligned with the variable region of the heavy chain. Antibody heavy chains can contain two or more additional CH domains (e.g., CH2, CH3, etc.) and may include a hinge region between the CH1 and CH2 constant domains. Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or a subclass. For example, an antibody could be a human antibody, its deimmunized form, germline form, or its affinity mature form.

[0099] The terms "full-length antibody" or "whole antibody" are used synonymously to refer to the substantially intact form of an antibody, as opposed to an antigen-binding fragment of the antibody. Specifically, whole antibodies include antibodies that have heavy and light chains, such as a constant region. The constant region may be the wild-type sequence constant region (e.g., the human wild-type sequence constant region) or an amino acid sequence variant thereof.

[0100] As used herein, the term “variable region” refers to a portion of the light chain and / or heavy chain of an antibody as defined herein, or a portion of an antibody consisting only of the heavy chain that can specifically bind to an antigen (e.g., a camelid antibody or a cartilaginous fish immunoglobulin neoantigen receptor (IgNAR)), and includes the amino acid sequences of the complementary determining region “CDR”; i.e., CDR1, CDR2, and CDR3, and the framework region “FR”. FR is a variable region residue other than the CDR residues. For example, the variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) along with the three CDRs. H This refers to the variable region of the heavy chain. L This refers to the variable region of the light chain.

[0101] As used herein, the term “complementarity-determining regions” (synonym CDR, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the variable regions of an antibody, the presence of which is a major contributor to specific antigen binding. Each variable region typically has three CDR regions, which are identified as CDR1, CDR2, and CDR3. Each complementarity-determining region may contain amino acid residues of the “complementarity-determining region” as defined by Kabat et al. (1987 and / or 1991). For example, in the heavy chain variable region, CDRH1 is located at residues 31–35, CDRH2 at residues 50–65, and CDRH3 at residues 95–102. In the light chain, CDRL1 is located at residues 24–34, CDRL2 at residues 50–56, and CDRL3 at residues 89–97. These CDRs may also include numerous inserts, as described, for example, in Kabat (1987 and / or 1991). This disclosure covers FRs and CDRs defined by all numbering systems, including, but not limited to, the Kabat numbering system, canonical numbering systems, or the numbering systems of Chothia and Lesk (1987); Chothia et al. (1989); and / or Al-Lazikani et al. (1997); Honnegher and Plukthun (2001); the IMGT system discussed by Giudicelli et al. (1997); or the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). In one example, the CDRs and / or FRs are defined according to the Kabat numbering system, as shown in bold in Figures 9A–9D, for example. Optionally, the heavy chain CDR2, as assigned by the Kabat numbering system, may not contain any of the five C-terminal amino acids described herein, or one or more of those amino acids may be substituted with other naturally occurring amino acids. Additionally or alternatively, the light chain CDR1 may not contain any of the four N-terminal amino acids described herein, or one or more of those amino acids may be substituted with other naturally occurring amino acids.In this regard, Padlan et al. established in 1995 that the five C-terminal amino acids of the heavy chain CDR2 and / or the four N-terminal amino acids of the light chain CDR1 are generally not involved in antigen binding. For example, CDRs and / or FRs are defined according to Chothia's numbering system, as depicted in the underlined text in Figures 9A-9D, for example.

[0102] In this specification, the term “Kabat numbering system” refers to the scheme described in Kabat (Kabat et al., 1987 and / or 1991) for numbering the variable regions of antibodies and identifying the CDR (hypervariable region).

[0103] In this specification, the term "Chothia numbering system" refers to the scheme described by Chothia and Lesk (Chothia and Lesk, 1987) or Al-Lazikani (Al-Lazikani et al., 1997) for numbering the variable regions of antibodies and identifying CDRs (structural loops).

[0104] As used herein, the term “antigen-binding domain” means a region of a compound that can specifically bind to an antigen (e.g., HER2). In one embodiment, the compound is a protein. In one embodiment, the protein is an antibody or a fragment thereof. In one embodiment, the antibody fragment is F as described herein. V , V H , or V L Includes one or more of the following.

[0105] As used herein, the terms “bind” or “binding” in relation to the interaction between a protein or its antigen-binding domain and an antigen mean that the interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, an antibody recognizes and binds to a specific protein structure, rather than a general protein. If an antibody is bound to epitope “A”, the presence of a molecule and antibody containing epitope “A” (or free, unlabeled “A”) in a reaction involving labeled “A” reduces the amount of labeled “A” that binds to the antibody.

[0106] When used herein, “specifically bind,” “bind specifically,” or similar phrases mean that the proteins of this disclosure react to or associate with a particular antigen (such as HER2) or a cell expressing it more frequently, more rapidly, more persistently, and / or with higher affinity than alternative antigens or cells. For example, a protein that specifically binds to one antigen will bind to that antigen with higher affinity (e.g., 20, 40, 60, 80 to 100, 150, 200 or more affinity), avidity, ease, and / or longer persistence than a protein that binds to another antigen. Also, by reading this definition, it will be understood that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen. Since such “specific binding” does not necessarily require exclusive or undetectable binding to another antigen, this is meant by the term “selective binding.”

[0107] In some embodiments, one targeting agent is covalently linked to the dendrimer. In some embodiments, two or more targeting agents are covalently linked to the dendrimer. In some embodiments, two targeting agents are covalently linked to the dendrimer. In some embodiments, three targeting agents are covalently linked to the dendrimer. In some embodiments, four targeting agents are covalently linked to the dendrimer.

[0108] The targeting agent is attached to the remainder of the complex via a spacer. In some embodiments, the covalent attachment or linkage between the targeting agent and the spacer group is formed by a reaction between a complementary reactive functional group present on the targeting agent precursor and the spacer group precursor, and the spacer group precursor may or may not be attached to the remainder of the complex at the time of reaction with the HER2 targeting partial precursor, depending on the process used to prepare the complex.

[0109] In some embodiments, the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting moiety.

[0110] The covalent attachment sites for the targeting agent precursor may be, for example, cysteine, lysine, N-terminal amines, tyrosine, carbohydrates, unnatural amino acids, or transaminases (e.g., transglutaminase or saltase A) or recognition sequences. Binding sites for covalent attachment to proteins are known in the art (e.g., Milla P. et al., 2012). In silico crystal structure prediction can be used to identify the position of the targeting agent within the polypeptide sequence to position the covalent attachment site structurally suitable for the conjugate (e.g., away from paratopes, CDRs, functionally important for correct folding and conformation, and / or residues with high charge, polarity, or bulky side chain groups). From these candidate positions, empirical screening can be performed to determine substitution sites that allow for a robust conjugate while preserving maximum functionality. In one embodiment, the 2D3 position is selected from the group consisting of positions 13, 16, 42, and 126 (c-terminus). In some embodiments, the targeting agent precursor includes a non-natural amino acid residue for attachment to the spacer. The non-natural amino acid residue may have a side chain having a reactive functional group complementary to the reactive functional group of the spacer group precursor. In some embodiments, the non-natural amino acid residue includes an azide group, for example, a 4-azidophenylalanine residue, for example, [ka] The azide group may undergo cycloaddition with an alkyne group that may be present in the spacer precursor group. In some embodiments, the non-natural amino acid is a diene-containing amino acid, for example, [ka] These are spirocyclopentadiene-containing amino acids, such as the following. The diene group can undergo cycloaddition reactions with alkene groups, such as those found in maleimides (e.g., Diels-Alder reaction). Further examples of unnatural amino acids that can be used to attach to spacers include those containing carbonyl groups, such as ketones, and those containing methylcyclopropylene groups. Additional examples of unnatural amino acids include: [ka] These are some examples.

[0111] In some embodiments, the targeting agent comprises or consists of any of the amino acid sequences defined herein.

[0112] Spacer base As used herein, the term “spacer group” refers to a chemical entity that plays a role in attaching a targeting agent to a dendrimer. That is, the spacer group binds the targeting agent to the dendrimer (e.g., via covalent attachment). The purpose of the spacer group is to position the targeting agent so that it can bind to the HER2 receptor without excessive adverse influence from other components of the dendrimer.

[0113] As described above, targeting agents can be covalently attached to dendrimers, for example, via the dendrimer core. In some embodiments, spacer groups are covalently attached to the core unit. In some embodiments, the targeting agent is covalently attached to the dendrimer via the dendrimer core. That is, targeting agents such as antibody fragments are covalently attached to the dendrimer core by spacer groups. Covalently attaching the targeting agent to the dendrimer via spacers attached to the dendrimer core can be beneficial when the dendrimers are sterically densely packed, and the spacer groups can be long enough to protrude beyond the surface of the dendrimer, thereby enabling the targeting agent to bind to its receptor or the like in vivo.

[0114] In some other embodiments, the targeting agent is covalently attached to the dendrimer via the surface constituent units of the dendrimer. In some embodiments, the spacer group is covalently attached to the surface constituent units. For example, the targeting agent may be linked to the surface nitrogen of the lysine residue via the spacer group, such as by an amide bond formed between the amine group of the lysine residue and the carboxylic acid group present on the intermediate containing the targeting agent and the spacer group. In one embodiment, the targeting agent is covalently attached to the surface nitrogen of the lysine residue via the spacer group, thereby forming an amide bond between the amine group of the lysine residue and the carboxylic acid group present on the intermediate containing the targeting agent and the spacer group.

[0115] Examples of spacer groups include polyethylene glycol (PEG), polypropylene glycol, polyallyls, peptides, amino acids, alkyl chains, alkenyl chains, and saccharides (mono, oligo, poly) or residues thereof. In some embodiments, the spacer group comprises a PEG chain with 2 to 60 ethyleneoxy repeating units, e.g., 2 to 20 or 20 to 48 repeating units. In one embodiment, the PEG has 8 to 36 repeating units. In further embodiments, the PEG has 12, 16, 20, 24, or 36 repeating units.

[0116] In some embodiments, the spacer group comprises multiple PEG groups sandwiched between other functional groups. For example, the spacer group may include PEG groups linked via, for example, an amide group or other functional groups useful for connecting portions of the spacer group.

[0117] To attach the spacer group to the targeting agent and / or dendrimer, the spacer group precursor may contain one or more reactive functional groups. In certain embodiments, the reactive functional groups include active esters such as hydroxy, carboxy, NHS, or pentafluorophenol esters; amino, azide, maleimide (such as sulfomaleimide); diene (cyclopentadiene, e.g., spiro[2.4]hepta-4,6-diene group); alkyne-containing groups such as tetrazine, citracomide, BCN (bisicle[6.1.0]non-4-in-9-yl); DBCO (dibenzocyclosin-amine); activated alkenes such as methyl-cyclopropylene-containing moieties; carbonyl groups such as thiols, aldehydes, and ketones; alkoxyamines, haloacetates, biotin, tetrazine, TCO (trans-cyclooctene); methyl-cyclopropylene groups; and PTAD or other tyrosine-reactive groups.

[0118] For example, in some embodiments, the spacer group intermediate may contain two reactive groups (e.g., one at each orthogonal end). That is, at least one of the reactive groups can react with a complementary group present on either the intermediate containing the targeting agent or the intermediate containing the dendrimer, under conditions where the other reactive group is stable and substantially unreactable, to attach the spacer group to the components of the complex. This allows the spacer to attach (e.g., covalently) to either the dendrimer or the targeting agent, and then the other reactive groups can react with complementary groups present on the remaining components to link the targeting agent to the dendrimer.

[0119] In some embodiments, the spacer group is attached to the targeting agent (e.g., by covalent bonding) by the reaction of precursors containing an alkyne group and an azide group, respectively (for example, the intermediate containing the targeting group may contain an azide group, and the intermediate containing the spacer group may contain an alkyne group). Such a reaction involves a triazole-containing group, for example, [ka] This brings about the formation, The following structure: [ka] It can be formed by reacting a precursor having [a certain characteristic].

[0120] As another example, a spacer group is a triazole-containing group, for example, [ka] It can be attached via the formation of the following structure: [ka] It can be formed by reacting a precursor having [a certain characteristic].

[0121] In some embodiments, the spacer group is attached to the targeting agent (e.g., by covalent bonding) by the reaction of precursors containing an alkene (e.g., a strained alkene such as trans-cyclooctene) and a tetrazine group, respectively. Such reactions involve pyridazine-containing groups with nitrogen protrusions, for example, [ka] This leads to the formation of the following structure: [ka] It can be formed by the reaction of a precursor having [a certain characteristic].

[0122] In some embodiments, the spacer group is attached to the targeting agent (e.g., by covalent bonding) by the reaction of precursors containing an alkene (e.g., a strained alkene such as methylcyclopropene) and a tetrazine group, respectively. Such reactions result in the formation of a pyridazine-containing group with a nitrogen protrusion.

[0123] To attach the spacer group to the dendrimer (for example, by covalent bonding), the spacer group precursor may contain additional functional groups. For example, the spacer group precursor may contain a carboxylic acid group that can react with an amino group that forms part of the core unit precursor (for example, to form an amide linkage).

[0124] In some embodiments, the spacer group is attached to the dendrimer (e.g., by covalent bonding) by the reaction of a precursor containing a carboxylic acid group and an amine group. For example, the spacer group intermediate may contain a carboxylic acid group that is present as part of the core unit or reacts with an amine group extending from the core unit to form, for example, an amide linkage, and is attached to the targeting agent by the reaction of a precursor containing an alkyne group and an azide group, respectively (for example, the intermediate containing the target group may contain an azide group, and the intermediate containing the spacer group may contain an alkyne group).

[0125] As described above, the targeting agent precursor may contain a non-natural amino acid residue. In some embodiments, the targeting agent contains a non-natural amino acid residue. This non-natural amino acid residue may be, for example, any non-natural amino acid capable of exhibiting a reactive side chain, the reactive side chain holding a functional group complementary to a functional group present on the spacer group precursor. In this way, the complementary functional groups react, resulting in the attachment of the targeting partial precursor to the spacer group precursor. In some embodiments, the non-natural amino acid residue is a 4-azidophenylalanine residue. In some embodiments, the spacer group precursor contains an alkyne group for conjugation to the targeting partial precursor containing the non-natural amino acid residue. In some embodiments, the spacer group precursor contains an alkyne group, which is a dibenzylcyclooctane group, for conjugation to the targeting partial precursor containing the 4-azidophenylalanine residue. In some embodiments, one end of the spacer group forms part of the DBCO group and the targeted portion (for example, below: [ka] By cycloaddition with the azide moiety on a 4-phenylalanine residue (forming a triazole-containing group such as the following), or by a BCN((bisicle 6.1.0]non-4-in-9-yl) group forming part of the targeting moiety (for example, below: [ka] It attaches to the target site by reacting with the azide moiety on the 4-phenylalanine residue (which forms a triazole-containing group like the one shown).

[0126] In some embodiments, one end of the spacer group is attached to the dendrimer (e.g., by covalent bonding) by amidation reactions (e.g., by the reaction of an activated ester) between amino groups present on the core or on the surface constituent unit and between carboxyl groups present on the spacer group. In some embodiments, the spacer group precursor includes a tetrazine group. In some embodiments, the spacer group precursor includes a maleimide group for conjugation to a diene (e.g., cyclopentadiene, e.g., spiro[2,4]hepta-4,6-diene group).

[0127] In some embodiments, the spacer group precursor comprises a PEG chain having a reactive carboxyl group for bonding to an amine in the dendrimer core and an azide group for conjugating to a targeting agent precursor containing a reactive alkyne moiety. In some embodiments, the spacer group precursor comprises a PEG chain having a reactive amine group for bonding to a carboxyl group in the dendrimer core and an azide group for conjugating to a targeting agent precursor containing a reactive alkyne moiety. In some embodiments, the spacer group precursor comprises a PEG chain having a reactive carboxyl group for bonding to an amine in the dendrimer core and a maleimide group for conjugating to a targeting agent precursor containing a reactive thiol moiety. In some embodiments, the spacer group precursor comprises a PEG chain having a reactive amine group for bonding to a carboxyl group in the dendrimer core and a thiol or masked thiol group for conjugating to a targeting agent precursor containing a reactive maleimide moiety. In some embodiments, the spacer group precursor comprises a PEG chain having a reactive carboxyl group for binding to an amine in the core of the dendrimer, and a tetrazine group for conjugating to a targeting agent precursor containing a reactive alkene moiety. In some embodiments, the spacer group precursor comprises a PEG chain having a reactive carboxyl group for binding to an amine in the core of the dendrimer, and a maleimide group for conjugating to a targeting agent precursor containing a reactive diene (cyclopentadiene, e.g., spiro[2,4]hepta-4,6-diene group).

[0128] In some embodiments, linking of a targeting agent to a dendrimer can be achieved by attaching a first spacer group to a targeting agent intermediate and a second spacer group to the dendrimer (e.g., to the core of the dendrimer) (e.g., by covalent bond), and then reacting complementary functional groups present on the first and second spacer groups together to link the targeting agent and the dendrimer. Such an approach may be provided to facilitate the linking of the dendrimer and the targeting. For example, the first spacer group intermediate may include a first reactive group at one end that is complementary to a reactive group on the targeting agent (e.g., an alkyne group complementary to an azide group that can react together to form a triazole group) and a second reactive group complementary to a reactive group on the second spacer group (e.g., a tetrazine-containing group complementary to the reaction with a trans-cyclooctene-containing group). The second spacer group intermediate may contain, for example, a third reactive group at one end that is complementary to the reaction with the reactive group on the dendrimer (e.g., a carboxylic acid group complementary to the reaction with an amine group), and a fourth reactive group at the other end that is complementary to the reaction with the reactive group on the first spacer group intermediate (e.g., a trans-cyclooctene-containing group complementary to the reaction with a tetrazine group). For example, the first group and the spacer group may be attached via a group produced by reacting a trans-cyclooctene group with a tetrazine group, structure: [ka] It may include.

[0129] In some embodiments, the targeted portion may be linked to the dendrimer via a spacer group formed by the reaction of an azide portion present on the targeting agent with an alkyne-containing group (e.g., DBCO, BCN) at one end of a spacer group, and the reaction of a tetrazine portion attached to the dendrimer with a strained alkene group at the other end of a spacer group (e.g., transcyclooctene).

[0130] A targeting agent precursor is reacted with a spacer group precursor to form attachment of the targeting agent to the spacer group. Means for attaching the spacer group precursor to the targeting agent precursor are known in the art. For example, sites for covalent attachment to the targeting agent precursor include, but are not limited to, cysteine ​​residues, lysine residues, C-terminal amino acid residues, N-terminal amines, tyrosine residues, carbohydrates, suitable non-natural amino acid residues, or transaminases or recognition sequences. Binding sites for covalent attachment to proteins such as targeting agent precursors are known in the art (e.g., Milla P et al., 2012). For example, the spacer group precursor may be reacted with the targeting agent precursor at the C-terminus of the targeting agent so that the spacer group attaches to the targeting agent via the C-terminus. In some embodiments, the targeting agent is attached to the spacer group via the C-terminus of the targeting agent. In one embodiment, the targeting agent is covalently attached to or linked to the spacer group via the C-terminus of the targeting agent.

[0131] Therapeutic drugs As used herein, the term “therapeutic agent” refers to an agent that exerts a therapeutic effect in vivo against, for example, HER-2 expressing cancer cells. Examples of therapeutic agents include, but are not limited to, anticancer agents, antineoplastic agents, and cytotoxic agents, also called chemotherapeutic agents. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent. As used herein, the term “cytotoxic agent” refers to an agent that exhibits chemotherapeutic properties. Examples of anticancer agents include, but are not limited to, DNA alkylating and crosslinking agents (e.g., platinum-containing agents such as cisplatin and oxaliplatin), DNA damaging agents, for example, PARP (poly-ADP-ribose polymerase) inhibitors (e.g., olaparib, rucaparib, niraparib, talazoparib), antimicrotubule agents (e.g., paclitaxel, docetaxel, cabazitaxel), and topoisomerase inhibitors (e.g., irinotecan, topotecan, SN-38, Examples of anticancer agents include nemorabicin, proteasome inhibitors (e.g., bortezomib), tyrosine protein kinase inhibitors (e.g., imatinib, nilotinib), EGF receptor inhibitors (e.g., gefitinib, erlotinib), cytotoxic antibiotics (e.g., anthracyclines, bleomycin), ribonucleotide reductase inhibitors (e.g., gemcitabine), antimetabolites (e.g., methotrexate, pemetrexate, etc.), and anti-division agents. Such anticancer agents may be used in the treatment of cancer with the aim of curing it (i.e., eliminating the cancer patient) or to alleviate / relieve symptoms that improve the quality of life and prolong the life of cancer patients. Anticancer agents may be used in combination with other non-chemotherapy treatments for cancer (i.e., radiation therapy, surgery / biopsy).

[0132] In one embodiment, the cytotoxic agent is nemorubicin. The structure of nemorubicin is as follows: [ka] That is the case.

[0133] In one embodiment, the cytotoxic agent is cabazitaxel. The structure of cabazitaxel is as follows: [ka] That is the case.

[0134] In one embodiment, the cytotoxic agent is docetaxel. The structure of docetaxel is as follows: [ka] That is the case.

[0135] In one embodiment, the cytotoxic agent is SN-38. The structure of SN-38 is: [ka] That is the case.

[0136] In one embodiment, the cytotoxic agent is irinotecan. The structure of irinotecan is: [ka] That is the case.

[0137] In one embodiment, the cytotoxic agent is topotecan. The structure of topotecan is as follows: [ka] That is the case.

[0138] In one embodiment, the cytotoxic agent is gemcitabine. The structure of gemcitabine is as follows: [ka] That is the case.

[0139] In some embodiments, the therapeutic agent is a hypercytotoxic agent. As used herein, “hypercytotoxic agent” refers to an agent that exhibits very potent chemotherapeutic properties but is so highly toxic that it cannot be administered alone as an anticancer agent. In other words, hypercytotoxic agents exhibit chemotherapeutic properties, but the harmful and toxic side effects outweigh the benefits of chemotherapy, and therefore they cannot generally be safely administered to the target.

[0140] Examples of hypercytotoxic agents include nemorubicin, dorastatins (e.g., dorastatin-10, dorastatin-15), auristatins (e.g., monomethyl auristatin-E, monomethyl auristatin-F), meitansinoids (e.g., meitansin, meltansin / emtansin (DM1, labtansin (DM4))), calichiamicins (e.g., calichiamicin γ1), esperamycins (e.g., esperamycin A1), and pyrrolobenzodiazepines (PDB).

[0141] In some embodiments, the therapeutic agent is auristatin. In some embodiments, the therapeutic agent is monomethyl auristatin. In one embodiment, the therapeutic agent is monomethyl auristatin E (MMAE). In one embodiment, the therapeutic agent is monomethyl auristatin F (MMAF). Both MMAE and MMAF are understood to inhibit cell division by blocking tubulin polymerization.

[0142] The chemical structure of MMAE is as follows: [ka] That is the case.

[0143] The chemical structure of MMAF is as follows: [ka] That is the case.

[0144] In some embodiments, the hypercytotoxic agent is a meitansinoid. In one embodiment, the hypercytotoxic agent is meitansin. In one embodiment, the hypercytotoxic agent is ansamitosin. In one embodiment, the hypercytotoxic agent is emtansine / meltansine (DM1). In one embodiment, the hypercytotoxic agent is labtansine (DM4). It is understood that meitansinoids inhibit microtubule assembly by binding to tubulin.

[0145] The chemical structure of meitansine is as follows: [ka] That is the case.

[0146] The chemical structures of emtansine / meltansine are as follows: [ka] That is the case.

[0147] In some embodiments, supracylotoxic agents are used to in vitro IC against cancer cell lines (e.g., SKBR3 cells and / or HEK293 cells and / or MCF7 cells). 50 However, the concentrations are less than 100 nM, or less than 10 nM, or less than 5 nM, or less than 3 nM, or less than 2 nM, or less than 1 nM, or less than 0.5 nM. The in vitro activity of MMAE and emtansine has been studied by Abdollahpour-Alitappeh et al. (2017) and Oroudjev et al. (2010), respectively.

[0148] It will be understood that dendrimer-targeting agent conjugates can have a distribution of therapeutic agents per targeting agent. For example, a dendrimer-targeting agent conjugate could potentially have up to approximately eight or more therapeutic agents per targeting agent.

[0149] From this, it can be understood that the drug-to-targeting ratio (DTR) for each dendrimer-targeting agent conjugate species can vary depending on the population. Typically, it is desirable to produce dendrimer-targeting agent conjugates with high DTRs. Generally, this results in a more effective treatment. However, if the DTR is too high, for example, if the drug is a hypercytotoxic agent, it can lead to undesirable toxicity.

[0150] In some embodiments, the dendrimer-targeting agent complex has a DTR greater than 2. In some embodiments, the dendrimer-targeting agent complex has a DTR greater than 4. In some embodiments, the dendrimer-targeting agent complex has a DTR greater than 8. In some embodiments, the dendrimer-targeting agent complex has a DTR greater than 14. In some embodiments, the dendrimer-targeting agent complex has a DTR greater than 26.

[0151] In some embodiments, the dendrimer-targeting agent complex has a DTR of about 1 to about 32. In some embodiments, the dendrimer-targeting agent complex has a DTR of about 7 to about 32. In some embodiments, the dendrimer-targeting agent complex is a G5 dendrimer and has a DTR of about 26 to about 32. In some embodiments, the dendrimer-targeting agent complex is a G4 dendrimer and has a DTR of about 14 to about 16. In some embodiments, the dendrimer-targeting agent complex is a G3 dendrimer and has a DTR of about 6 to about 8.

[0152] When more than two targeting agents are conjugated to a dendrimer, the DTR is lower compared to a dendrimer-targeting agent complex with only the targeting agent. In some embodiments, the dendrimer-targeting agent complex has a DTR of about 2 to about 16. In some embodiments, the dendrimer-targeting agent complex has a DTR of about 8 to about 16. In some embodiments, the dendrimer-targeting agent complex is a G5 dendrimer and has a DTR of about 4 to about 16. In some embodiments, the dendrimer-targeting agent complex is a G5 dendrimer and has a DTR of about 6 to about 16. In some embodiments, the dendrimer-targeting agent complex is a G4 dendrimer and has a DTR of about 4 to about 8. In some embodiments, the dendrimer-targeting agent complex is a G4 dendrimer and has a DTR of about 4 to about 8. In some embodiments, the dendrimer-targeting agent complex is a G3 dendrimer and has a DTR of about 2 to about 4.

[0153] The ability of dendrimer-based therapeutics to carry more than one therapeutic agent may eliminate the need for supercytotoxic agents. For example, the ability to target the delivery of multiple cytotoxic agents, such as docetaxel, on a single dendrimer may result in equivalent or superior therapeutic efficacy compared to the delivery of a single supercytotoxic agent, such as MMAE. The absence of supercytotoxic agents may also be an advantage.

[0154] Linker In some embodiments, the therapeutic agent is attached to the dendrimer via a linker. The linker is preferably used to retain the desirable properties of the therapeutic agent and to maintain substantially intact and non-toxicity in systemic circulation. The linker group can be used, for example, to provide a suitable group for attaching the pharmaceutically active agent to the dendrimer when the available functionality of the pharmaceutically active agent is not suitable for direct attachment to the constituent units. Alternatively, the linker group can also be used to facilitate the controlled release of the pharmaceutically active agent from the dendrimer scaffold, thereby providing a therapeutically effective concentration and a desirable pharmacokinetic profile of the pharmaceutically active agent for a suitable period (e.g., a long period).

[0155] One end of the linker adheres to the therapeutic agent, and the other end of the linker adheres to the dendrimer. The attachment point of the linker to the dendrimer may be, for example, a surface constituent unit of the dendrimer.

[0156] Those skilled in the art will understand that any one of several suitable linkers may be used. The linker should provide sufficient stability during systemic circulation while, for example, enabling the rapid and efficient release of the active form of a cytotoxic drug at its site of action once internalized in cancer cells.

[0157] The linker may be a non-cleavable linker or a cleavable linker. In one embodiment, the linker is a non-cleavable linker. In one embodiment, the linker is a cleavable linker. The cleavable linker, either by itself or in conjunction with linkage to a pharmaceutically active agent, comprises one or more of the following cleavable moieties: an ester group, a hydrazone group, an oxime group, an imine group, or a disulfide group. In some embodiments, the linker is cleavable in a tumor environment, unstable to acid, unstable in a reducing environment, unstable to hydrolysis, or unstable to proteases.

[0158] Chemically unstable linkers include, but are not limited to, acid-unstable linkers (i.e., hydrazones) and disulfide linkers. Enzymatically cleavable linkers include, but are not limited to, peptide linkers and β-glucuronide linkers. Examples of peptide linkers include, but are not limited to, Val-Ala, Val-Cit, Phe-Lys, Phe-Arg, Phe-Cit, Val-Arg, Val-Cit, Ala-Arg, and Ala-Cit. Because lysosomal proteolytic enzymes have very low activity in the blood, peptide linkers and their peptide bonds are expected to have favorable serum stability. Val-Ala, Val-Cit, Phe-Lys, Phe-Arg, Phe-Cit, Val-Arg, Val-Cit, Ala-Arg, and Ala-Cit linkers are rapidly hydrolyzed by cathepsin B. In some embodiments, the linker is an enzymatically cleavable linker. For example, in some embodiments, the linker contains amino acid residues that can be recognized and cleaved by enzymes.

[0159] In some embodiments, the linker includes a peptide group. In some embodiments, the linker has, for example, the structure: [ka] It contains a valine-citrulline-para-aminobenzyl alcohol-containing group (Val-Cit-PAB) having the following properties.

[0160] For example, the PAB group may be covalently bonded to the amine group present in the therapeutic portion via a carbonyl group to form a carbamate linkage, or it may be bonded to the amine group present in the outer constituent unit via a diacyl linker that forms an amide bond with the valine amino group and the amine group present in the outer constituent unit.

[0161] In some embodiments, the linker is structured as follows: [ka] It contains, or consists of, a glutaric acid-valine-citrulline-para-aminobenzyl alcohol group having the following properties.

[0162] In some embodiments, the linker is structured as follows: [ka] It contains or consists of a valine-alanine-paraaminobenzyl alcohol group having the following properties.

[0163] In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, and the residue of the pharmaceutically active agent is attached to the linker via the oxygen atom of the hydroxyl group. In this approach, it is possible to attach to the linker via an ester group, and such ester groups have been found to be cleavable in vivo, releasing the pharmaceutically active agent at a desired rate.

[0164] In some embodiments, the core unit is formed from a core unit precursor containing an amino group, the constituent units are lysine residues or analogs thereof, the pharmaceutically active agent contains a hydroxyl group, the residues of the pharmaceutically active agent are attached via the oxygen atom of the hydroxyl group, and the cleavable linker is a diacyl linker, thereby resulting in an ester linkage between the residues of the pharmaceutically active agent and the linker, and an amide linkage between the linker and the nitrogen atom present on the outermost constituent unit. In some embodiments, the pharmaceutically active agent contains a hydroxyl group, the residues of the pharmaceutically active agent are attached via the oxygen atom of the hydroxyl group, and the cleavable linker is of formula: [ka] It is a diacyl linker group. In the formula, A is a C2-C group suspended by O, S, SS, NH, or N(Me). 10A is either an alkylene group or a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine, and N-methylpyrrolidine.

[0165] As used herein, the term “alkyl” refers to a monovalent linear or branched saturated hydrocarbon group. For example, an alkyl group may have 1 to 10 carbon atoms (i.e., C 1~10 Alkyl compounds include alkyl groups. For example, alkyl groups have 1 to 6 carbon atoms (i.e., C 1~6 It includes alkyl groups. Examples of alkyl groups include methyl, ethyl, propyl (e.g., n-propyl, iso-propyl), butyl (e.g., n-butyl, sec-butyl, tert-butyl), pentyl, and hexyl groups.

[0166] As used herein, the term "alkylene" refers to a divalent linear (i.e., linear) or branched saturated hydrocarbon group. For example, an alkylene group has 2 to 10 carbon atoms (i.e., C 2~10 It contains alkylene. For example, an alkylene group has 2 to 6 carbon atoms (i.e., C 2~6 It contains alkylenes. Examples of alkylene groups include -CH2CH2-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, and -CH2CH(CH3)CH2-.

[0167] Such linkers are particularly suitable for linking to the therapeutic agent via hydroxyl groups present in the therapeutic agent. In such cases, the complex includes an ester linkage between the linker and the therapeutic agent portion, and an amide linkage between the linker and the dendrimer portion.

[0168] In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, and the residues of the pharmaceutically active agent are attached via the oxygen atom of the hydroxyl group, and the cleavable linker is of the formula: [ka] The diacyl linker group is C2-C, interrupted by O, S, NH, or N(Me). 10 It is an alkylene group.

[0169] In some embodiments, the pharmaceutically active agent has a hydroxyl group, and the residue of the pharmaceutically active agent is attached via the oxygen atom of the hydroxyl group, and the diacyl linker is [ka] That is the case.

[0170] Certain types of cleavable linkers include a disulfide moiety. Such linkers are susceptible to cleavage by glutathione. For example, this type of linker may contain two acyl groups linked via an alkyl chain interrupted by the disulfide moiety.

[0171] In some embodiments, the linker comprises an alkyl chain interrupted by a disulfide moiety, where one or both carbon atoms adjacent to the disulfide group are substituted with one or more methyl groups. For example, one of the carbon atoms adjacent to the disulfide moiety may be substituted with a gem-dimethyl group, e.g., the linker has that group: [ka] It may include.

[0172] Another specific type of cleavable linker is one that contains a diphosphate moiety. Such linkers are susceptible to hydrolysis by lysosomal acid pyrophosphatases and acid phosphatases. In some embodiments, the linker has a pyrophosphate moiety: [ka] It contains an alkyl chain interrupted by a diphosphate moiety, also known as a diphosphate moiety.

[0173] Another specific type of cleavable linker is one that contains a carbamate moiety. Such linkers have high hydrolytic stability before activation and efficient cleavage through different intramolecular mechanisms. In some embodiments, the linker is a carbamate moiety: [ka] It contains an alkyl chain that has been interrupted by [something].

[0174] Another specific type of severable linker has an orthoester portion: [ka] It includes.

[0175] A non-cleavable linker is a linking group that is inert to cleavage, or substantially inert to cleavage, even when exposed to in vivo conditions for the required period of time. Non-cleavable linkers are not cleaved under biological conditions.

[0176] For example, alkylene groups or cycloalkylene groups (e.g., C 1-10 Alkylene group or C 3-10 Examples include diacyl linkers crosslinked by cycloalkylene groups. Further examples of non-cleavable linkers include thioether linkers. A specific example of a non-cleavable linker is one formed using SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). SMCC can be used to form thioether links by reacting the maleimide functionality with thiol groups present in or attached to the therapeutic portion. The carboxylic acid functionality can be used to react with amino groups present on the outer constituent unit.

[0177] In some embodiments, the linker includes a Val-Cit-PAB group. In some embodiments, the linker includes a Val-Arg-PAB group. In some embodiments, the linker includes a Val-Arg-trigger group. In some embodiments, the linker is a Val-Ala-PAB-P-trigger group. In some embodiments, the linker is a Val-Ala-PNB-P group.

[0178] In one embodiment, the linker contains a Val-Cit-PAB group, the therapeutic portion is MMAE, and the Val-Cit-PAB group is attached to the therapeutic portion as follows (i.e., Val-Cit-PAB-MMAE): [ka] .

[0179] In one embodiment, the linker is a Val-Cit-PAB group, the therapeutic portion is MMAF, and the Val-Cit-PAB group is attached to the therapeutic portion as follows (i.e., Val-Cit-PAB-MMAF): [ka] .

[0180] In one embodiment, the linker is a glutaric acid-Val-Cit-PAB group, the therapeutic portion is MMAE, and the glutaric acid-Val-Cit-PAB group is attached to the therapeutic portion as follows (i.e., glutaric acid-Val-Cit-PAB-MMAE): [ka] .

[0181] In one embodiment, the linker is DGA (diglycolic acid) or a glutaric acid group, and the therapeutic portion is MMAF or an ester thereof, for example: [ka] That is the case.

[0182] In some embodiments, the therapeutic agent is covalently linked to the surface constituent units of the dendrimer via connector groups. For example, the therapeutic agent may be covalently linked to the surface constituent units of the dendrimer via connector groups in addition to linker groups. The connector groups allow the therapeutic agent to be anchored to the surface of the dendrimer at different lengths. For example, connector groups may be used to extend the distance of the therapeutic agent from the surface of the dendrimer. This can avoid any potential problems related to steric hindrance that may hinder access to the cleavable linker and reduce the therapeutic agent's ability to interact with the target. In some embodiments, the dendrimer-targeting agent complex includes a therapeutic agent covalently linked to the surface of the dendrimer via linker groups and connector groups. In some embodiments, the order of connection is dendrimer-connector-linker-therapeutic agent. In some embodiments, the order of connection is dendrimer-linker-connector-therapeutic agent.

[0183] The connector group may contain any chemical moieties that play a role in extending the distance of the therapeutic agent from the surface of the dendrimer. In some embodiments, the connector group contains one or more hydrophilic polymer groups. In some embodiments, the connector group contains about 1 to about 50 hydrophilic polymer groups. In some embodiments, the connector group contains a PEG group. In some embodiments, the connector group contains a PEOX group. In some embodiments, the connector group contains a polysarcosine group. In some embodiments, the connector group contains 1 or more, 2 or more, 4 or more, 10 or more, 20 or more, 30 or more, 40 or more, or 50 or more PEG moieties. In some embodiments, the connector group contains about 1 to about 50 PEG moieties. In some embodiments, the connector group contains 4 PEG moieties. In some embodiments, the connector group contains 12 PEG moieties. In some embodiments, the connector group contains 24 PEG moieties. In some embodiments, the connector group contains 48 PEG moieties.

[0184] In some embodiments, the linker includes a connector group (PEG) X This is the -Val-Arg-PAB-P-trigger- section. As mentioned above, X can be a number between approximately 1 and approximately 50.

[0185] In one embodiment, the linker includes a connector base and a (PEG)9-Val-Arg-PAB-P-trigger-NHCO portion: [ka] That is the case.

[0186] In one embodiment, the linker includes a connector group, and the (PEG)9-Val-Arg-PAB-P-trigger-N(CH3)CO portion: [ka] That is the case.

[0187] In some embodiments, the linker includes a connector group (PEG) x This is the -OP(O)OH-OP(O)OH-O- part. Here again, as mentioned above, X can be a number between approximately 1 and approximately 50.

[0188] In one embodiment, the linker includes a connector group, (PEG) x -OP(O)OH-OP(O)OH-O- section: [ka] That is the case.

[0189] In some embodiments, the linker includes a connector group (PEG) X This is the -Val-Ala-PAB-P-trigger- portion. X can be a number from about 1 to about 50, as described above. In one embodiment, the linker includes a connector base and is the (PEG)9-Val-Ala-PAB-P-trigger-NHCO portion.

[0190] For example, (PEG) X -Val-Arg-PAB-P-Trigger- or (PEG) X The release of therapeutic agents can be achieved by using specific linkers such as -OP(O)OH-OP(O)OH-O-.

[0191] (PEG) X -Val-Arg-PAB-P-Trigger-NHCO, (PEG) X In examples such as -Val-Arg-PAB-P-trigger-N(CH3)CO, the linker is initially activated by cleavage of the dipeptide portion by cellular enzymes such as cathepsin B, revealing a self-destructive 2-(aminomethyl)pyrrolidine connector. Following intramolecular cyclization, the hydroxyl-containing therapeutic agent disappears. Exemplary hydroxyl-containing therapeutic agents include cabazitaxel, docetaxel, and SN-38.

[0192] (PEG) x In the case of the -OP(O)OH-OP(O)OH-O- moiety, the linker not only specifically and cleanly cleaves the moiety, yielding hydroxyl-containing drugs such as cabazitaxel, docetaxel, and SN-38, but also has the additional advantage of providing drug monophosphates that are active species of nucleotide inhibitors, such as gemcitabine.

[0193] Pharmacokinetic modification group This complex contains multiple hydrophilic polymer groups covalently bonded to the surface constituent units of the dendrimer, thereby modifying the pharmacokinetic properties of the complex. For example, the hydrophilic polymer groups contribute to improved pharmacokinetic properties.

[0194] In some embodiments, the dendrimer comprises a plurality of hydrophilic polymer groups covalently linked to the surface constituent units of the dendrimer. The term “hydrophilic polymer group” typically refers to a polymer group having a solubility in water of at least 25 mg / ml, more preferably at least 50 mg / ml, and even more preferably at least 100 mg / ml at 25°C.

[0195] In some embodiments, the hydrophilic polymer group comprises repeating units of amino acids, alkyloxy, or alkyl(acyl)amino groups. In some embodiments, the hydrophilic polymer group comprises repeating units of amino acids such as sarcosine. In some embodiments, the hydrophilic polymer group comprises repeating units of alkyloxy groups (for example, the hydrophilic polymer is a PEG group). In some embodiments, the hydrophilic polymer comprises repeating units of alkyl(acyl)amino groups (for example, the hydrophilic polymer is a PEOX group).

[0196] In some embodiments, the hydrophilic polymer group comprises at least 10 monomer units. In some embodiments, the hydrophilic polymer group comprises up to 100 monomer units. In some embodiments, the hydrophilic polymer group comprises 10 to 100, or 10 to 50 monomer units.

[0197] In some embodiments, the hydrophilic polymer group is a PEG group. A PEG group is a polyethylene glycol group, i.e., a group containing repeating units of the formula -CH2CH2O-. The PEG material used to produce the dendrimers of this disclosure typically comprises a mixture of PEGs having some variability in molecular weight (i.e., ±10%), and therefore, when a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEG composition. For example, "PEG ~2100 The term "PEG" refers to an average molecular weight of approximately 2100 daltons, i.e., ±10% (PEG). 1890 ~PEG 2310 This refers to polyethylene glycol containing PEG. ~2300 "This refers to an average molecular weight of approximately 2300 daltons, or ±10% (PEG). 2070 ~PEG 2530This refers to polyethylene glycol having polyethylene glycol of ) . Three commonly used methods for calculating the MW average are the number average, weight average, and Z average molecular weight. As used herein, the term “molecular weight” is intended to refer to the weight average molecular weight that can be measured using techniques well known in the art, such as NMR, mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF), gel permeation chromatography, or other liquid chromatography techniques, light scattering techniques, ultracentrifugation, and viscosity measurement, but is not limited to these.

[0198] In some embodiments, the PEG group has an average molecular weight in the range of 500 to 2500 daltons. In some embodiments, the PEG group has an average molecular weight in the range of 800 to 1200 daltons. In some embodiments, the PEG group has an average molecular weight in the range of 900 to 1100 daltons. In some embodiments, the PEG group has an average molecular weight in the range of 1500 to 2500 daltons. In some embodiments, the PEG group has an average molecular weight in the range of 1900 to 2300 daltons. In some embodiments, the PEG group has an average molecular weight in the range of 2100 to 2500 daltons.

[0199] In some embodiments, the PEG group has an average molecular weight of about 1100 daltons. In some embodiments, the PEG group has an average molecular weight of about 2000 daltons. In some embodiments, the PEG group has an average molecular weight of about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2100, about 2200, about 2300, about 2400, or about 2500 daltons.

[0200] In some embodiments, the PEG group has a polydispersity index (PDI) of about 1.00 to about 1.50, about 1.00 to about 1.25, or about 1.00 to about 1.10. In some embodiments, the PEG group has a polydispersity index (PDI) of about 1.05. The term "polydispersity index" is an indicator of the distribution of molecular weight in a polymer sample. The polydispersity index (PDI) is the weight-average molecular weight (M w ) number average molecular weight (M n The polydispersity index (PDI) is calculated by dividing by ) and indicates the distribution of individual molecular weights within a batch of polymers. The polydispersity index (PDI) has a value of 1 or greater, but as the polymer approaches a uniform change in length and average molecular weight, the polydispersity index (PDI) approaches 1.

[0201] The PEG group may be linear or branched. End-capped PEG groups may be used if desired. In some embodiments, the PEG group is methoxy-terminated PEG.

[0202] In some embodiments, the hydrophilic polymer group is a PEOX group. In some embodiments, the dendrimer contains multiple PEOX groups covalently bonded to the surface constituent units of the dendrimer portion. A PEOX group is a polyethyloxazoline group, i.e., formula: [ka] It is a base that contains repeating units.

[0203] The PEOX group is so named because it can be produced by polymerization of ethyl oxazoline. The PEOX material used to produce the dendrimers of this disclosure typically comprises a mixture of PEOX having some variability in molecular weight (i.e., ±10%), and therefore, where a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEOX composition. In some embodiments, the second terminal group comprises a PEOX group having an average molecular weight of at least 750 daltons, at least 1000 daltons, or at least 1500 daltons. In some embodiments, the second terminal group comprises a PEOX group having an average molecular weight in the range of 750 daltons to 2500 daltons, or 1000 daltons to 2000 daltons. End-capped PEOX groups may be used if desired. In some embodiments, the PEOX group is methoxy-terminated PEOX.

[0204] In some embodiments, the hydrophilic polymer group consists of a polysarcosine group. In some embodiments, the polysarcosine group has an average molecular weight of at least 750 daltons, at least 1000 daltons, or at least 1500 daltons. In some embodiments, the hydrophilic polymer group includes a polysarcosine group having an average molecular weight in the range of 750 to 2500 daltons, or 1000 to 2000 daltons.

[0205] The hydrophilic polymer groups may be attached to the outer structural units by any preferred means. In some embodiments, linking groups are used to attach the hydrophilic polymer groups to the outer structural units.

[0206] Hydrophilic polymer groups are typically attached via the use of precursors containing reactive groups that react with amine groups, such as reactive acyl groups (which can form amide bonds) or aldehydes (which can form amine groups under reductive amination conditions).

[0207] In some embodiments, the PEG group is covalently attached to the PEG linking group (L1) via an ether linkage formed between the carbon atom present in the PEG group and the oxygen atom present in the PEG linking group, and each PEG group is covalently attached to the constituent unit via an amide linkage formed between the nitrogen atom present in the constituent unit and the carbon atom of the acyl group present in the PEG linking group. In some embodiments, the L1-PEG groups are each, [ka] The PEG group is a methoxy-terminated PEG with an average molecular weight in the range of approximately 500 to 2500 daltons.

[0208] In some embodiments, the PEOX group is covalently attached to the PEOX linking group (L1') via a bond formed between the nitrogen atom present in the PEOX group and the carbon atom present in the PEOX linking group, and each PEOX group is covalently attached to the constituent unit via an amide bond formed between the nitrogen atom present in the constituent unit and the carbon atom of the acyl group present in the PEOX linking group. In some embodiments, the L1'-PEOX groups are, [ka] That is the case.

[0209] In some embodiments, the hydrophilic polymer group is a polysarcosine group, i.e., formula: [ka] Includes a base that contains a repeating unit.

[0210] In some embodiments, the polysarcosine group is attached to the constituent unit via an amide linkage formed between a nitrogen atom present in the constituent unit and a carbon atom of the acyl group present in the polysarcosine group. In some embodiments, the hydrophilic polymer group comprises a polysarcosine group having an average molecular weight of at least 750 daltons, at least 1000 daltons, or at least 1500 daltons. In some embodiments, the second terminal group comprises a polysarcosine group having an average molecular weight in the range of 750 to 2500 daltons, or 1000 to 2000 daltons.

[0211] In many cases, a population of dendrimers functionalized on their surface contains a random stoichiometry and topology of the functional groups. For example, reacting a population of dendrimer-targeting agent complexes containing, for instance, 64 reactive surface groups with one or more reactive functional groups results in a diverse population of functionalized dendrimers, where some dendrimers may contain more functional groups than others. If there are multiple different surface groups available to react with reactive functional groups, a broad distribution of dendrimers with different surface topologies can also be obtained.

[0212] The dendrimer-targeting agent complex, intermediates, and processes of the present invention may enable high loading of therapeutic agents by covalent attachment to surface constituent units. Such dendrimer-targeting agent complexes facilitate the release of therapeutically effective levels of the therapeutic agent over a long period after administration, thereby potentially reducing the required administration frequency and / or number of doses. Furthermore, the dendrimer-targeting agent complexes of the present invention may enable targeted delivery of the therapeutic agent to its site of action. As a result, such dendrimer-targeting agents may reduce off-target activities, such as cytotoxic activity.

[0213] Internalization of dendrimer-targeting agent complexes Dendrimer-targeting agent conjugates provide targeted anticancer drug therapies. The beneficial properties of the conjugates are understood to be at least in part related to the observation that exemplary conjugates containing MMAE therapeutic agents are rapidly internalized into target cells. In some embodiments, dendrimer-targeting agent conjugates are internalized into HER2-expressing cells.

[0214] As used herein, the term “internalization” refers to the endocytosis of a dendrimer-targeting agent complex into a cell. The ability of a dendrimer-targeting agent complex to specifically locate and bind to the HER2 receptor on targeted HER2-expressing cancer cells and rapidly internalize it means that a reduced amount of the complex is circulating in the plasma. Thus, therapeutic agents such as hypercytotoxic moieties like MMAEs are less likely to undergo undesirable cleavage from the dendrimer-targeting agent complex outside of HER2-overexpressing sites (e.g., tumor cells), and the potential for side effects or toxicity associated with the nonspecific release of the therapeutic agent in the subject is reduced. In contrast, administering therapeutic agents such as hypercytotoxic moieties at equivalent doses can cause extreme toxicity to the point where the therapeutic agent cannot be safely administered alone. For example, MMAEs and MMAFs are known to be too toxic to be safely administered to subjects alone. Therefore, dendrimer-targeting agent complexes are considered to provide a safer means of delivering therapeutic agents such as hypercytotoxic agents like MMAEs and MMAFs.

[0215] In some embodiments, administration of the complex results in reduced side effects compared to administration of an equivalent dose of the free therapeutic agent. In some embodiments, administration of the complex results in a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the plasma concentration of the therapeutic agent (e.g., Cmax, or concentration at specific time points after administration (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours)) compared to administration of an equivalent dose of the free therapeutic agent.

[0216] In some embodiments, the therapeutic agent is hypercytotoxic (e.g., MMAE, MMAF), and administration of the dendrimer-targeting agent conjugate results in a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the plasma concentration of the therapeutic agent (e.g., Cmax, or concentration at specific time points after administration (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 48 ​​hours)) compared to administration of an equivalent dose of the free therapeutic agent.

[0217] Accordingly, a method is provided for killing HER2-expressing cells, the method comprising contacting a complex as defined herein with HER2-expressing cells, thereby internalizing the complex into the cells, and thereby causing the therapeutic agent to kill the HER2-expressing cells. In some embodiments, the therapeutic agent is hypercytotoxic (e.g., MMAE, MMAF). In some embodiments, the complex (e.g., hypercytotoxic-containing complex) is internalized into the cells by endocytosis.

[0218] The complexes of this disclosure have been shown to be effective in in vivo cancer studies, i.e., xenograft studies. Their relatively small size (e.g., low molecular weight complexes overall) is thought to facilitate penetration into tumor tissue and support the efficacy of this therapy. Accordingly, in some embodiments, the molecular weight of the complexes ranges from approximately 120 kDa, 100 kDa, 80 kDa, 60 kDa, 50 kDa, 45 kDa, and 40 kDa to a maximum.

[0219] composition In some embodiments, the dendrimer-targeting agent complex is presented as a composition, preferably a pharmaceutical composition.

[0220] It will be understood that, as a result of the nature of the synthetic process for producing dendrimers, there may be some variation in the molecular composition of dendrimer-targeting agent complexes present in a given composition. For example, as described above, one or more synthetic steps used to produce a dendrimer-targeting agent complex may not proceed completely, resulting in dendrimer-targeting agent complexes that all contain the same number of therapeutic moieties, or contain no targeting agent, or have incomplete generations of constituent units.

[0221] In one example, the composition comprises multiple dendrimers, each not containing an equal number of therapeutic agents. In another example, the composition comprises multiple dendrimers, each not containing an equal number of targeting agents. In some embodiments, the composition comprises multiple dendrimer-targeting agent complexes, each having an average of 1 targeting agent covalently bonded to the dendrimer. In some embodiments, the composition comprises multiple dendrimer-targeting agent complexes, each having an average of 1.2 targeting agents covalently bonded to the dendrimer. In some embodiments, the composition comprises multiple dendrimer-targeting agent complexes, each having an average of 1 to 1.5 targeting agents covalently bonded to the dendrimer. In some embodiments, the composition comprises multiple dendrimer-targeting agent complexes, each having an average of 0.5 to 5 targeting agents covalently bonded to the dendrimer. In some embodiments, the composition comprises multiple dendrimer-targeting agent complexes, each having an average of 1 to 4 targeting agents covalently bonded to the dendrimer. In some embodiments, the composition comprises multiple dendrimer-targeting agent complexes, each having an average of 1 or more targeting agents covalently bonded to the dendrimer. In some embodiments, the composition comprises a plurality of dendrimer-targeting agent complexes, wherein the average number of covalently bonded targeting agents to the dendrimers is 1 to 1.5. Thus, compositions comprising a plurality of dendrimer-targeting agent complexes or pharmaceutically acceptable salts thereof are provided, where the dendrimer-targeting agent complexes are as defined herein. In some embodiments, compositions for therapeutic use are provided, comprising dendrimer-targeting agent complexes and therapeutically acceptable excipients. In some embodiments, the composition is formulated for parenteral delivery. In some embodiments, the composition is formulated for intravenous delivery. In some embodiments, the composition is formulated for subcutaneous delivery. In some embodiments, the composition is formulated for intramuscular injection.

[0222] The Disclosure also provides pharmaceutical formulations or compositions for both veterinary and human medical applications, which comprise the complex or a pharmaceutically acceptable salt thereof of the Disclosure together with one or more pharmaceutically acceptable carriers and optionally any other therapeutic components, stabilizers, etc.

[0223] Accordingly, compositions for pharmaceutical use are also provided, comprising i) the complex as defined herein, and ii) pharmaceutically acceptable excipients.

[0224] Excipients(s) must be pharmaceutically acceptable in the sense that they are compatible with the other components of the formulation / composition and do not unduely adversely affect the recipient.

[0225] In some embodiments, the composition comprises a pharmaceutically acceptable solvent, such as water for injection and / or a pharmaceutically acceptable organic solvent.

[0226] The compositions of this disclosure may include, for example, polymeric excipients / additives or carriers, such as polyvinylpyrrolidone, derivatized cellulose, such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficol (a polymeric sugar), hydroxyethyl starch (HES), dextrates (for example, cyclodextrins (such as 2-hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin)), polyethylene glycol, and pectin.

[0227] The composition may further include diluents, buffers, citrates, trehalose, binders, disintegrants, thickeners, lubricants, preservatives (such as antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (phospholipids such as lecithin, and other phosphatidylcholine, phosphatidylethanolamine, fatty acids, and fatty acid esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc, and other suitable cations). Other pharmaceutical excipients and / or additives suitable for use in the compositions of this disclosure are described in "Remington: The Science & Practice of Pharmacy," 19th sup.th ed., Williams & Williams, (1995), "Physician's Desk Reference," 52nd sup.nd ed., Medical Economics, Montvale, NJ, (1998), and "Handbook of Pharmaceutical Excipients" (3rd edition), ed., AHKibbe, Pharmaceutical Press, 2000.

[0228] The dendrimer-targeting agent complexes of this disclosure can be formulated into compositions suitable for, for example, inhalation into the lungs, aerosol administration, or parenteral administration (such as intraperitoneal, intravenous, subcutaneous, or intramuscular injection). The compositions may be conveniently provided in unit dosage forms or prepared by any method well known in the field of pharmaceuticals. All methods involve the step of associating the dendrimer-targeting agent complex with a carrier comprising one or more accessory components. Generally, compositions are prepared by associating the dendrimer-targeting agent complex with a liquid carrier to form a solution or suspension, or by associating the dendrimer-targeting agent complex with a formulation component suitable for forming a solid, optionally particulate product, and, where appropriate, shaping the product into a desired delivery form. When the solid dosage forms of this disclosure are micronized, they typically contain particles ranging in size from about 1 nanometer to about 500 microns. Generally, for solid dosage forms intended for intravenous administration, the particles range in diameter from about 1 nm to about 10 microns. The composition may include the dendrimer-targeted subcomplex of the Disclosure, which is a nanoparticle having a particle size of 1000 nm or less, for example, 5 to 1000 nm, particularly 5 to 500 nm, more particularly 5 to 400 nm, for example 5 to 50 nm, and particularly 5 to 20 nm. As an example, the composition includes a dendrimer-targeted subcomplex with an average diameter of 5 to 20 nm. In some embodiments, the dendrimer-targeted subcomplex is polydisperse in the composition, and the PDI is 1.01 to 1.8, particularly 1.01 to 1.5, and more particularly 1.01 to 1.2. In one example, the dendrimer-targeted subcomplex is monodisperse in the composition.

[0229] In some preferred embodiments, the composition is formulated for parenteral delivery. For example, in one embodiment, the formulation may be a sterile lyophilized composition that is suitable for reconstitution with an aqueous vehicle before injection.

[0230] In some embodiments, formulations suitable for parenteral administration conveniently include a sterile aqueous preparation of the dendrimer-targeting agent complex, which can be formulated, for example, to be isotonic with the recipient's blood.

[0231] In some embodiments, the composition is formulated for parenteral infusion as part of a chemotherapy regimen.

[0232] In some embodiments, the composition is formulated for intraperitoneal delivery. Any suitable delivery means may be used. For example, in some embodiments, delivery may be by lavage solution or aerosol. In one embodiment, the composition is formulated for intraperitoneal delivery and is for the treatment of intraperitoneal cancers such as malignant epithelial tumors (e.g., ovarian cancer) and peritoneal carcinomatosis (e.g., gastrointestinal, particularly colorectal cancer, gastric cancer, gynecological cancers and primary peritoneal neoplasms).

[0233] Furthermore, pharmaceutical formulations suitable for administration by inhalation as an aerosol are also provided. These formulations comprise a solution or suspension of a desired dendrimer or a salt thereof. The desired formulation may be placed in a small chamber and nebulized. Nebulization can be performed using compressed air or ultrasonic energy to form multiple droplets or solid particles containing the dendrimer or a salt thereof.

[0234] As described later, the dendrimer-targeting agent conjugates of this disclosure can be administered, for example, in combination with one or more additional pharmaceutically active agents. For example, the dendrimer-targeting agent conjugates can be administered by being included in a composition together with further pharmaceutically active agents. Further examples of pharmaceutically active agents include chemotherapeutic agents, cytotoxic agents, small molecule cytotoxic agents, tyrosine kinase inhibitors, checkpoint inhibitors, EGFR inhibitors, antibody therapies, taxanes, and aromatase inhibitors.

[0235] The dendrimer-targeting agent conjugates of this disclosure can be administered not only together with other chemotherapeutic agents, but may also be included in a composition with other pharmaceuticals as appropriate.

[0236] How to use Furthermore, this specification also provides a method for treating cancer, comprising administering a therapeutically effective amount of the complex as defined herein, or a pharmaceutical composition containing the complex as defined herein, to a subject in need thereof.

[0237] Furthermore, this specification provides for the use of the complexes defined herein, or compositions comprising the complexes defined herein, in the manufacture of pharmaceuticals for the treatment of cancer.

[0238] Furthermore, this specification provides a complex as defined herein, or a pharmaceutical composition containing a complex as defined herein, for use in therapeutic applications. This specification also provides a complex as defined herein, or a pharmaceutical composition containing a complex as defined herein, for use in the treatment of cancer.

[0239] In some embodiments, dendrimer-targeting agent conjugates are used in methods for treating or preventing cancer, such as inhibiting tumor growth. In some embodiments, dendrimer-targeting agent conjugates are intended for use in the treatment of cancer.

[0240] In some embodiments, cancer is a solid tumor. Cancer can be a primary or metastatic tumor. In some embodiments, cancer is a primary tumor. In some embodiments, cancer is a metastatic tumor.

[0241] In some embodiments, the cancer is characterized by abnormal or overexpression of HER2 (also known as ERBB2). Such abnormal or overexpression of HER2 is known to occur, for example, in breast cancer, testicular cancer, ovarian cancer, stomach cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., severe endometrial cancer). In some embodiments, the cancer is selected from the group consisting of breast cancer, testicular cancer, ovarian cancer, stomach cancer, lung adenocarcinoma, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., severe endometrial cancer). In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is salivary duct cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is uterine cancer.

[0242] Accordingly, methods, uses, dendrimer-targeting agent conjugates, or compositions for use are also provided, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, gastric cancer, endometrial cancer, or another cancer characterized by abnormal or overexpression of HER2 (i.e., ERBB2).

[0243] combination Drugs are often administered in combination with other drugs, especially during chemotherapy. Therefore, in some embodiments, the dendrimer-targeting agent conjugate is administered in combination with one or more additional pharmaceutically active agents, such as one or more additional anticancer agents / drugs. The dendrimer-targeting agent conjugate and one or more additional pharmaceutically active agents can be administered simultaneously, subsequently, or separately. For example, they may be administered as part of the same composition or as separate compositions. One or more additional pharmaceutically active agents may be, for example, anticancer agents for the treatment of colorectal cancer, gastric cancer, pancreatic cancer, prostate cancer, or breast cancer. Examples of additional pharmaceutically active agents include chemotherapeutic agents, cytotoxic agents, small molecule cytotoxic agents, tyrosine kinase inhibitors, checkpoint inhibitors, EGFR inhibitors, antibody therapies, taxanes, and aromatase inhibitors.

[0244] dose The term “therapeutic effective dose” will be understood to refer to a dendrimer-targeting agent complex administered in an amount sufficient to alleviate or prevent, to some extent, one or more symptoms of the disorder or condition being treated. A therapeutic effective dose of a dendrimer-targeting agent complex may be named, for example, based on the amount of the dendrimer-targeting agent complex administered. Alternatively, it may be determined based on the amount of therapeutic agent that can theoretically be delivered to the administered dendrimer-targeting agent complex, for example, the amount of therapeutic agent added to the dendrimer-targeting agent complex.

[0245] The dendrimer-targeting agent complex may be administered by any preferred route, for example, intravenously. In some embodiments, the dendrimer-targeting agent complex is delivered as an IV bolus. In some embodiments, the dendrimer-targeting agent complex is administered IV over a period of 0.5 to 60 minutes, or 0.5 to 15 minutes, or 0.5 to 5 minutes. In another example, the dendrimer-targeting agent complex may be administered intraperitoneally. The route of administration may target, for example, a disease or disorder in the subject. For example, in some embodiments, the disease or disorder may be an intraperitoneal malignancy such as gynecological cancer or gastrointestinal cancer, and the dendrimer-targeting agent complex may be administered intraperitoneally. In some embodiments, the dendrimer-targeting agent conjugate may be for the treatment of cancers of the peritoneal cavity, such as malignant epithelial tumors (e.g., ovarian cancer) or peritoneal carcinomatosis (e.g., gastrointestinal cancer, particularly colorectal cancer, gastric cancer, gynecological cancers, and primary peritoneal neoplasms), and the dendrimer-targeting partial conjugate is administered intraperitoneally.

[0246] In some embodiments, the dose of the dendrimer-targeting agent complex is 2 to 100 mg of activator / m². 2 , 2-50 mg of activator / m 2 , 2-40 mg of activator / m 2 , 2-30 mg of activator / m 2 , 2-25 mg of activator / m 2 , 2-20 mg of activator / m 2 , 5-50 mg of activator / m 2 , 10-40 mg of activator / m 2 , 15-35 mg of activator / m 2 , 10-20 mg / m² 2 , 20-30 mg / m² 2 , or 25-35 mg of activator / m 2 This is sufficient to deliver the active ingredient. The mouse dose of 10 mg / kg of the active ingredient is equivalent to the human dose of 30 mg / m². 2 This should roughly correspond to the dose (FDA guidance 2005). (The human mg / kg dose is mg / m 2To convert it, you can multiply by 37 (FDA guidance 2005).

[0247] In some embodiments, a therapeutically effective dose of the dendrimer-targeting agent complex is administered to a subject in need at a predetermined frequency. In some embodiments, the dendrimer-targeting agent complex is administered to a subject in need according to a dosing regimen administered once every 1 to 4 weeks. In some embodiments, the dendrimer-targeting agent complex is administered to a subject in need according to a dosing regimen administered once every 3 to 4 weeks.

[0248] The targeted conjugates of this disclosure deliver a therapeutic agent in a controlled manner. By controlling the release of the therapeutic agent from the conjugate, the level of the circulating therapeutic agent (e.g., hypercytotoxicity) can also be controlled. For example, the maximum plasma concentration of the therapeutic agent can be considerably lower than the result obtained from administering an equivalent amount of free therapeutic agent. In some embodiments, administration of the conjugate reduces the maximum plasma concentration of the released therapeutic agent by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% compared to administering an equivalent dose of free therapeutic agent.

[0249] Preparation of the complex Dendrimer-targeting agents can be prepared using any suitable synthetic route, such as those described in the examples.

[0250] In some embodiments, the dendrimer-targeting agent complex is It is produced by a process that involves reacting a first intermediate containing a HER2 targeting agent with a second intermediate which is a dendrimer: The first intermediate has a molecular weight of up to approximately 80 kDa and is a peptide moiety containing an antigen-binding site, the targeting agent is covalently attached to the spacer precursor group, and the spacer precursor group contains the first reactive group, The second intermediate is i) Core unit (C), and ii) comprising a constituent unit (BU), where each constituent unit is a lysine residue or an analog thereof, A dendrimer is formed in which a core unit is covalently attached to at least two constituent units via amide linkages, and each amide linkage is formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit; A therapeutic agent covalently bonded to the surface constituent units of a dendrimer; It includes hydrophilic polymer groups covalently bonded to the surface constituent units of the dendrimer, The second intermediate contains a second reactive group, The first and second reactive groups are complementary to each other so as to be suitable for reacting with each other in order to covalently link the HER2 target agent to the dendrimer.

[0251] Furthermore, this disclosure provides intermediates useful for the synthesis of dendrimer-targeting agent complexes. Accordingly, intermediates are also provided for preparing dendrimer-targeting agent complexes having a molecular weight of up to approximately 80 kDa and containing a HER2 targeting agent which is a peptide portion containing an antigen-binding site, wherein the targeting agent is covalently attached to a spacer precursor group, and the spacer precursor group contains a reactive group suitable for reaction with complementary reactive groups present on the dendrimer intermediate.

[0252] In some embodiments, the targeting agent is covalently attached to the spacer precursor group via a non-natural amino acid residue. In some embodiments, the targeting agent is covalently attached to the spacer precursor group via a triazole moiety (e.g., produced by the reaction of an azide-containing non-natural amino acid such as azidophenylalanine with an alkyne-containing group such as DBCO [dibenzylcyclooctin]).

[0253] In some embodiments, the spacer precursor group comprises one or more oligomer or polymer groups, such as PEG, PEOX, or polyamino acid (e.g., polysarcosine) groups. For example, it may contain PEG groups with 2 to 100 -CH2CH2O- units.

[0254] In some embodiments, the reactive group present on the spacer precursor group is an alkene group, for example, a reactive alkene suitable for reaction with a tetrazine-containing group. In some embodiments, the reactive group is a trans-cyclooctene group.

[0255] This disclosure also relates to the following numbered clauses:

[0256] 1. A dendrimer-targeting agent conjugate, a) i) Core unit (C), and ii) A dendrimer comprising a constituent unit (BU), wherein each constituent unit is a lysine residue or an analog thereof, A core unit is covalently attached to at least two constituent units via amide linkages, and each amide linkage is formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit, forming a dendrimer; b) A HER2 targeting agent having a maximum molecular weight of approximately 80 kDa and containing an antigen-binding site, which is covalently linked to a dendrimer by a spacer group; c) A therapeutic agent covalently linked to the surface constituent units of a dendrimer; d) A PEG or PEOX group covalently bonded to the surface constituent unit of the dendrimer. 2. The complex according to Clause 1, wherein the peptide portion is selected from a heavy chain antibody, Fab, Fv, scFv, or single-domain antibody. 3. The peptide portion is heavy chain variable (V H ) containing domains or heavy chain variable (V H A complex consisting of domains as described in Clause 1 or 2. 4. The peptide portion is light chain variable (V L ) containing domains or light chain variable (V L A complex consisting of domains as described in any one of clauses 1 to 3. 5. A complex according to any one of clauses 1 to 4, wherein the targeting agent has a molecular weight of approximately 5 kDa to approximately 30 kDa. 6. The complex according to Clause 5, wherein the targeting agent has a molecular weight of approximately 5 kDa to approximately 15 kDa. 7. The complex according to Clause 6, wherein the targeting agent has a molecular weight of approximately 10 kDa to approximately 16 kDa. 8. A complex according to any one of clauses 1 to 7, wherein the targeting agent comprises fewer than 120 amino acid residues. 9. A complex according to any one of the clauses 1 to 8, wherein the targeting agent comprises or consists of any of the amino acid sequences defined herein. 10. The complex according to any one of clauses 1 to 9, wherein the covalent linkage between the targeting agent and the spacer group is formed by a reaction between complementary reactive functional groups present on the targeting agent precursor and the spacer group precursor. 11. The complex according to Clause 10, wherein the targeting agent precursor comprises a non-natural amino acid residue, and the non-natural amino acid residue has a side chain containing a reactive functional group. 12. The complex according to Clause 11, wherein the non-natural amino acid residue is a 4-azidophenylalanine residue. 13. The complex according to any one of clauses 1 to 12, wherein the targeting agent is covalently linked to a spacer group via the C-terminus of the targeting agent. 14. The complex according to any one of the clauses 10 to 13, wherein the spacer group precursor comprises a reactive functional group which is an alkyne group. 15. The complex according to Clause 14, wherein the alkyne group is a dibenzocyclooctin group. 16. A complex according to any one of the clauses 1 to 15, wherein the therapeutic agent is a hypercytotoxic agent. 17. The complex according to Clause 16, wherein the therapeutic agent is auristatin or a meitansinoid. 18. The complex according to Clause 17, wherein the therapeutic agent is monomethyl auristatin E. 19. The complex according to Clause 17, wherein the therapeutic agent is monomethyl auristatin F. 20. The complex according to any one of the clauses 1 to 19, wherein the therapeutic agent is covalently linked to the surface constituent units of a dendrimer via a linker. 21. The composite according to any one of the clauses 1 to 20, wherein the therapeutic agent is covalently linked to the surface constituent units of a dendrimer via a cleavable linker. 22. The composite according to Clause 21, wherein the cleavable linker contains a Val-Cit-PAB group. 23. A composite according to any one of clauses 1 to 22, comprising a PEG group covalently bonded to a surface constituent unit of a dendrimer. 24. The complex according to Clause 23, wherein the PEG group has an average molecular weight in the range of approximately 500 to approximately 2500 g / mol. 25. The complex according to any one of the clauses 1 to 24, wherein the spacer group comprises a PEG group. 26. The core unit is structured [ka] A composite including any one of the clauses 1 to 25. 27. A complex according to any one of the clauses 1 to 26, wherein the dendrimer has constituent units of one to five generations. 28. The complex according to Clause 27, wherein the dendrimer has three generations of constituent units. 29. Each of the constituent units is, [ka] A composite, as described in any one of the clauses 1 to 28. 30. A complex described in any one of clauses 1 to 29, for administration in combination with a further activator. 31. The complex described in any one of the clauses 1 to 30, which is internalized within HER2-expressing cells. 32. A complex described in any one of clauses 1 to 31, wherein administration of the complex results in a reduction of side effects compared to administration of an equivalent dose of the free therapeutic agent. 33. The complex described in any one of Clauses 1 to 32, wherein administration of the complex results in a reduction of at least 50% of the maximum plasma concentration of the released therapeutic agent compared to administration of an equivalent dose of the free therapeutic agent. 34. A composition comprising multiple complexes as described in any one of the clauses 1 to 33. 35. i) A complex as described in any one of clauses 1 to 34; ii) Pharmacopoeia-acceptable excipients, A pharmaceutical composition containing the following: 36. The composition according to clause 34 or 35, wherein the composition is formulated for parenteral delivery. 37. A complex according to any one of Clauses 1 to 33, or a composition according to any one of Clauses 34 to 36, for use in the treatment of cancer. 38. A method for treating cancer, comprising administering a therapeutically effective amount of the complex described in any one of clauses 1 to 33 or the composition described in any one of clauses 34 to 37 to a subject in need thereof. 39. Use of a complex described in any one of Clauses 1 to 33, or use of a composition described in any one of Clauses 34 to 38, in the manufacture of a drug for the treatment of cancer. 40. The method, use, or complex or composition for use described in any one of the clauses 37 to 39, wherein the cancer is ovarian cancer, breast cancer, gastric cancer, uterine cancer, or another cancer characterized by abnormal expression of the ERBB2 gene. 41. A method for killing HER2-expressing cells, A method comprising contacting a complex described in any one of clauses 1 to 33 with HER2-expressing cells, thereby causing the complex to be internalized into the cells, and thereby causing the therapeutic agent to kill the HER2-expressing cells.

[0257] This disclosure is further illustrated by the following embodiments. It should be understood that the following description is intended solely to illustrate specific embodiments and is not intended to limit the scope of the above description.

[0258] Examples In this specification, the following nomenclature is used in relation to the synthesis of dendrimer complexes. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0259] HPLC / MS (mass spectrometer) and NMR instrument details: HPLC-Waters 2795, 2996 diode array detector (DAD) MS-Waters ZQ4000 (with ESI probe), the inlet flow is branched to approximately 50 μL / min to the MS.

[0260] Mass spectrometry data were obtained in positive or negative electrospray ionization modes, as shown. The raw data were deconvolved using the Maximum Entropy algorithm (MaxEnt) implemented in MassLynx software v4.0 provided by Waters Corporation. The data reported in the experimental details correspond to the observed values ​​after deconvolution to the theoretical zero-charge state. NMR-300MHz Bruker.

[0261] The preparation of carboxy-reactive dendrimer scafolds is described in particular in International Publications 2007 / 082331, 2008 / 017125, 2012 / 167309, and 2015 / 184510. Those skilled in the art can use these methods to adapt and prepare the various dendrimers outlined herein.

[0262] In the following example, [Lys] in the formula refers to the lysine constituent unit located in the surface layer of the dendrimer.

[0263] Example 1 General Procedure A. Boc Deprotection To a Boc compound (1.0 equivalent) / aqueous suspension, which had been cooled on ice and stirred, TFA (40-200 equivalents / Boc group) was added. After 5 minutes, the ice bath was removed, and the reaction mixture was stirred at room temperature overnight. Volatile substances were removed by vacuum, and the remaining aqueous solution was further diluted with water, freeze-dried, and the deprotected product was obtained in quantitative yield.

[0264] General procedure B. Addition of a lysine layer to the dendrimer surface. Under a nitrogen atmosphere, TEA (6.0 equivalents / NH2) was added to a TFA dendrimer (1.0 equivalent) / DMF stirred solution, followed by DBL-oPNP (2.0 equivalents / NH2). The resulting reaction mixture was then stirred at room temperature overnight. Volatile substances were removed by vacuum, and the resulting crude product was purified using standard methods.

[0265] General procedure: PEGylation of dendrimer surfaces using a C.HO-Lys-(α-NHBoc)(ε-NHPEG1100) wedge. PyBOP (2.0 equivalents / NH2) and DIPEA (8.0 equivalents / NH2) were added to a TFA dendrimer (1.0 equivalent) / DMF stirred solution under a nitrogen atmosphere. After 10 minutes, HO-Lys-(α-NHBoc)(ε-NHPEG 1100 (1.35 equivalents / NH2) / DMF solution was added, and the resulting reaction mixture was stirred overnight at room temperature. Volatile substances were removed by vacuum, and the resulting crude product was purified using standard methods.

[0266] The general procedure involves capping the dendrimer surface with a D.HO-Lys-(α-NHBoc)(ε-NHFmoc) wedge, followed by Fmoc deprotection. Step 1: PyBOP (1.4 equivalents / NH2) was added to a stirred solution of HO-Lys-(α-NHBoc)(ε-NHFmoc) (1.5 equivalents / NH2) and NMM (2.5 equivalents / NH2) / DMF. The resulting reaction mixture was then stirred at room temperature for 15 minutes, and TFA-dendrimer (1.0 equivalent) and NMM (2.5 equivalents / NH2) / DMF solution were added. The resulting reaction mixture was then stirred at room temperature for 1 hour and allowed to stand, after which it was slowly added to ice-cold MeCN and stirred for 15 minutes. The resulting solid was collected by filtration, washed with MeCN (3 times), and then freeze-dried.

[0267] Step 2: Piperidine (21 eq / Fmoc) was added to a DMF solution containing Fmoc / Boc dendrimer (1.0 equivalent). This solution was stirred at room temperature for 90 minutes, then slowly added to ice-cold Et2O. After 15 minutes, the precipitated solid was collected by filtration, washed with Et2O, dissolved in H2O, and frozen for storage.

[0268] General procedure E. Capping of dendrimer surfaces with DGA-doxorubicin or DGA-nemorubicin DGA-3'-NH-doxorubicin

[0269] Step 1: A DMAP (2.5 equivalents) / DMF solution was added to a stirred Dox.HCl (1.0 equivalent) / DMF solution at room temperature. After 5 minutes, a DGA (0.9 equivalents) / DMF solution was added. The reaction was completed in 3 hours.

[0270] Step 2: Add PyBOP (2.0 equivalents / dendrimer NH2) to the above reaction mixture, then add TFA-dendrimer (azide-PEG). 24 -CO[N(PN)2[Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )4 or Azide-PEG 24-CO[N(PN)2[Lys]8[(α-NH2.TFA)(ε-NHPEG 1100 )]8) (1.0 equivalent) and DIPEA (6.0 equivalents / dendrimer NH2) / DMF solution were added. The resulting reaction mixture was then stirred and left to stand overnight at room temperature. Volatile substances were removed by vacuum, and the resulting crude material was purified by SEC.

[0271] DGA-14-O-Nemorubicin Step 1: A DMAP (2.5 equivalents) / DMF solution was added to a stirred nemorubicin (1.0 equivalent) / DMF solution at room temperature. After 5 minutes, a DGA (0.9 equivalents) / DMF solution was added. The reaction was completed in 3 hours.

[0272] Step 2: Add PyBOP (2.0 equivalents / dendrimer NH2) to the above reaction mixture, then add TFA-dendrimer (azide-PEG). 24 -CO[N(PN)2[Lys]2[Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )4 or Azide-PEG 24 -CO[N(PN)2[Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NHPEG 1100 )]8) (1.0 equivalent) and DIPEA (6.0 equivalents / dendrimer NH2) / DMF solution were added. The resulting reaction mixture was then stirred and left to stand overnight at room temperature. Volatile substances were removed by vacuum, and the resulting crude material was purified by SEC.

[0273] The dendrimer surface is capped using the general procedure F.Glu-vc-PAB-MMAE or DGA-MMAF(OMe). Azido-PEG 24 -CO[N(PN)2[Lys]8[(α-NH2.TFA)(ε-NHPEG 570 / 1100 / 2000 )]8 (1.0 equivalent) was dissolved in a mixture of DMF and NMM (5.0 equivalents / NH2) at room temperature. This solution was added to HO-Glu-vc-PAB-MMAE or DGA-MMAF(OMe) (1.2 equivalents / NH2) and PyBOP (2.0 equivalents / NH2) and left at room temperature. The resulting crude material was purified by SEC.

[0274] General Procedure G: Conjugation of affibody to Dox / Pt(IV)-acetate / MMAE / MMAF dendrimer Step 1: A solution of affibody protein (1.0 mg / mL in PBS) was treated with TCEP (50 mM, 39.0 eq.), and the reaction mixture was shaken at 650 rpm for 2 hours at room temperature. The resulting solution was purified by SEC.

[0275] Step 2: The collected permeate was treated with a solution of maleimide-bicyclo[6.1.0]nonine (Mal-BCN) (20.0 eq.) in DMSO. Subsequently, the resulting reaction mixture was shaken at 650 rpm for 2 hours at room temperature. The resulting solution was purified by SEC.

[0276] Step 3: The affibody-BCN solution was reacted with azido-PEG 24 -CO[N(PN)₂[Lys]₄[(α-DGA-3’-NH-Dox)(ε-NHPEG 1100 )]₄ solution (858 μM in PBS) or azido-PEG 24 -CO[N(PN)₂[Lys]₈[(α-DGA-3’-NH-Dox)(ε-NHPEG 1100 )]₈ (580 μM in PBS) or azido-PEG 24 -CO[N(PN)₂][Lys]₈[(α-Pt(IV)-acetate)(ε-NHPEG 1100 )]₈ (903 μM in PBS) or azido-PEG 24 -CO[N(PN)₂[Lys]₈[(α-Glu-vc-PAB-MMAE)(ε-NHPEG 570 / 1100 / 2000 )]₈ (240 μM in PBS) or azido-PEG 24 -CO[N(PN)₂[Lys]₈[(α-DGA-MMAF(OMe))(ε-NHPEG 570 / 1100 / 2000)] was treated with 8 (365 μM / PBS) (1.3 equivalents affibody-BCN / dendrimer). The resulting mixture was subsequently shaken at 650 rpm at room temperature overnight, then treated with a 9.38 mM (30% EtOH / water) solution of DBCO agarose (5.0 equivalents / dendrimer). The resulting suspension was subsequently shaken at 1200 rpm at room temperature overnight. This suspension was purified by SEC.

[0277] General Procedure H. Conjugation of Nanobodies to MMAE Dendrimers Step 1: A linker (1 mg) was dissolved in 20:80 (DMSO / 10 mM PBS, 1 mL) to prepare a solution of the linker (BCN-PEG2NH-Glu-NHPEG 24 CO-NHPEG3-TCO or DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO).

[0278] Step 2: A TCO-linker solution (1 equivalent) was added to a solution of tetrazine-functionalized dendrimer (e.g., Tz-MMAE-dendrimer or BHA-Tz-MMAE-dendrimer) (1.0 equivalent, 8 mg / mL) / PBS (1). The reaction mixture was left to stand at room temperature for 30 minutes. Completion of the reaction was indicated by the disappearance of the pink tetrazine color. This reaction was monitored by HPLC.

[0279] Step 3: After the reaction was completed, the contents were diluted with PBS (to a final volume of 0.5 mL). A portion of BCN / DBCO-MMAE-dendrimer (1.0 equivalent) was added to a solution of nanobody-azide (1.0 equivalent, 9.2 mg / ml) in Tris buffer (20 mM, 1 mL). The resulting solution was left to stand at RT for 7 hours, then at 4° C overnight. Purification of the nanobody-dendrimer construct was performed by anion exchange chromatography followed by SEC.

[0280] Example 1a Synthesis of Intermediates 1.1 Azido-PEG 24 -CO[N(PNBoc)2] Compound 1 Under N2 atmosphere, azido-PEG24 - To a stirred solution of acid (2.00 g, 1.71 mmol) and PyBOP (1.33 g, 2.56 mmol) / DMF (20 mL), NMM (563 μL, 5.12 mmol) was added. After 10 minutes, a DMF (5 mL) solution containing N(PNBoc)2 (622 mg, 1.88 mmol) was added, and the resulting reaction mixture was stirred at room temperature overnight. After removing volatile components by vacuum, the resulting oil was dissolved in MeCN and purified by preparative HPLC (27-50-70% MeCN, Rt 47-50 min) to obtain a pale yellow oily solid (1.37 g, 54%). 1H-NMR(300MHz,CD3OD)δ(ppm): 1.44(m,18H);1.65-1.84(m,4H);2.63(t,J6.3Hz,2H);3.05(dt,J6.9and14.7Hz,4H);3.36-3.41(m,6H);3.60-3.78(m,98H). LCMS (hydrophilic method, formic acid buffer) R t =9.32 min ESI MS(+ve)1486.3[M] + ;C 67 H 132 N6O 29 [M] + m / z calculated value: 1486.8

[0281] 1.2 Azide-PEG 24 -CO[N(PNH2.TFA )2 ], compound 2 Azido-PEG 24 -CO[N(PNBoc)2](1.37g, 922μmol) was used to prepare the solution according to general procedure A. The freeze-dried product was obtained as a pale yellow oil (1.67g, 119%). 1H-NMR(300MHz,D2O)δ(ppm): 1.88-2.06(m,4H);2.74(t,J6.0Hz,2H);2.96(t,J7.2Hz,2H);3.04(a apparent t,J7.5Hz,2H);3.42-3.52(m,6H);3.67-3.95(m,93H). LCMS (hydrophilic method, TFA buffer) R t =8.47 min, ESI MS(+ve) 1286.0[M] + ;C 57 H 116 N6O25 [M]+ m / z calculation value = 1286.6.

[0282] 1.3 Azide-PEG 24 -CO[N(PN)2][Lys]2[Boc]4, G1, compound 3 Azido-PEG 24 -CO[N(PNH2.TFA)2](186 mg, 145 μmol) was used to prepare the material according to general procedure B. The crude material was dissolved in MeCN and preparative HPLC (30-80% MeCN, R t The mixture was refined for 33.5 to 36 minutes to obtain a pale yellow oil (224 mg, 80%). 1H-NMR(300MHz,CD3OD)δ(ppm):1.22-1.87(m,56H);2.64(t,J6.0Hz,2H);3.03(t,J6.6Hz,4H);3.13-3.23( m,4H),3.36-3.45(m,6H);3.60-3.69(m,100H);3.77(t,J6.0Hz,2H);3.85-3.88(m,1H);3.92-4.02(m,2H). LCMS (hydrophobic method, formic acid buffer) R t =6.74 minutes;ESI MS(+ve)1942.4,[M]+;C 89 H 172 N 10 O 35 + [M+H] + The calculated m / z value is 1942.4.

[0283] 1.4 Azide-PEG 24 -CO[N(PN)2][Lys]2[NH2.TFA]4, G1, compound 4 Azido-PEG 24 Prepared using CO[N(PN)2[Lys]2[Boc]4 (220 mg, 113 μmol) according to general procedure A. The lyophilized product was obtained as a pale yellow oil (251 mg, 111%). LCMS (hydrophilic method, formate buffer) R t =6.49 min, ESI MS(+ve) 1542.1[M] + ;C 69 H 140 N 10 O 27 [M] +The calculated m / z value is 1541.90.

[0284] 1.5 Azide-PEG 24 -CO[N(PN)2][Lys]4[(α-Boc)(ε-NHPEG 1100 )]4, G2, compound 5 Azido-PEG 24 -CO[N(PN)2[Lys]2[NH2.TFA]4 (120 mg, 60.1 μmol) was used to prepare the product according to general procedure C. The crude material was dissolved in MeCN / H2O (1:1) and purified by preparative HPLC (20-70% MeCN, Rt 31-32.5 min) to obtain the product as a pale yellow oil (244 mg, 59%). 1 H-NMR(300MHz,CD3OD)δ(ppm): 1.24-1.91(m,74H);2.42-2.47(m,8H);2.62-2.66(m,2H);3.13-3.25(m,12H);3.36(s ,12H);3.52-3.78(m,490H);3.85-3.88(m,4H);3.94-4.11(m,4H);4.25-4.31(m,2H). LCMS (hydrophilic method, formic acid buffer) Rt=8.70 min; ESI MS(+ve)1714.0[M+4H] 4+ / 4,1371.7[M+5H] 5+ / 5;1143.0[M+6H] 6+ / 6,980.0[M+7H] 7+ Convert to / 7.6852.

[0285] 1.6 Azide-PEG 24 -CO[N(PN)2][Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )]4, G2, compound 6 Azido-PEG 24 -CO[N(PN)2[Lys]4[(α-Boc)(ε-NHPEG 1100 Prepared using )4 (244 mg, 35.6 μmol) according to general procedure A. The freeze-dried crude product was redissolved in water and preparative HPLC (22-70% MeCN, 0.01% TFA, R tThe product was purified by (27 minutes) and obtained as a pale yellow viscous solid (173 mg, 67%). 1 H-NMR(300MHz,D2O)δ(ppm): 1.31-1.98(m,40H);2.51-2.56(m,8H);2.72(broadt,J6.0Hz,2H);3.16-3.30(m,16H);3.4 0(s,12H);3.46-3.53(m,4H);3.62-3.97(m,490H);4.03(t,J6.6Hz,2H);4.24-4.29(m,2H). LCMS (hydrophilic method, TFA buffer) Rt=9.85 min; ESI MS(+ve)1614.1[M+4H] 4+ / 4,1291.6[M+5H] 5+ / 5;1076.5[M+6H] 6+ Convert / 6 to 6452.

[0286] 1.7 Azide-PEG 24 -CO[N(PN)2][Lys]4[Boc]8, G2, compound 7 Azido-PEG 24 Prepared using CO[N(PN)2][Lys]2[NH2.TFA]4 (117 mg, 58.6 μmol) according to general procedure B. The crude material was obtained as a pale yellow oil (167 mg, 100%). LCMS (hydrophobic method 4.1a, formate buffer) R t =8.35 minutes;ESI MS(+ve)1328.6[M+2H] 2+ / 2-Boc;C 133 H 252 N 18 O 47 [M]+ m / z calculated value = 2855.6.

[0287] 1.8 Azide-PEG 24 -CO[N(PN)2][Lys]4[NH2.TFA]8, G2, compound 8 Azido-PEG 24-CO[N(PN)2][Lys]4[Boc]4 (167 mg, 58.6 μmol) was used to prepare the solution according to general procedure A. The crude aqueous solution was purified by preparative HPLC (10-60% MeCN, 0.1% TFA buffer; Rt 27-29 min) to obtain the product as a very pale yellow viscous solid (124 mg, 71% in step 2). 1 H-NMR(300MHz,D2O)δ(ppm): 1.30-1.96(m,42H);2.71(t,J6.0Hz,2H);2.98-3.04(m,8H);3.13-3.30(m,8H);3.36-3.53( m,7H);3.68-3.84(m,100H);3.93(t,J6.6Hz,2H);4.04(t,J6.6Hz,2H);4.25(t,J7.2Hz,2H). LCMS (hydrophilic method, TFA buffer) Rt=7.76 min, ESI MS (+ve)1028.3[M+2H] 2+ / 2,685.9[M+3H] 3+ / 3;C 93 H 190 N 18 O 31 2+ [M+2H] 2+ m / z calculation value for / 2: 1028.3, C 93 H 191 N 18 O 313+ [ M+3H ]3+ / m / z calculation value for 3: 685.9.

[0288] 1.9 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG1100)]8,G3,Compound 9 Azido-PEG 24 -CO[N(PN)2][Lys]4[NH2.TFA]8 (123 mg, 41.5 μmol) was used to prepare the crude material according to general procedure C, and the crude material was obtained as a brown oil. LCMS (hydrophilic method, formate buffer) R t =11.52 minutes,ESI MS (+ve)2113[M+6H] 6+ / 6,1812[M+7H] 7+ / 7,1585[M+8H] 8+ / 8,1409[M+9H]9+ / 9,1268[M+10H] 10+ / 10,1153[M+11H] 11+ / 11,1057[M+12H] 12+ Convert / 12 to 12 673.

[0289] 1.10 Azido-PEG24-CO[N(PN)2][Lys]8[(α-TFA)(ε-NHPEG 1100 )]8, G3, compound 10 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 1100 )]8 (526 mg, 41.5 μmol) was used to prepare the product according to general procedure A. The crude aqueous solution was purified by preparative HPLC (3-60% MeCN, 0.1% TFA buffer; Rt 38-39 min) to obtain the product as a pale yellow viscous solid (359 mg, 68% in step 2). It was converted to 11880 by LC-MS (hydrophilic method, TFA buffer) Rt = 10.27 min.

[0290] 1.11 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-Fmoc)]8,G3, Compound 11 Azido-PEG 24 -CO[N(PN)2][Lys]4[NH2.TFA]8 (105 mg, 35.4 μmol) was used to prepare the solution according to general procedure D. The product was obtained as a white solid (166 mg, 83%). 1 H-NMR(300MHz,d6-DMSO)δ(ppm): 1.23-1.49(m,160H);2.73-2.95(m,36H);3.44-3.60(m,94H);3.83(m,8H);4.05-4.28(m,29H);6.33-6.90(m,8H,NH);7.24-7.87(m,84H).

[0291] 1.12 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NH2)]8,G3, Compound 12 Azido-PEG 24-CO[N(PN)2][Lys]8[(α-Boc)(ε-Fmoc)]8 (169 mg, 29.9 μmol) was used to prepare a cottony solid according to general procedure D (95 mg, 82%). 1 H-NMR(300MHz,d4-MeOH)δ(ppm): 1.46-1.49(m,160H);2.69(brs,14H);3.09-3.19(m,18H);3.38-3.41(m,8H);3.55-3.78(m,96H),3.89-4.33(m,14H).

[0292] 1.13 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 570 )8, G3, Compound 13 and Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NHPEG 570 )]8,G3, compound 14 mPEG 570 -CO2H (205 mg, 348 μmol), NMM (60 μL, 546 μmol), and PyBOP (171 mg, 329 μmol) in DMF (1.5 mL) / DMF solution, with azide-PEG added. 24 -CO[N(PN)2[Lys]8[(α-Boc)(ε-NH2)]8 (0.5 mL) / DMF was added. The resulting reaction mixture was then stirred overnight at room temperature, then concentrated under vacuum, dissolved in water, treated with TFA, and stirred overnight at room temperature. After the mixture was concentrated and incorporated into water, it was purified using a Millipore Centrifugation filtration unit (3K MWCO regenerated cellulose) to obtain a lyophilized product (68% in 2 steps) as an off-white cottony material. 1H-NMR(300MHz, D2O)δ(ppm): 1.33-1.90(m,88H);2.51-2.55(m,16H);2.67-2.75(m,4H),3.16-3.23 (m,32H);3.40-3.53(m,32H),3.62-4.03(m,470H);4.21-4.39(m,7H). LCMS (hydrophilic method, formic acid buffer) R t =7.50 minutes.

[0293] 1.14 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 2000 )]8, G3, compound 15 Azido-PEG 24 -CO[N(PN)2[Lys]8[(α-Boc)(ε-NH2)]8 (95.0 mg, 24.5 μmol) / DMF (4 mL) was stirred, to which DIPEA (85 μL, 488 μmol) was added, followed by mPEG2000-NHS (720 mg, 313 μmol). The resulting reaction mixture was then stirred overnight at room temperature. The crude residue was dissolved in water and purified by ultrafiltration (5 K, Pall PES membrane). The retaining solution was collected and freeze-dried to obtain an off-white cottony material (76%). 1 H-NMR (300MHz,D2O)δ(ppm): 1.33-1.63(m,160H);3.05-3.15(m,35H);3.29(s,24H);3.35-3.96(m,1370H);4.13-4.19(m,6H). LCMS (hydrophilic method, formic acid buffer) R t =11.24 minutes

[0294] 1.15 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NHPEG 2000 )]8, G3, compound 16 Follow general procedure A, Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 2000 Prepared using )8 (40.0 mg, 1.87 μmol), the product was obtained as an off-white, cotton-like material (35 mg, 88%). 1 H-NMR(300MHz,D2O)δ(ppm): 1.28-1.79(m,88H);2.51-2.58(m,4H);3.04-3.18(m,35H);3.28(s,24H);3.35-3.97(m,1348H),4.14-4.25(m,6H). LCMS (hydrophilic method, formic acid buffer) Rt =9.12 minutes

[0295] 1.16(MeTzPh)-PEG 24 -CO[N(PNBoc)2] Compound 46 (MeTzPh)-PEG 24 -CO2H (0.402 g, 0.305 mmol), PyBOP (0.205 g, 0.394 mmol), and NMM (130 μL, 1.18 mmol) / DMF (3 mL) were stirred together, and NH(PNBoc)2 (0.147 g, 0.444 mmol) was added under an N2 atmosphere. The resulting reaction mixture was then stirred at room temperature and left to stand overnight. Volatile components were removed by vacuum, and the resulting oil was purified by silica chromatography (5%~10% MeOH / DCM) to obtain the desired product, compound 106, as a red residue (0.474 g, 95%). 1 HNMR(300MHz,CD3OD)δ(ppm):1.43-1.44(m,18H);1.64-1.80(m,4H);2.63(t,J6.0Hz,2H);3.00(s,3H);3.02-3.09(m,4 H);3.34-3.40(m,4H),3.58-3.77(m,99H);3.88-3.91(m,2H);4.25-4.28(m,2H);7.15-7.20(m,2H);8.46-8.51(m,2H). LCMS (hydrophobic method, formic acid buffer) RT = 6.20 minutes. ESI MS(+ve)1631.0[M+H]+;C 76 H 140 N7O 30 [M+H] + The calculated value in m / z is 1631.0.

[0296] 1.17 (MeTzPh)-PEG 24 -CO[N(PNH2.HCl)2] Compound 47 (MeTzPh)-PEG in ice / water bath 24 -CO[N(PNBoc)2] Compound 1 (0.420 g, 0.258 mmol) was slowly mixed with 1.25 M HCl / MeOH solution (8 mL, 10.0 mmol). After 5 minutes, the ice bath was removed, and the resulting reaction mixture was stirred overnight at room temperature. Volatile components were removed by vacuum, and the product, Compound 107, was obtained as a red residue (0.388 g, 100%). 1 HNMR(300MHz,CD3OD)δ(ppm): 1.91-2.07(m,4H);2.68(t,J6.0Hz,2H);2.94-3.09(m,7H);3.53-3.80(m,108H );3.88-3.91(m,2H);4.25-4.28(m,2H);7.16-7.20(m,2H);8.47-8.51(m,2H). LCMS (hydrophilic method, formic acid buffer) RT=8.12 min.ESI MS(+ve)1430.9[M+H]+;C66H124N7O26[M+H] + The calculated value in m / z is 1430.8.

[0297] 1.18 (MeTzPh)-PEG4CO-NH-PEG 24 -CO[N(PNH2.HCl)2] Compound 48 H2N-PEG 24 To a stirred solution of -CO[N(PNBoc)2](0.418g, 0.286 mmol) / DMF(2.0 mL), (MeTzPh)-PEG4-CO2H(0.15g, 0.344 mmol), PyBOP(0.178g, 0.342 mmol), and NMM(80 μL, 0.727 mmol) were added. The resulting reaction mixture was then stirred at room temperature overnight. After removing volatile components by vacuum, the resulting oil was cooled in an ice / water bath, and then 1.25 M HCl / MeOH solution(10.0 mL, 12.5 mmol) was slowly added. After 5 minutes, the ice bath was removed, and the resulting reaction mixture was then stirred at room temperature overnight. The volatile components were removed by vacuum, and the resulting oil was dissolved in MeCN / H2O (8 mL, 1:1). It was then purified by preparative HPLC (10-50% MeCN, 0.1% formate buffer, RT 35 min) to obtain the red solid compound 108 (1.37 g, 54%). 1HNMR(300MHz,CD3OD)δ(ppm): 1.91-2.04(m,4H);2.43(t,J6.0Hz,2H);2.68(t,J6.0Hz,2H);2.94-3.07(m,4H);3.00(s,3H);3.35(t,J6. 0Hz,2H);3.51-3.80(m,119H);3.88-3.91(m,2H);4.25-4.28(m,2H);7.16-7.20(m,2H);8.46-8.51(m,2H). LCMS (hydrophilic method, formic acid buffer) RT=8.15 min.ESIMS(+ve)1677.9[M+H]+;C 77 H 145 N8O 31 [M+H] + The calculated value in m / z is 1678.0.

[0298] 1.19 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NHBoc]4G1 Compound 49 Under an N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PNH2.HCl)2] Compound 48 (0.195 g, 0.111 mmol) and DBL-oPNP (0.146 g, 0.312 mmol) / DMF (3.0 mL) were stirred together, and NMM (125 μL, 0.864 mmol) was added. The resulting reaction mixture was then stirred at room temperature overnight. Volatile components were removed by vacuum, and the resulting oily residue was purified by column chromatography (5%-10%-15% MeOH / DCM) on silica gel to obtain the desired product, Compound 109, as a red oil (0.186 g, 72%). 1HNMR(300MHz,CD3OD)δ(ppm): 1.28-1.87(m,56H);2.43(t,J6.0Hz,2H);2.63(t,J6.0Hz,4H);3.00(s,3H);3.02-3.06(m,4H);3.10-3.21(m,4H);). 3.35-3.41(m,6H);3.51-3.78(m,110H);3.84-3.99(m,4H);4.25-4.28(m,4H);7.15-7.20(m,2H);8.47-8.51(m,2H). LCMS (hydrophobic method, formic acid buffer) RT=6.68 min; ESI MS (+ve)2335.3, [M+H] + ;C 109 H 201 N 12 O41+[M+H] + The calculated m / z value is 2335.4.

[0299] 1.20 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NH2.HCl]4G1 Compound 50 Ice-cooled (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NHBoc]4 Compound 49 (0.215 g, 0.0921 mmol) was slowly mixed with a solution of 1.25 M HCl / MeOH (6.0 mL, 7.50 mmol). After 5 minutes, the ice bath was removed, and the resulting reaction mixture was stirred overnight at room temperature. The volatile matter was removed by vacuum, and the product, Compound 110, was obtained as a red oil (0.208 g, 108%). 1 HNMR(300MHz,CD3OD)δ(ppm): 1.51-1.99(m,20H);2.47(t,J6.0Hz,2H);2.67(t,J6.0Hz,4H);2.90-3.04(m,7H);3.35-3.41(m,7H);3.51-3.78(m,10 6H);4.25-4.28(m,2H);7.17-7.20(m,2H);8.47-8.51(m,2H);LCMS(hydrophilic method, formic acid buffer)RT=7.28min,ESIMS(+ve)1934.9[M+H] + ;C 89 H 169 N 12 O33 [M] + The calculated m / z value is 1935.2.

[0300] 1.21 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]4[NHBoc]8G2 Compound 51 Under an N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NH2.HCl]4 Compound 50 (0.192 g, 0.0822 mmol) and DBL-oPNP (Reference 1) (0.215 g, 0.460 mmol) / DMF (3.0 mL) were stirred together, and NMM (215 μL, 0.1.96 mmol) was added. The resulting reaction mixture was then stirred at room temperature and left to stand overnight. Volatile components were removed by vacuum, and the resulting oily residue was purified by silica chromatography (5%-10%-15% MeOH / DCM) to obtain the desired product, Compound 51, as a red oil (0.231 g, 87%). 1 HNMR(300MHz,CD3OD)δ(ppm): 1.28-1.87(m,140H);2.44(t,J6.0Hz,2H);2.63(t,J6.0Hz,2H);3.00(s,3H);3.02-3.06(m,10H);3.10-3.21(m,16kH);). 3.33-3.40(m,8kH);3.51-3.77(m,120H);3.84-3.99(m,14H);3.94-4.10(m,4H);4.25-4.28(m,4H);7.15-7.20(m,2H);8.47-8.52(m,2H). LCMS (hydrophobic method, formic acid buffer) RT=8.15 min; ESI MS (+ve) [M+2] + =1624.5;[(M-3Boc)+3]=1016.6;C 153 H 281 N 20 O 53 + [M+H] + The calculated m / z value is 3247.99.

[0301] 1.22 (MeTzPh)-PEG4CO-NHPEG 24-CO[N(PN)2][Lys]4[NH2.HCl]8G2 Compound 52 Ice-cooled (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]4[NHBoc]8 compound 51 (0.231 g, 0.0711 mmol) was slowly mixed with a solution of 1.25 M HCl / MeOH (9.0 mL, 11.3 mmol). After 5 minutes, the ice bath was removed, and the resulting reaction mixture was stirred overnight at room temperature. The volatile matter was removed by vacuum, and the product, compound 51, was obtained as a red oil (0.226 g, 100%). 1 HNMR(300MHz,CD3OD)δ(ppm): 1.51-1.96(m,40H);2.53(t,J6.0Hz,2H);2.96-3.04(m,10H);3.15-3.20(m,8H);3.39-3.45(m,7H);3.54 -4.10(m,102H);4.25-4.28(m,2H);7.17-7.20(m,2H);8.48-8.51(m,2H);LCMS (hydrophilic method, formic acid buffer) RT=6.38 min, ESI MS(+ve)2447.6[M+H] + ;C 113 H 217 N 20 O 37 [M+H] + The calculated m / z value is 2447.6.

[0302] 1.23 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]8[(α-NH2.HCl)(ε-NH-COPEG1100)]8G3 Compound 53 Under N2 atmosphere, HO-Lys(α-NHBoc)(ε-NH-COPEG 1100 (MeTzPh)-PEG4CO-NHPEG was added to a stirred solution of (MeTzPh) (0.541 g, 0.402 mmol) and PyBOP (0.195 g, 0.375 mmol) / DMF (2 mL). After 10 minutes, the solution was prepared by adding (MeTzPh)-PEG4CO-NHPEG. 24-CO[N(PN)2][Lys]4[NH2.HCl]8 compound 52 (0.109 g, 0.0398 mmol) / DMF (1 mL) was added. The resulting reaction mixture was then stirred at room temperature overnight. Volatile components were removed by vacuum, and the resulting crude product was cooled in an ice / water bath. 1.25 M (8.0 mL, 10 mmol) was slowly added to the cooled residue. After 10 minutes, the cold water bath was removed, and the reaction mixture was stirred at room temperature overnight. Volatile components were removed by vacuum, and the mixture was dissolved in H2O (16 mL). This solution was centrifuged using Millipore Amicon Ultra-15 Centrifugal Filter Units (4 units x 10 kDa MWCO units). * The compound was purified by [method]. The holding solution was freeze-dried overnight to obtain the product, compound 53 (0.327 g, 65%), as a red solid. 1 HNMR(300MHz,CD3OD)δ(ppm):1.40-1.87(m,88H);2.49-2.56(m,18H);2.68-2.72(m,2H);3.08(s,3H);3.17-3.30(m30H);3.37-3.51 (m,10H);3.41(s,24H);3.59-4.01(m,816H);4.25-4.40(m,8H);7.29-7.33(m,2H);8.44-8.49(m,2H);LCMS (hydrophilic method, TFA buffer) RT=10.28 min

[0303] *[The unit was pre-rinsed with H2O (5 mL) and rotated at 4000 rpm for 5 minutes. This process was repeated. The crude solution was filtered through a 0.45 μm syringe filter and placed in a centrifuge. The unit was then rotated at 4000 rpm for 15 minutes. The retained solution was then diluted with H2O (4 mL) and rotated again at 4000 rpm for 15 minutes. This process was repeated 8 times.]

[0304] 1.24 MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]8[NH2.HCl] 16 G3 compound 54 Under an N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24-CO[N(PN)2][Lys]4[NH2.HCl]8 compound 52 (0.098 g, 0.0358 mmol) and DBL-oPNP (0.207 g, 0.443 mmol) / DMF (2.0 mL) were stirred together, and NMM (190 μL, 1.973 mmol) was added. The resulting reaction mixture was then stirred at room temperature overnight. Volatile components were removed by vacuum, and the resulting oily residue was purified by silica chromatography (5%-10%-15% MeOH / DCM) to obtain a Boc-protected product as red oil (0.128 g, 70%). After cooling the purified material in an ice / water bath, a solution of 1.25 M HCl / MeOH (6.5 mL, 8.5 mmol) was slowly added. After 5 minutes, the ice bath was removed, and the resulting reaction mixture was then stirred at room temperature overnight. Volatile substances were removed under vacuum, and the product, compound 54, was obtained as a red oil (0.107 g, 100%). LC-MS (hydrophilic method, TFA buffer) RT=7.48 min, ESI MS (+ve) 3471[M+H] + ;[M+H] + The calculated value in m / z is 3472.

[0305] 1.25 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 16 [(α-NH2.HCl)(ε-NH-CO PEG 1100 )] 16 G4 compound 55 Under N2 atmosphere, HO-Lys(α-NHBoc)(ε-NH-COPEG 1100 (MeTzPh)-PEG4-PEG (0.107 g, 0.132 mmol) and PyBOP (0.065 g, 0.125 mmol) / DMF (1.5 mL) were stirred together, and NMM (50 μL, 0.455 mmol) was added. After 10 minutes, the solution was mixed with (MeTzPh)-PEG4-PEG. 24 -CO[N(PN)2][Lys]8[NH2.HCl] 16Compound 54 (0.0221 g, 0.00545 mmol) was added. The resulting reaction mixture was then stirred overnight at room temperature. Volatile components were removed by vacuum, and the resulting crude product was cooled in an ice / water bath. 1.25 M HCl / MeOH (1.5 mL, 1.88 mmol) was slowly added to the cooled residue. After 10 minutes, the cold water bath was removed, and the reaction mixture was stirred overnight at room temperature. Volatile components were removed by vacuum, and the mixture was dissolved in H2O (5 mL). This solution was purified by centrifugation using a Millipore Amicon Ultra-15 Centrifugal Filter Unit (10 kDa MWCO). The retaining solution was freeze-dried overnight and further purified using Gilson filtration (single gradient 60 min, 10>60% ACN (0.1% TFA buffer, RT 36 min)) to obtain the product, compound 55 (0.021 g, 25%), as a red solid. 1 HNMR(300MHz,CD3OD)δ(ppm): 1.28-1.87(m,184H);2.42-249(m,35H);3.00(s,3H);3.12-3.23(m60H);3.34-3.40(m,63H);3.52-4.0 1(m,826H);4.25-4.39(m,16H);7.17-7.20(m,2H);8.48-8.51(m,2H);LCMS (hydrophilic method, TFA buffer) RT=8.96 min.ESI MS(+ve)2095[M+7H] + ;1834[M+8H] + ;1630[M+9H] + .

[0306] 1.26 MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 16 [NH2.TFA] 32 G4 Compound 56 Compound 56 was prepared in the same manner as compound 54, deprotected with TFA / AcOH, and obtained as a red solid (0.078 g, 78%). LC-MS (hydrophilic method, TFA buffer) RT=7.74 min. ESI MS (+ve) 2095 [M+7H] + ;1834[M+8H] + ;1630[M+9H] + .

[0307] 1.27 MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 32 [(α-NH2.TFA)(ε-NH-COPEG 1100 )] 32 G5 Compound 57 Under N2 atmosphere, HO-Lys(α-NHBoc)(ε-NH-COPEG 1100 (MeTzPh)-PEG4CO-NHPEG (0.476 g, 0.354 mmol) and PyBOP (0.170 g, 0.327 mmol) / DMF (3.0 mL) were stirred together, and NMM (180 μL, 0.500 mmol) was added. After 10 minutes, the solution was prepared by adding (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 16 [NH2.TFA] 32 Compound 56 (0.078 g, 0.00850 mmol) was added. The resulting reaction mixture was then stirred at room temperature overnight. Volatile components were removed by vacuum, and the resulting crude product was dissolved in H2O (1.0 mL), to which TFA (1.0 mL) was slowly added. This reaction was stirred at room temperature overnight. The reaction mixture was diluted with H2O (12 mL) and purified by centrifugation using a Millipore Amicon Ultra-15 Centrifugal Filter Unit (10 kDa MWCO). The retaining solution was freeze-dried overnight to obtain compound 57 (0.374 g, 91%) as a pink solid. 1 HNMR(300MHz,D2O)δ(ppm): 1.41-1.87(m,376H);2.52-2.56(m,64H);3.09(s,3H);3.17-3.24(m,126H);3.41(s,93H)3.48-3.98(m,29 60H); 4.27-4.39 (m, 32H); 7.30-7.33 (m, 2H); 8.46-8.49 (m, 2H); HPLC analysis (hydrophilic method, ammonium formate as buffer) RT = 8.60 min.

[0308] 1.28 (MeTzPh)PEG4CO-NHPEG 24CO-[N(PN)2][Lys]8[((α-NHCy5)1(α-NHAc)7)(ε-NH-COPEG1100)8], G3, Compound 37 A solution of Cy5-NHS ester (1 mg / mL DMF solution, 1.0 mL; 1.0 mg, 1.59 μmol) is used in (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[(α-NH2.HCl)8)(ε-NH-COPEG 1100 Compound 53 (20 mg, 1.59 μmol) / DMF (0.5 mL) was added to a vial. NMM (10 μL, 91.0 μmol) was added to this solution, and the resulting reaction mixture was then protected from light and stirred under RT. After 3.5 hours, acetic anhydride (20 μL, 212 μmol) was added, and the reaction mixture was stirred and left to stand overnight. The reaction mixture was concentrated under reduced pressure, then taken up with MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. When the sample entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 19.3 mg (93%) of the title product as a bright blue powder. HPLC (C8X Bridge, 3x100 mm) gradient. 5% ACN / H2O (0~1 min), 5~80% ACN (1~7 min), 80% ACN (7~12 min), 80~5% ACN (12~13 min), 5% ACN (13~15 min), 214 nm, 0.4 mL / min, Rt (min) = 8.30.

[0309] 1.29 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 16 [((α-NHCy5)1(α-NHAc) 15 )(ε-NH-COPEG 1100 ) 16 ],G4,Compound 38 A solution of Cy5-NHS ester (520 μL of 1 mg / m L DMF solution; 0.52 mg, 844 nmol) is used to (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 16 [((α-NH2.HCl) 16)(ε-NH-COPEG 1100 ) 16 The compound 55 (20 mg, 844 nmol) / DMF (1.0 mL) was added to a vial. NMM (10 μL, 94.5 μmol) was added to this solution, and the resulting reaction mixture was then protected from light and stirred under RT. After 3.5 hours, acetic anhydride (20 μL, 230 μmol) was added, and the reaction mixture was stirred and left to stand overnight. The reaction mixture was concentrated under reduced pressure, then taken up with MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. When the sample entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 19.9 mg (98%) of the title product as a bright blue powder. HPLC (C8X Bridge, 3 x 100 mm) gradient. 5% ACN / H2O (0~1 min), 5~80% ACN (1~7 min), 80% ACN (7~12 min), 80~5% ACN (12~13 min), 5% ACN (13~15 min), 214 nm, 0.4 mL / min, Rt (min) = 8.40

[0310] 1.30 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 32 [((α-NHCy5)1(α-NHAc) 31 )(ε-NH-COPEG 1100 ) 32 ], G5, compound 39 A solution of Cy5-NHS ester (300 μL of 1 mg / m LDMF solution; 0.30 mg, 487 nmol) is used to prepare (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 32 [((α-NH2.HCl) 32 )(ε-NH-COPEG 1100 ) 32Compound 57 (20 mg, 435 nmol) / DMF (1.2 mL) was added to a vial. NMM (11 μL, 97.5 μmol) was added to this solution, and the resulting reaction mixture was then protected from light and stirred under RT. After 3.5 hours, acetic anhydride (22 μL, 237 μmol) was added, and the reaction mixture was stirred and left to stand overnight. The reaction mixture was concentrated under reduced pressure, then taken up with MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. Upon entering the column bed, the sample was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 18.7 mg (91%) of the title product as a bright blue powder. HPLC (C8X Bridge, 3 x 100 mm) gradient. 5% ACN / H2O (0~1 min), 5~80% ACN (1~7 min), 80% ACN (7~12 min), 80~5% ACN (12~13 min), 5% ACN (13~15 min), 214 nm, 0.4 mL / min, Rt (min) = 8.50.

[0311] 1.31 Cabazitaxel (CTX)-2'-OCOO-oPNP ester, compound 58 To a solution of cabazitaxel (CTX, 111.0 mg, 0.132 mmol) in DMF (3 mL), triethylamine (37.0 μL, 0.264 mmol), DMAP (1.64 mg, 0.013 mmol), and p-nitrophenyl chloroformate (24.0 mg, 0.12 mmol) were added. The mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and the resulting residue was purified using column chromatography on silica gel with a gradient of 0:100 to 50:50 (ethyl acetate:hexane) to obtain the title product as a white solid (28 mg, 21%). LC-MS (hydrophilic method, TFA buffer) Rt = 6.55 min. ESI MS (+ve) 100 1[M] + ;C 52 H 60 N2O 18 [M]+ m / z calculated value: 1001

[0312] 1.32 Fmoc-Val-Ala-PAB-P-Trigger-(NMeBoc), compound 59 This compound can be obtained by the method described by Dal Corso et al., Angew. Chem Int. ed. 2020, 59, 4176-4181. Alternatively, it may be prepared as follows: Fmoc-Val-Ala-PAB-O-oPNP ester (Iris Biotech, 127.0 mg, 0.186 mmol) and (S)-tert-butylmethyl (pyrrolidine-2-ylmethyl) carbamate (Ascension Chemical, 42.0 mg, 0.195 mmol) were placed in a round-bottom flask, followed by the addition of THF (5 mL). The reaction mixture was stirred at room temperature for 2 hours under an inert atmosphere. LC-MS analysis of the reaction mixture showed the formation of the title compound. The solvent was removed under reduced pressure, and the resulting product was used in the next reaction without purification. LC-MS (hydrophilic method, TFA buffer), Rt = 6.60 min. ESI MS (+ve) 756[M]+1;C 42 H 53 Calculated m / z value for N5O8[M]+1: 756]

[0313] 1.33 TFA.NH2-Val-Ala-PAB-P-Trigger-(NMeBoc), Compound 60 To a stirred solution of compound 59 (141.0 mg, 0.186 mmol) in DMF (3 mL), piperidine (0.6 mL, 6.07 mmol) was added at 0°C, and the reaction mixture was stirred for 1 hour. The solvent was removed under reduced pressure, and the resulting residue was dissolved in acetonitrile. Purification was performed using preparative HPLC (40-90% MeCN, 0.05% TFA buffer, RT approximately 45 minutes), and the title compound was obtained as a colorless liquid after lyophilization (63.0 mg, 52%). LC-MS (hydrophilic method, TFA buffer) ESI MS (+ve) 556[M]+Na;C 27 H 43 Calculated m / z value of N5O6[M]+Na: 556; 1H-NMR(300MHz,MeOD)δ(ppm):7.62-7.51(m,2H),7.44-7.25(m,2H),5.18-4.98(bs,2H),4.56(q,J=9.0Hz,1H),4.13(bs,1H),3.72(d,J= 6.0Hz,1H),3.51-3.36(m,2H),2.89(s,1H),2.73(bs,1H),2.35-2.13(m,1H),2.05-1.70(m,4H),1.59-1.36(m,11H),1.18-1.00(m,6H).

[0314] 1.34 COOH-PEG9-Val-Ala-PAB-P-Trigger-(NMeBoc), Compound 61 To a stirred solution of compound 60 (52.0 mg, 0.081 mmol) in acetonitrile (2 mL), DIPEA (87.2 μL, 0.50 mmol) was added, and the reaction mixture was cooled to 0°C. COOH-PEG9-NHS ester (Iris Biotech; 86.0 mg, 0.139 mmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 16 hours. LC-MS analysis of the reaction mixture showed product formation. LC-MS (hydrophilic method, TFA buffer; 5-60% acetonitrile for 15 minutes or more), Rt = 11.32 mins. ESI MS (+ve) 1048[M] + H2O; C 49 H 83 N5O 18 [M] + H2O m / z calculation value: 1048]. Remove the solvent under reduced pressure, and use the obtained product in the next reaction without purification.

[0315] 1.35 COOH-PEG9-Val-Ala--PAB-P-Trigger-(NMe.TFA), Compound 62 To a stirred solution of compound 61 (84.0 mg, 0.081 mmol) in dichloromethane (2 mL), TFA (0.5 mL, 6 mmol) was added at 0°C, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the resulting residue was dissolved in acetonitrile. The mixture was purified using preparative HPLC (5-70% acetonitrile, 0.05% TFA buffer, RT 32-33 min), and after lyophilization, the title compound was obtained as a concentrated colorless liquid (87.0 mg, 100%). LC-MS (hydrophilic method, FA buffer) ESI MS(+ve)930[M]+1;C 45 H 77 N5O 15 [M]+1 m / z calculation value: 930; 1 H-NMR(300MHz,MeOD)δ(ppm): 7.64(d,J=9.0Hz,2H),7.37(d,J=9.0Hz,2H),5.13(d,J=9.0Hz,1H),4.61-4.40(m ,2H),4.19(d,J=6.0Hz,1H),3.85(t,J=6.0Hz,1H),3.79-3.69(m,4H),3.69-3.51 (m,35H),3.17-3.05(m,2H),2.85(s,1H),2.76-2.68(m,3H),2.92(t,J=6.0Hz,1H ),2.62-2.45(m,4H),2.27-1.84(m,4H),1.46(d,J=9.0Hz,2H),1.08-0.92(m,6H).

[0316] 1.36 COOH-PEG9-Val-Ala-PAB-P-Trigger-NMeCO-CTX, Compound 63 Compound 46 (28.0 mg, 0.027 mmol) / DMF (3 mL) solution was mixed with DIPEA (9.40 μL, 0.54 mmol) and Compound 51 (27.3 mg, 0.027 mmol). The mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure, and the resulting residue was dissolved in acetonitrile (2 mL). This solution was purified by preparative HPLC (5-60% ACN, Rt 32.2-34.3 min), and after lyophilization, the title product was obtained as a white solid (26 mg, 56%). LC-MS (hydrophilic method, TFA buffer) Rt = 5.57 min. ESI MS (+ve) 1792[M]+;C 90 H 130 N6O31 [M]+ m / z calculated value: 1792

[0317] Example 1b Synthesis of BCN and DBCO linker 1.37 BCN-PEG2-Glu-CO-NHPEG 24 CO2H, compound 31 NH2-PEG 24 Sodium bicarbonate (15.2 mg, 0.181 mmol) was added to a solution of water / THF (1:1, 4 mL) containing -COOH (93.7 mg, 0.082 mmol). The mixture was stirred at room temperature for 5 minutes, and then a BCN-PEG2-NHS ester (50 mg, 0.093 mmol) / THF (2 mL) solution was added. The resulting reaction mixture was stirred in RT for 15 hours. After that, volatile components were removed under reduced pressure, and ACN (2.5 mL) was added to the resulting aqueous suspension. This solution was purified by preparative HPLC (5-60% ACN, Rf 32.2-34.3 min), and after lyophilization, the title product was obtained as a white solid (42 mg, 33%). 1 H-NMR (300MHz, D2O) δ (ppm). 0.85-0.92(m,2H);1.25-1.35(m,1H);1.43-1.57(m,2H);1.73-1.83(m,2H);2.09-2.25(m, 9H);2.39(t,J=9.0Hz,2H);3.21-3.31(m,6H);3.35-3.85(m,106H);4.10(d,J=9.0Hz,2H). LCMS (hydrophilic method, formic acid buffer) Rt=10.28 min; ESI MS(+ve)m / z 1566.7[M]+.

[0318] 1.38 BCN-PEG2-Glu-CO-NHPEG 24 CO-NHPEG3-TCO, compound 32 BCN-PEG2-Glu-CO-NHPEG 24To a stirred solution of -COOH compound 31 (10.0 mg, 0.006 mmol) / DMF (3.0 mL), PyBOP (3.64 mg, 0.007 mmol), NMM (1.31 μL, 0.012 mmol), and then TCO-PEG3-NH2 (2.41 mg, 0.007 mmol) were added. The resulting reaction mixture was then stirred overnight at room temperature. The solvent was then removed under reduced pressure, and the resulting residue was dissolved in ACN (2 mL) and filtered through a 0.45 μm filter. The recovered filtrate was purified by preparative HPLC (30-50% ACN Rt 40-42 min), and the fraction containing the product was concentrated under reduced pressure to remove the ACN. The remaining aqueous solution was freeze-dried overnight to obtain the title product as a white solid (4.7 mg, 39%). 1 H-NMR(300MHz,CD3OD)δ(ppm): 0.89-1.05(m,2H),1.26-1.48(m,2H),1.52-1.79(m,5H),1.83-2.06(m,6H),2.11-2.39(m,12H),2.48(t,2H,J=6.0Hz),3.35-3. 44(m,6H),3.47-3.79(m,102H),3.83-3.92(m,1H),4.16(d,2H,J=6.0Hz),4.26-4.41(m,1H),4.58(bs,10H),5.39-5.74(m,3H). LCMS (hydrophilic method, formic acid buffer) Rt=11.0 min ESI MS(+ve)1894.0[M]+;C 90 H 165 N5O 36 [M]+ m / z calculation value: 1894.2

[0319] 1.39 DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO, compound 33 DBCO-Glu-NHPEG 24To a stirred solution of COOPFP (50.0 mg, 0.031 mmol) / DMF (3 mL), TCO-PEG3-NH2 (10.7 mg, 0.031 mmol) was added, followed by NMM (4.08 μL, 0.037 mmol). The resulting reaction mixture was then stirred at room temperature for 3 hours and then concentrated under reduced pressure. The resulting residue was dissolved in ACN (2 mL), filtered through a 0.45 μm filter, and the filtrate was purified by preparative HPLC (40-70% ACN, Rt 27-29 min). The fraction containing the product was concentrated under reduced pressure to remove ACN, and the remaining aqueous solution was freeze-dried overnight to obtain the title product as a colorless viscous liquid (25.0 mg, 42%). 1 H-NMR(300MHz,CD3OD)δ(ppm): 1.31-1.62(m,4H),1.60-1.83(m,6H),1.92-2.16(m,4H),2.25(t,2H,J=6.0Hz),2.91-3.03(m,4H),3.12-3. 58(m,78H),3.59-3.69(m,1H),4.01-4.18(m,1H),4.92(d,2H,J=15H),5.15-5.49(m,3H),6.95-7.59(m,8H). LCMS (hydrophilic method, formic acid buffer) Rt=7.0 min.ESI MS(+ve)1775.0.0[M]+;C 88 H1 48 N4O 32 [M]+ m / z calculated value: 1775.12

[0320] 1.40 MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys]8[(α-Cy5)1(α-NHAc)7(ε-NHPEG 1100 )8], G3, compound 37 A solution of Cy5-NHS ester (1.0 mL of 1 mg / m LDMF solution; 1.0 mg, 1.59 μmol) is added to MeTzPh-PEG4PEG. 24 -CO[N(PN)2][Lys]8(α-NH2)8(ε-NHPEG 1100)8] was added to a vial containing (20 mg, 1.59 μmol) / DMF (0.5 mL). NMM (10 μL, 91.0 μmol) was added to this solution, and the resulting reaction mixture was then protected from light and stirred under RT. After 3.5 hours, acetic anhydride (20 μL, 212 μmol) was added, and the reaction mixture was stirred and left to stand overnight. The reaction mixture was concentrated under reduced pressure, then taken up with MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. When the sample entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 19.3 mg (93%) of the title product as a bright blue powder. HPLC (C8X Bridge, 3 x 100 mm) gradient. 5% ACN / H2O (0~1 min), 5~80% ACN (1~7 min), 80% ACN (7~12 min), 80~5% ACN (12~13 min), 5% ACN (13~15 min), 214 nm, 0.4 mL / min, Rt (min) = 8.30.

[0321] 1.41 MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 16 [(α-Cy5)1(α-NHAc) 15 (ε-NHPEG 1100 ) 16 ], G4, compound 38 A solution of Cy5-NHS ester (520 μL of 1 mg / m LDMF solution; 0.52 mg, 844 nmol) is used in MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 16 [(α-NH2) 16 (ε-NHPEG 1100 ) 16The solution was placed in a vial containing (20 mg, 844 nmol) / DMF (1.0 mL). NMM (10 μL, 94.5 μmol) was added to this solution, and the resulting reaction mixture was then protected from light and stirred under RT. After 3.5 hours, acetic anhydride (20 μL, 230 μmol) was added, and the reaction mixture was stirred and left to stand overnight. After concentrating the reaction mixture under reduced pressure, it was taken into MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. Upon entering the column bed, the sample was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 19.9 mg (98%) of the title product as a vivid blue powder. HPLC (C8X Bridge, 3 x 100 mm) gradient. 5% ACN / H2O (0~1 min), 5~80% ACN (1~7 min), 80% ACN (7~12 min), 80~5% ACN (12~13 min), 5% ACN (13~15 min), 214 nm, 0.4 mL / min, Rt (min) = 8.40.

[0322] 1.42 MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 32 [(α-Cy5)1(α-NHAc) 31 (ε-NHPEG 1100 ) 32 ], G5, compound 39 A solution of Cy5-NHS ester (300 μL of 1 mg / m LDMF solution; 0.30 mg, 487 nmol) is used in MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 32 [(α-NH2) 32 (ε-NHPEG 1100 ) 32The mixture was placed in a vial containing (20 mg, 435 nmol) / DMF (1.2 mL). NMM (11 μL, 97.5 μmol) was added to this solution, and the resulting reaction mixture was then protected from light and stirred under RT. After 3.5 hours, acetic anhydride (22 μL, 237 μmol) was added, and the reaction mixture was stirred and left to stand overnight. After concentrating the reaction mixture under reduced pressure, it was taken into MQ water (5 mL) and divided into two parts, which were purified using two (pre-equilibriumized) PD10 columns. Upon entering the column bed, the sample was eluted with 3.5 mL of MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying yielded 18.7 mg (91%) of the title product as a bright blue powder. HPLC (C8X Bridge, 3 x 100 mm) gradient. 5% ACN / H2O (0~1 min), 5~80% ACN (1~7 min), 80% ACN (7~12 min), 80~5% ACN (12~13 min), 5% ACN (13~15 min), 214 nm, 0.4 mL / min, Rt (min) = 8.50.

[0323] 1.43 BHA[Lys]4[((α-NH-COPEG 24 NH-COPEG4(PhTzMe)) 1-3 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 0-2 )(ε-NH-COPEG 1000 )4],G2, Compound 99 BHA[Lys]4[(α-NH-COPEG 24 NH-COPEG4(PhTzMe)) 1-3 (α-NH2) 1-3 )(ε-NH-COPEG 1000 The compounds were prepared according to general procedure C using compound 107 (3.0 mg, 0.414 μmol) and compound 108 (0.56 mg, 0.414 μmol) of HO-Lys[(α-Cy5)(ε-DFO)], and purified by spin column (10 kDa MW cutoff, 10 x 450 μL MQ water washing) to obtain the target compound 99 (final concentration of 3.56 mg in 300 μL MQ water).

[0324] 1.44 BHA[Lys]8[((α-NH-COPEG24 NH-COPEG4(PhTzMe)) 1-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 3-6 )(ε-NH-COPEG 412 )8],G3,Compound 100 BHA[Lys]8[((α-NHCOPEG 24 NH-COPEG4(PhTzMe)) 1-4 (α-NH2) 4-7 ))(ε-NH-COPEG 412 Compound 109 (2.97 mg, 0.452 μmol) and HO-Lys[(α-NHCy5)(ε-NHDFO)] compound 40 (0.61 mg, 0.452 μmol) were prepared according to general procedure C and purified by spin column (10 kDa MW cutoff, 10 x 450 μL MQ water washing) to obtain the product compound 100 (final concentration of 3.60 mg in 300 μL MQ water).

[0325] 1.45 BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)6)(ε-NH-COPEG 1000 )8],G3, Compound 101 BHA[Lys]8[((α-NHCOPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NHCOPEG 1000 Compound 110 (3.14 mg, 0.234 μmol) and HO-Lys(α-NHCy5)(ε-NHDFO) compound 108 (0.32 mg, 0.234 μmol) were prepared according to general procedure C, purified by spin column (washing 10 kDa MW cutoff with 10 x 450 μL of MQ water), and purified again by spin column (washing 10 kDa MW cutoff with 10 x 450 μL of MQ water) to obtain the product, compound 27 (300 μL of MQ water, final concentration 3.45 mg).

[0326] 1.46 BHA[Lys] 16 [((α-NH-COPEG24 NH-COPEG4(PhTzMe))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 14 )(ε-NH-COPEG 1000 )16],G4, compound 102 BHA[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)1(α-NH2) 15 )(ε-NH-COPEG 1000 ) 16 Compound 28 was prepared according to general procedure C using G4, compound 111 (11.8 mg, 0.454 μmol), and HO-Lys(α-NHCy5)(ε-NHDFO) compound 108 (0.62 mg, 0.454 μmol), and purified by spin column (washed with 10 x 450 μL MQ water at a 10 kDa MW cutoff) to obtain 9.3 mg of compound 28 as a blue solid (after lyophilization).

[0327] 1.47 BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)) 1-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 27-30 )(ε-NH-COPEG 1000 ) 32 ],G5,Compound 103 BHA[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)) 1-4 (α-NH2) 27-30 )(ε-NH-COPEG 1000 ) 32 Using [ ], G4, compound 112 (12.9 mg, 0.271 μmol), and HO-Lys(α-Cy5)(ε-DFO) compound 108 (0.37 mg, 0.271 μmol) were prepared according to general procedure C, purified by spin column (washed with 10 x 450 μL MQ water at a 10 kDa MW cutoff), and after lyophilization, 8.4 mg of the product, compound 103, was obtained as a blue solid.

[0328] 1.48 BHA[Lys]4[((α-Lys(α-NHCy5)(α-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4],G2, compound 105 BHA[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24 To a stirred solution of NH-COPEG4(PhTzMe)4], G2, compound 113 (11.0 mg, 0.0015 mmol) / DMF (3 mL), NMM (3.32 μL, 0.039 mmol), HO-Lys[(α-NHCy5)(ε-NHDFO)] compound 108 (2.06 mg, 0.0015 mmol) and PyBOP (1.18 mg, 0.0022 mmol) were added by RT. After 18 hours, volatile matter was removed under reduced pressure, the blue solid residue was dissolved in MQ water, and the solution was filtered (0.45 μm Acrodisc syringe filter). The filtrate was concentrated by spin column (Amicon Ultra, 0.5 mL, 3 kDaMW cutoff), and the retained solution was repeatedly washed with MQ water (10 x 450 μL) to obtain compound 24 (concentration 10 mg / mL in MQ water; 1.3 mL). LCMS (hydrophilic method, TFA buffer), gradient 20-90, acetonitrile, 8 min; Rt = 5.64 min; 1 HNMR (300MHz,D2O)δ(ppm): 8.52(d,8H,J=9.0Hz),8.32(s,1H),8.29-8.17(m,2H),7.78-7.70(m,2H),7.69-7.62(m,2H),7.57-7.47(m,2H) ,7.48-7.25(m,16H),7.21(d,8H,J=9.9Hz),7.15-7.08(m,1H),7.06-7.00(m,1H),6.70-6.60(m,2H),6.21-6.12 (m,2H),4.37-4.20(m,14H),4.18-4.06(m,8H),3.98-3.50(m,480H),3.48-3.34(m,15H),3.27-3.06(m,18H),3 .06-2.97(m,19H),2.88(s,8H),2.54-2.40(m,19H),2.08-1.98(m,28H),1.93-1.12(m,99H),1.00-0.82(m,8H).

[0329] 1.49 BHA[Lys]4[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)3)(ε-NH-COPEG1 000 )4],G2, compound 107 BHA[Lys]4[(α-NH2.TFA)4(ε-NH-COPEG 1000 )4](50.0mg,0.007mmol) and HOOCPEG 24 Using NH-COPEG4(PhTzMe) (Click Chemistry Tools, 13.39 mg, 0.010 mmol), methanol was used as the eluent, except that the reaction vessel was wrapped in foil to block out light. The residue was purified using SEC (Sephadex® LH-20) to obtain the product, compound 107 (44.00 mg, 77%). 1 HNMR(300MHz,D2O)δ(ppm). 8.42-8.26(m,2H),7.44-7.11(m,12H),6.04(bs,1H),4.44-4.07(m,8H),4.07-3.37(m,556H),3.33(s,12H) ),3.25-2.90(m,17H),2.62-2.43(m,2H),1.96-0.97(m,48H);HPLC(C8XBridge,3x100mm) gradient (formate buffer): 5%ACN / H2O(0-1min), 5-80%ACN(1-7min), 80%ACN(7-12min), 80-5%ACN(12-13min), 5%ACN(13-15min), 214nm, 0.4mL / min, Rt(min)=8.08-9.01.

[0330] 1.50 BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NH-COPEG 412 )8],G3, compound 109 BHA[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 412 )8](50.0 mg, 0.008 ammol) and HOOC-PEG 24Using NH-COPEG4(PhTzMe) (Click Chemistry Tools; 11.2 mg, 0.008 mmol), the reaction vessel was wrapped in foil to protect it from light, and the mixture was prepared according to general procedure C. The residue was purified using SEC (Sephadex® LH-20) with methanol as the eluent to obtain the product, compound 109 (29 mg, 55%). 1 HNMR(300MHz,D2O)δ(ppm):8.43-8.20(m,2H),7.50-7.09(m,10H),6.03(bs,1H),4.41-4. 05(m,10H),4.01-3.41(m,258H),3.31(s,16H),3.24-2.83(m,28H),2.41(bs,13H),2.00-0 .98(m,75H); HPLC (C8XBridge, 3x100mm) Gradient (Formic Acid Buffer): 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rt=8.16-8.93 min.

[0331] 1.51 BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NH-COPEG 1000 )8], G3, compound 110 BHA[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1000 )8](100.0mg,0.007mmol) and HOOCPEG 24 Using NH-COPEG4(PhTzMe) (Click Chemistry Tools; 15.97 mg, 0.010 mmol), the reaction vessel was wrapped in foil to protect it from light, and the mixture was prepared according to general procedure C. Methanol was used as the eluent, and the residue was purified with SEC (Sephadex® LH-20) to obtain the product, compound 19 (SPL-9248) (69 mg, 66%). 1HNMR(300MHz,D2O)δ(ppm)8.45-8.25(m,2H),7.47-7.07(m,12H),6.07(bs,1H),4.45-4.05(m,12H),4.04-3.37(m,936H) ),3.32(s,25H),3.23-2.88(m,32H),2.59-2.32(m,6H),1.90-0.98(m,90H);HPLC(C8XBridge,3x100mm) gradient (formate buffer): 5% ACN / H2O(0-1 min),5-80% ACN (1-7 min),80% ACN (7-12 min),80-5% ACN(12-13 min),5% ACN(13-15 min),214 nm,0.4mL / min, Rt = 8.21-9.15 minutes.

[0332] 1.52 BHA[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)1(α-NH2) 15 )(ε-NH-COPEG 1000 ) 16 ],G4,Compound 111 BHALys[Lys]2[Lys]4[Lys]8[Lys] 16 [(α-NH2.TFA) 16 (ε(-NH-COPEG 1000 ) 16 (Reference 1) (100.0 mg, 0.004 mmol) and HOOCPEG 24 Using NH-COPEG4(PhTzMe) (Click Chemistry Tools; 6.74 mg, 0.005 mmol), preparation was carried out according to general procedure C (except for wrapping the reaction vessel in foil to protect it from light), methanol was used as the eluent, and the residue was purified with SEC (Sephadex® LH-20) to obtain the product, compound 111 (63 mg, 65%). 1HNMR(300MHz,D2O) δ(ppm)8.45-8.28(m,2H),7.47-7.07(m,12H),6.03(bs,1H),4.40-4.08(m,23H),4.06-3.38(m,1906H),3.33(s ,56H),3.29-2.91(m,78H),2.63-2.45(m,3H),1.98-0.98(m,200H);HPLC(C8XBridge,3x100mm) gradient (formate buffer): 5% ACN / H2O (0-1 min),5-80% ACN (1-7 min),80% ACN (7-12 min),80-5% ACN (12-13 min),5% ACN (13-15 minutes), 243nm, 0.4mL / min, R t =8.51-9.02 minutes.

[0333] 1.53 BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe)) 1-4 (α-NH2) 28-31 )(ε-NH-COPEG 1000 ) 32 ],G5, compound 112 BHA[Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1000 ) 32 ](100.0 mg, 0.002 mmol) and HOOCPEG 24 Using NH-COPEG4(PhTzMe) (Click Chemistry Tools; 3.38 mg, 0.005 mmol), the reaction vessel was wrapped in foil to protect it from light, and the mixture was prepared according to general procedure C. The residue was purified using SEC (Sephadex® LH-20) with methanol as the eluent to obtain the product, compound 21 (68 mg, 72%). 1HNMR(300MHz,D2O)δ(ppm):8.44-8.29(m,2H),7.44-7.09(m,12H),6.02(bs,1H),4.37-4.11(m,31H),4.08-3.37(m,2937H) ),3.32(s,83H),3.27-2.88(m,116H),2.59-2.42(m,3H),2.18-0.92(m,313H);HPLC(C8XBridge,3x100mm) gradient (formate buffer): 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rt=8.77 min.

[0334] 1.54 BHA[Lys]4[(α-NHBoc)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4],G2, compound 113 BHA[Lys]4[(α-NHBoc)4(ε-NH2)4](46.0 mg, 0.031 mmol) / DMF stirred solution, NMM (68.0 μL, 0.620 mmol), HOOCPEG, RT. 24 NH-COPEG4 (PhTzMe) (Click Chemistry Tools; 43.0 mg, 0.155 mmol) and PyBOP (81.0 mg, 0.155 mmol) were added. After 16 hours, the reaction mixture was dissolved in ACN:MQ water (3 mL, 1:1 v / v), and the solution was filtered (0.45 μm Acrodisc syringe filter). The filtrate was purified by preparative HPLC. 30-80% ACN, 60 min, mobile phase: MQ water and acetonitrile, R t Compound 22 was obtained as a pink solid of 52 mg (22%) at 33.0-36.0 minutes. LC-MS (hydrophilic method, TFA buffer) Rt = 5.66 minutes; 1HNMR(300MHz,D2O) δ(ppm): 8.52(d,8H,J=9.0Hz),7.40-7.26(m,10H),7.20(d,8H,J=9.0Hz),6.23-6.17(m,1H),4.36-4.21(m,10H),3.99-3.83(m,1 3H),3.82-3.46(m,487H),3.46-3.32(m,22H),3.27-3.02(m,15H),3.02(s,12H),2.54-2.38(m,17H),1.92-1.14(m,89H).

[0335] 1.55 BHA[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 114 BHA[Lys]4[(α-NHBoc)4(ε-NH-COPEG 24 To a stirred solution of NH-COPEG4(PhTzMe)4), G2, compound 113 (48.0 mg) / methanol (2 mL), a 3 M hydrochloric acid / methanol (2 mL) solution was added, and the reaction mixture was stirred at room temperature for 20 hours. Volatile substances were removed under reduced pressure, and BHALys[Lys]2[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24 Compound 23, [NH-COPEG4(PhTzMe)4],G2, was obtained as a pink solid of 41.0 mg (91%). LC-MS (hydrophilic method, TFA buffer), gradient: 20-90 minutes, acetonitrile, 8 minutes; R t =5.27 minutes.

[0336] 1.56 HO-Lys[(α-NHCy5)(ε-NHDFO)], Compound 108 To a stirred solution of compound 115 (20.0 mg, 0.032 mmol) in DMSO (5 mL), DIPEA (32 μL, 0.224 mmol) was added, followed by p-SCN-deferoxamine (Macrocyclics, 24.0 mg, 0.032 mmol). The reaction mixture was stirred at room temperature for 4 hours, and then concentrated by blowing a nitrogen gas stream over the solution for several hours. The resulting residue was then purified by preparative HPLC. Compound 108, the product, was obtained as a blue solid of 15 mg (34%) by 30-60% ACN / MQ water + 0.1% TFA (60 min, Rt 39-42 min). LC-MS (hydrophilic method, TFA buffer) R t =5.93 minutes;ESI MS(+ve)1364[M]+;C 71 H 103 N 12 O 11 Calculated m / z value of S2[M]+ = 1364. 1HNMR (300MHz, D2O) δ (ppm). 8.25(t,2H,J=12.0Hz),7.56-7.18(m,9H),6.65(t,1H,J=12.0Hz),6.37-6.15(m,2H),4.43-4.32(m,1H),4.11(t,2H,J=6.0Hz),3. 70-3.46(m,8H),3.22-3.10(m,3H),2.86-2.69(m,3H),2.56-2.38(m,3H),2.31(t,2H,J=6.0Hz),2.11(s,2H),1.96-1.18(m,32H).

[0337] 1.57 HO-Lys[(α-NHCy5)(ε-NH2)], compound 115 To a DMF (4 mL) stirred solution containing compound 116 (36 mg; 0.043 mmol), piperidine (1.5 mL) was added, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by preparative HPLC; 20-90% ACN / MQ water + 0.1% formic acid (60 min, Rt 32.0-33.0 min) to obtain compound 59 as a blue solid of 12 mg (44%). LC-MS (hydrophilic method, TFA buffer) R t =7.65 min; ESI MS(+ve)611[M] + ;C 38 H 51 N4O3[M]+ The calculated m / z value is 611.

[0338] 1.58 Synthesis of HO-Lys[(α-NHCy5)(ε-NHDFO)] wedge, compound 116 To a stirred solution of HO-Lys[(α-NH2.TFA)(ε-NHFmoc)](57.0 mg, 0.118 mmol) / DMF(5 mL), NMM(52 μL, 0.472 mmol) and cyanine 5 NHS ester (Lumiprobes, 40.0 mg, 0.059 mmol) were added. The reaction mixture was stirred overnight at room temperature, and volatile components were removed by vacuum. The residue was dissolved in ACN:MQ water(3 mL, 1:1 v / v), and the solution was filtered (0.45 μm Acrodisc syringe filter). The filtrate was separated and subjected to HPLC; 20-90% ACN / MQ water + 0.1% formic acid (60 min, R) t The compound was purified by 39.0-42.0 mins, yielding 58 33 mg (67%) as a blue solid. LC-MS (hydrophilic method, TFA buffer) R t =10.54 min; ESI MS (+ve) 833[M] + ;C 53 H 61 The calculated m / z value for N4O5[M]+ is 833.46. 1 HNMR(300MHz,D2O)δ(ppm):8.21(t,2H,J=15.0Hz),7.79(d,2H,J=6.0Hz),7.63(d,2H,J= 6.0Hz),7.49-7.24(m,10H),6.61(t,1H,J=12.0Hz),6.29-6.22(m,2H),4.49-4.26(m,2H ),.18(t,1H,J=6.0Hz),4.07(t,2H,J=6.0Hz),3.68-3.60(m,2H),3.60(s,3H),3.12(t,2 H,J=6.0Hz),2.28(t,2H,J=6.0Hz),1.94-1.61(m,6H),1.71(s,12H),1.62-1.19(m,6H).

[0339] Example 2 Synthesis of dendrimer drug conjugates 2.1 Azide-PEG 24-CO[N(PN)2][Lys]4[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100 )]4, G2, compound 17 Azido-PEG 24 -CO[N(PN)2][Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 Using )4 (50.0 mg, 7.24 μmol), preparation was carried out according to general procedure E, yielding a dark red oily solid (58 mg, 89%). 1 H-NMR(300MHz,CD3OD)δ(ppm):0.69-2.17(m,68H);2.30-2.50(m,8H);2.56-2.68(m,2H);3.02-3.24(m,8 H);3.38-3.41(m,24H);3.52-4.56(m,520H);4.69-4.77(m,12H);5.19-5.57(m,4H);7.15-8.14(m,12H). LCMS (hydrophilic method, formic acid buffer) R t =11.23 min.ESI MS(+ve) is converted to 9016.

[0340] 2.2 Azide-PEG 24 CO-[N(PN)2][Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4,G2, compound 64 Azido-PEG 24 CO-[N(PN)2][Lys]4[(α-NH2.TFA)(ε-NH-COPEG 1100 Using )4 (50.0 mg, 7.24 μmol), preparation was carried out according to general procedure E, yielding a dark red oily solid (58 mg, 89%). 1 H-NMR(300MHz,CD3OD)δ(ppm): 0.69-2.17(m,68H);2.30-2.50(m,8H);2.56-2.68(m,2H);3.02-3.24(m,8H);3.38-3.41( m,24H);3.52-4.56(m,520H);4.69-4.77(m,12H);5.19-5.57(m,4H);7.15-8.14(m,12H). LCMS (hydrophilic method, formic acid buffer) R t=11.23 min.ESI MS(+ve) is converted to 9016.

[0341] 2.3 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100 )]8, G3, compound 18 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NHPEG 1100 Using )8 (50.0 mg, 3.91 μmol), preparation was carried out according to general procedure E to obtain a dark red oily solid (50 mg, 75%). 1 H-NMR(300MHz,CD3OD)δ(ppm): 0.88-1.93(m,120H);2.33-2.51(m,16H);2.58-2.72(m,2H);3.04-3.24(m,8H); 3.35-3.41(m,76H);3.52-4.61(m,964H);5.27-5.45(m,8H);7.13-8.13(m,24H). LCMS (hydrophilic method, formic acid buffer) R t =11.30 minutes.

[0342] 2.4 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG1100)]8,G3, Compound 65 Azido-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1100 Using )8 (50.0 mg, 3.91 μmol), preparation was carried out according to general procedure E to obtain a dark red oily solid (50 mg, 75%). 1H-NMR(300MHz,CD3OD)δ(ppm): 0.88-1.93(m,120H);2.33-2.51(m,16H);2.58-2.72(m,2H);3.04-3.24(m,8H); 3.35-3.41(m,76H);3.52-4.61(m,964H);5.27-5.45(m,8H);7.13-8.13(m,24H). LCMS (hydrophilic method, formic acid buffer) R t =11.30 minutes.

[0343] 2.5 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG 570 )8], G3, compound 19 Azido-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 570 Using [8] (7.2 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol), preparation was carried out according to general procedure F to obtain a product concentration of 14.6 mg / 3.5 mL (240 μM).

[0344] 2.6 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG 1100 )8],G3, compound 20 Azido-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1100 Using [8] (10.8 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol), preparation was carried out according to general procedure F to obtain a product concentration of 18 mg / 3.5 mL (240 μM).

[0345] 2.7 Azide-PEG 24 CO-[N(PN)2[Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG 2000)8],G3, compound 21 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 2000 Using [8] (18.1 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol), a solution was prepared according to general procedure F to obtain a product concentration of 25.5 mg / 3.5 mL (240 μM).

[0346] 2.8 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH-DGA-MMAF(OMe))8(ε-NH-COPEG 1100 )8],G3, compound 22 Azido-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NH0-COPEG 1100 Using )8 (16.3 mg, 1.28 μmol) and DGA-MMAF(OMe) (10.6 mg, 12.3 μmol), preparation was carried out according to general procedure F to obtain a product concentration of 23.8 mg / 3.5 mL (365 μM).

[0347] 2.9 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-Pt(IV) Acetate)(ε-NH-COPEG 1100 )]8,G3, compound 23 Azido-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NH-COPEG 1100NMM (7.4 μL, 67.3 μmol) was added to a stirred solution of )8 (SPL19 and SPL32) (22.4 mg, 1.75 μmol) / DMF (0.5 mL). Next, the resulting solution was added to a stirred solution of diglycolic acid acetate-1R,2R-cyclohexane-1,2-diamine oxalatoplatinum(IV) (10.4 mg, 17.7 μmol) and PyBOP (8.74 mg, 16.8 μmol) / DMF (0.5 mL). The resulting reaction mixture was then protected from light, stirred overnight at room temperature, and purified by SEC to obtain the product as an off-white solid (24 mg, 86%). 1 H-NMR(300MHz,CD3OD)δ(ppm):1.28-1.89(m,128H),2.07(s,23H),2.24-3.01(m, 57H), 3.12-3.26(m, 22H), 3.37(s, 25H), 3.39-3.90(m, 768H), 4.04-4.69(m, 80H). LCMS (hydrophilic method, TFA buffer) R t =10.63 min. The ICP-OES determined Pt% 9.0% indicates that the macromolecule has 7 Pt-containing moieties. The actual molecular weight of the complex is determined to be 16.1 kDa.

[0348] 2.10 BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 (α-NH2) 4-7 )(ε-NH-COPEG 1000 )8], G3, compound 34 BHA[Lys]8[(αNH2.TFA)8(ε-NH-COPEG 1000 A stirred solution of (MeTzPh)PEG4CO-NHPEG was prepared in RT with (100 mg, 0.00786 mmol, 1.0 eq) / DMF (300 μL). 24CO2H (Click Chemistry Tools; 16 mg, 0.01 mmol, 1.3 equivalents), PyBOP (8 mg, 0.013 mmol, 1.6 equivalents), and DMF (200 μL) were added. After stirring the reaction mixture for 3 minutes, NMM (40 mg, 50 μL, 0.38 mmol, 48 equivalents) was added. The contents were protected from light and stirred overnight in RT. The reaction mixture was diluted with MQ water and frozen overnight. The lyophilized material was taken up in MeOH (1 mL) and purified by SEC (400 drops / tube, MeOH sephadex LH20, 35 drops / min). The product-containing fraction was identified by HPLC and collected in two different fractions. Each fraction was concentrated under reduced pressure, the resulting residue was taken up in MQ water, filtered (0.45 μm Acrodisc filter), and lyophilized to obtain the title product as a pink solid (69 mg, 66%). HPLC (C8 Xbridge, 3x100mm) gradient. 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rf (min) = 8.4 (broad peak). 1 H-NMR(300MHz,D2O)δ(ppm): 1.00-2.00(m,90H),2.51(t,3H),2.60(brs,3H),3.00-3.12(m,6H),3.12-3.35(brs,27H),3.35-3.45( m,26H),3.45-4.15(m,937H),4.15-4.45(m,12H),6.12(s,1H),7.15-7.50(m,12H),8.40-8.50(m,2H).

[0349] 2.11 BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG2-BCN) 1-4 (α-NH2) 4-7 )(ε-NH-COPEG 1000 )8],G3, compound 35 BCN-PEG2CO-NHPEG 24A stirred solution of -CO2H compound 31 (9.6 mg, 0.006 mmol, 1.3 equivalents) / DMF (200 μL) was prepared by RT. PyBOP (4 mg, 0.008 mmol, 1.6 equivalents) and NMM (23 mg, 25 μL, 0.226 mmol, 48 equivalents) were added to this solution, and after 5 minutes, BHA[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1000 )8](60 mg, 0.006 mmol, 1.0 equivalent) was added, followed by DMF (200 μL). The contents were protected from light and stirred overnight in RT. The reaction mixture was diluted with ACN (10 mL) and then purified by SEC (400 drops / tube, ACNsephadex LH20, 35 drops / min). The product-containing fraction was identified by HPLC, recovered, filtered (0.45 μm Acrodisc filter), concentrated under reduced pressure, and freeze-dried overnight to obtain the title compound as a pale yellow solid (58 mg, 92% yield). HPLC (C8 Xbridge, 3x100mm) gradient. 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rf (min) = 8.43 (broad peak). 1 H-NMR(300MHz,MeOD)δ(ppm):0.75-1.12(m,15H)1.12-2.15(m,95H),2.15-2.35(m,7H),2.55(brs,3H),3.00-3.35(m ,90H),3.35-3.38(s,17H),3.38-4.09(m,592H),4.13(d,2H),4.18-4.63(brs,7H),6.18(s,0.9H),7.12-7.48(m,7H).

[0350] 2.12 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH-Cy5) 1-4 (α-NH-Glu-Val-Cit-PAB-MMAE) 4-7 )(ε-NH-COPEG 1100 )8],G3, compound 36 Cy5-NHS (214 μL 4.2 mg / mL DMF solution; 1.43 μmol, 1.0 equivalent) solution is prepared using Neat (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 Compound 8 (18 mg, 1.43 μmol) was obtained. After complete dissolution, NMM (10 μL, 91.0 μmol) was added, and the resulting reaction mixture was stirred and protected from light. After 2 hours, NMM (10 μL, 91.0 μmol) and PyBOP (7.3 mg, 14.0 μmol) were added to the dendrimer solution, and then to the Glu-VC-PAB-MMAE / DMF solution (290 μL, 60 mg / mL solution, 14.0 μmol, 9.8 equivalents). The resulting reaction mixture was then protected from light and stirred overnight at RT.

[0351] The reaction mixture was diluted with PBS (2.0 mL) to a final volume of 2.5 mL. The diluted solution was then passed through a PD10 desalting column (pre-equilibrated with PBS). After the entire solution entered the column bed, PBS (3.5 mL) was added to elute the product (which appeared as a blue band). The final theoretical concentration of the construct = 8.7 mg / mL / PBS. The materials were stored frozen at -80°C. HPLC (C8 Xbridge, 3x100 mm) gradient: 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rt (min) = 95-10 min (broad peak); 17% MMAE / MMAE-linker complex-related peak 83%.

[0352] 2.13(MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[ 3 H-Lys]4[Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG 1100 )8],G3, compound 40 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[3 H-Lys]4[Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 Solid PyBOP (24.7 mg, 47.5 μmol) was added to HO-Glu-VC-PAB-MMAE (40 mg, 32.3 μmol) / NMM / DMF (7.8 μL / 0.5 mL) (prepared using the method used for the synthesis of compound 53). After complete dissolution, this dendrimer solution was added to HO-Glu-VC-PAB-MMAE (40 mg, 32.3 μmol) / NMM / DMF (7.8 μL / 0.5 mL). The resulting reaction mixture was then protected from light and stirred overnight at RT.

[0353] The reaction mixture was diluted with PBS (4.0 mL) to a final volume of 5 mL. The diluted solution was then passed through two PD10 desalting columns (pre-equilibrated with PBS, 2.5 mL in each column). Once all the solution had entered the column bed, PBS (3.5 mL) was added to each column to elute the product. The resulting crude mixture was further purified using a regenerated cellulose Amicon Ultra-0.5 mL centrifugation unit (10K MWCO). The final theoretical concentration of the construct was 29-30 mg / mL / PBS. The material was stored frozen at -20°C. HPLC (C8 Xbridge, 3x100 mm) gradient. 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rt (min) = 9.5-12 min (broad peak); complex-related peaks account for 91.4% and MMAE / MMAE-linker-related peaks account for 8.6%.

[0354] 2.14(MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)6)(ε-NH-COPEG 1100 )8], G3 compound 66 A stirred solution of p-SCN-deferoxamine (2.1 mg, 2.79 μmol) / DMSO (100 μL) was prepared by RT. To this, (MeTzPh)PEG4CO-NHPEG was added.24 CO[N(PN)2][Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 Compound 53 (17.0 mg, 1.35 μmol) / DMF (200 μL) was added. The resulting reaction mixture was then stirred for 3 minutes, and NMM (10 μL, 91.0 μmol) was added. The resulting solution was protected from light and stirred at RT for 4 hours. PyBOP (7.0 mg, 13.5 μmol) was added, and after 5 minutes, the reaction mixture was added to neat HO-Glu-Val-Cit-PAB-MMAE (9.76 mg, 7.89 μmol). The resulting reaction mixture was then left to stand overnight. The reaction mixture was diluted with PBS buffer (4.5 mL), divided into four Amicon Ultra centrifugation filters (10K MWCO), and the filters were centrifuged (14K rcf, 15 min). The retained solution was dialyzed against PBS (400 μL, 14K rcf, 15 min x 10 times). The retained solution was mixed to obtain a pink solution. The concentration of compound 67 is approximately 16 mg in 2 mL. HPLC (C8X Bridge, 3x100mm) gradient. 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rt (min) = 8.7-9.8 min (broad peak).

[0355] 2.15 BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)5)(ε-NH-COPEG 1100 )8]G3, compound 67 A 100 μL stirred solution of p-SCN-deferoxamine (2.0 mg, 2.66 μmol) / DMSO was prepared by RT. To this, BHALys[Lys]8[(α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-NH2)7)(ε-NHPEG 1100Compound 34 (17.0 mg, 1.27 μmol) / DMF (200 μL) was added. The resulting reaction mixture was then stirred for 3 minutes, and NMM (10 μL, 91.0 μmol) was added. The resulting solution was protected from light and stirred at RT for 4 hours. PyBOP (7.0 mg, 13.5 μmol) was added, and after 5 minutes, the reaction mixture was added to neat HO-Glu-Val-Cit-PAB-MMAE (9.17 mg, 7.41 μmol). The resulting reaction mixture was then left to stand overnight. The reaction mixture was diluted with PBS buffer (4.5 mL), divided into four Amicon Ultra centrifugation filters (10K MWCO), and the filters were centrifuged (14K rcf, 15 min). The retained solution was dialyzed against PBS (400 μL, 14K rcf, 15 min x 10 times). The retained solution was mixed to obtain a pink solution. The concentration of compound 68 is approximately 16 mg in 2 mL. Gradient from HPLC (C8X Bridge, 3x100mm). 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rt (min) = 9.3-9.7 min (broad peak).

[0356] 2.16 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH-Cy5)1(α-NH-Glu-SN 38 )7)(ε-NH-COPEG 1100 )8],G3 Compound 68 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH2.HCl)8(ε-NH-COPEG 1100)8]NMM (4.0 μL, 36.0 μmol) and sulfoCy5 NHS ester (0.58 mg, 0.75 μmol) were added to a stirred solution of compound 53 (10 mg, 0.75 μmol) in DMF (2 mL). The reaction mixture was stirred at room temperature for 2 hours. SN38-Glu-COOH (WO2020 / 102852) (3.94 mg, 7.8 μmol) and PyBOP (4.05 mg, 7.8 μmol) were added to the reaction mixture, and the reaction mixture was stirred at room temperature for a further 20 hours. The solvent was removed under reduced pressure, and the resulting residue was purified by size exclusion chromatography (Sephadex LH 20) using acetonitrile as the mobile phase to obtain the title compound as a blue solid (6.0 mg). ULC-TOF analysis of the reaction mixture confirmed the random addition of sulfoCy5 NHS ester and SN-38-Glu-COOH to the dendrimer. UPLC-TOF (hydrophilic method, TFA buffer) Rt=84.80-5.80 minutes.

[0357] 2.17 BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 (α-NH2) 28-31 (ε-NH-COPEG 1100 ) 32 ],G5,Compound 69 BHALys[Lys] 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1000 ) 32 ](100.0 mg, 0.002 mmol) and HOOCPEG 24 Using NH-COPEG4(PhTzMe) (Click Chemistry Tools; 3.38 mg, 0.005 mmol), preparation was carried out according to general procedure C (except for wrapping the reaction vessel in foil to protect it from light), methanol was used as the eluent, and the residue was purified using SEC (Sephadex® LH-20) to obtain the product compound 69 (68 mg, 72%). 1HNMR(300MHz,D2O)δ(ppm): 8.44-8.29(m,2H),7.44-7.09(m,12H),6.02(bs,1H),4.37-4.11(m,31H),4.08-3.3 7(m,2937H),3.32(s,83H),3.27-2.88(m,116H),2.59-2.42(m,3H),2.18-0.92(m,3 13H); HPLC (C8XBridge, 3x100mm) HPLC (glucose buffer): 5% ACN / H2O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.4 mL / min, Rt = 8.77 min.

[0358] 2.18 BHA [Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 (α-NHCy5)1(α-NH-COPEG9-Val-Ala-PAB-P-Torion-NMeCO-CTX) 22 (α-NH2) 5-8 (ε-NH-COPEG 1100 ) 32 G5, Compound 70 BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 ((α-NH2) 28-31 (ε-NH-COPEG 1100 ) 32To a stirred solution of compound 69 (20.0 mg, 0.4 μmol) / DMF, NMM (8.0 μL, 72 μmol) was added, followed by Cy5-NHS ester (0.26 mg, 0.4 μmol). The reaction mixture was stirred overnight at room temperature. Compound 63 (25.7 mg, 14.4 μmol) and PyBOP were added to the reaction mixture, and the mixture was stirred for a further 20 hours at room temperature. The solvent was removed under reduced pressure, and the crude residue was dissolved in acetonitrile (2 mL). The resulting solution was filtered through a 0.45 μm filter. The filtrate was collected and purified using size exclusion chromatography (Sephadex LH20) with acetonitrile as the solvent to obtain the title compound as a blue solid (27 mg; 75%). 1 HNMR (300MHz, MeOD) δ (ppm). 8.52(d,2H,J=9.0Hz),8.25-7.05(m,329H),6.39-5.89(m,23H),5.73-4.94(m,141H),4.64-4.05(m,161H),4 .05-3.36(m,4026H),3.18-2.22(m,323H),2.18-0.56(m,1362H);HPLC(C8XBridge,3x100mm) Gradient (Formic Acid Buffer): 75% ACN / H2O (0-1 min), 75-90% ACN (1-7 min), 90% ACN (7-12 min), 90-75% ACN (12-13 min), 75% ACN (13-15 min), 214 min, 0.4 mL / min, Rt (min) = 8.90-9.10.

[0359] Example 3 Synthesis of Target Dendrimer Complex 3.1 Affibody-MPED-BCN-Triazolo-PEG 24 -CO[N(PN)2][Lys]4[(α-NH-DGA-3'NH-Dox) 1-4 (ε-NH-COPEG 1100 )]4, G2, compound 24 Affibody-BCN (900 μL) and Azide-PEG 24 -CO[N(PN)2][Lys]4[(α-NH-DGA-3'-NH-Dox) 1-4 (ε-NH-COPEG 1100)]4 (300 μL of 858 μM solution) was used to prepare the product according to general procedure G (aphibody-BCN:DGA-Dox dendrimer molar ratio: 1:3). SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 19 kDa (700 nm).

[0360] 3.2 Affibody-BCN / N3-PEG 24 CO-[N(PN)2][Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4,G2, compound 71 Affibody-BCN (900 μL) and Azide-PEG 24 CO-[N(PN)2][Lys]4[(α-NH-14-O-DGA-Nemo)(ε-NH-COPEG 1100 )]4 (300 μL of 858 μM solution) was used to prepare the product according to general procedure G (aphibody-BCN:DGA-Nemo dendrimer molar ratio: 1:3). SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 19 kDa (700 nm).

[0361] 3.3 Affibody-MPED-BCN-Triazolo-PEG 24 -CO[N(PN)2][Lys]8[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100 )]8, G3, compound 25 Affibody-BCN (105 μL) and Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100 The mixture was prepared using )8 (35 μL of 580 μM solution) according to general procedure G (aphibody-BCN:DGA-Dox dendrimer molar ratio: 1:1). SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 27 kDa (700 nm). The conjugate yield was estimated to be 80% by adding azide IR Dye 800CW and measuring fluorescence at 800 nm.

[0362] 3.4 Affibody-BCN / N3-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]8,G3, compound 72 Affibody-BCN (105 μL) and Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 The mixture was prepared using )8 (35 μL of 580 μM solution) according to general procedure G (aphibody-BCN:DGA-Nemo dendrimer molar ratio; 1:1). SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 27 kDa (700 nm). The conjugate yield was estimated to be 80% by adding azide IR Dye 800CW and measuring fluorescence at 800 nm.

[0363] 3.5 Affibody-BCN / N3-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG 1100 )8],G3, compound 26 Afibody BCN (2.0 mg, 286 nmol, 1.0 mg / mL PBS) and Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-vc-PAB-MMAE)(ε-NH-COPEG 1100 The material was prepared using )8 (1.0 mL of 240 μM solution) according to general procedure G. The purified material was freeze-dried to obtain a white, cottony powder (2.19 mg, 31%). SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 30 kDa (700 nm).

[0364] 3.6 Affibody-BCN / N3-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG2000 )8],G3, Compound 27 (Compound 78 / SPL40) Affibody BCN (1.0 mg, 143 nmol, 1.0 mg / mL PBS) and Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-vc-PAB-MMAE)(ε-NH-COPEG 2000 The sample was prepared using )8 (250 μL of 233 μM solution) according to general procedure G. SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 40 kDa (700 nm).

[0365] 3.7 Affibody-BCN / N3-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-Pt(IV)-acetate)(ε-NH-COPEG 1100 )]8,G3, compound 28 Affibody BCN (5.0 mg, 715 nmol, 1.0 mg / mL PBS) and Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-Pt(IV)-acetate)(ε-NH-COPEG 1100 The sample was prepared using )8 (600 μL of a 903 μM solution) according to general procedure G. The purified material was freeze-dried to obtain a white, cottony powder (5.90 mg, 47%). SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 30 kDa (700 nm).

[0366] 3.8 Fab-triazole DBCO-PEG 24 -CO[N(PN)2[Lys]4[(α-DGA-3'-NH-Dox)(ε-NHPEG 1100 ) 4G2-Dox / PEG1100 dendrimer, compound 29 Azido-PEG 24 -CO[N(PN)2[Lys]4[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100The solution of )4 (10 μL of 500 μM PBS solution) was added to the Fab-DBCO* solution (40 μL of 13.4 μM solution / HEPES buffer). The resulting reaction mixture was then shaken overnight at room temperature (650 rpm) and Fab-azide-PEG was added. 24 -CO[N(PN)2[Lys]4[(α-NH-DGA-3'NH-Dox)(ε-NH-COPEG 1100 )]4 was generated. SDS-PAGE analysis was performed, scanning at 700nm and 800nm ​​using an Odyssey scanner. In the SDS-PAGE scanned at 700nm, the DBCO-Fab band corresponding to 50kDa and the expected Fab-[dendrimer-Dox / PEG band at approximately 65kDa were observed. 1100 The band for compound 2 (compound 30) was observed.

[0367] 3.9 Fab-DBCO / N3-PEG 24 CO-[N(PN)2[Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4, compound 73 Azido-PEG 24 CO-[N(PN)2[Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 The solution of )4 (10 μL of 500 μM PBS solution) was added to the Fab-DBCO* solution (40 μL of 13.4 μM solution / HEPES buffer). The resulting reaction mixture was then shaken overnight at room temperature (650 rpm) and Fab-DBCO / N3-PEG was added. 24 CO-[N(PN)2[Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4 was generated. SDS-PAGE analysis was performed, scanning at 700nm and 800nm ​​using an Odyssey scanner. In the SDS-PAGE scanned at 700nm, the DBCO-Fab band corresponding to 50kDa and the expected Fab-[dendrimer-Nemo / PEG band at approximately 65kDa were observed. 1100 The band for compound 2 (compound 30) was observed.

[0368] 3.10 Affibody-BCN / N3-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH-DGA-MMAF(OMe)8(ε-NH-COPEG 1100 )8], G3, compound 30 Afibody BCN (2.0 mg, 286 nmol, 1.0 mg / mL PBS) and Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-MMAF(OMe))(ε-NH-COPEG 1100 The material was prepared using )8 (600 μL of a 365 μM solution) according to general procedure G. The purified material was freeze-dried to obtain a white, cottony powder (2.06 mg, 33%). SDS-PAGE analysis showed a band corresponding to the aphibody-dendrimer complex at around 30 kDa (700 nm).

[0369] 3.11 Nanobody-PEG 12 -TCO-MePhTz-PEG4-PEG 24 -CO[N(PN)2][Lys]8[(α-NH-Cy5)1(α-NH-Glu-VC-PAB-MMAE)7(ε-NH-COPEG 1100 )8], compound 41 DBCO-PEG 12 Nanobody constructs were prepared using TCO and nanobody 2D3 N-terminal tags, TEV, and C-terminal azides according to Example 4 below.

[0370] 3.12 Nanobody-PEG 12 -TCO-MePhTz-PEG4-PEG 24 -CO[N(PN)2][Lys]8[(α-NH-Cy5)1(α-NH-Glu-VC-PAB-MMAE)7(ε-NH-COPEG 1100 )8], compound 42 DBCO-PEG 12 Nanobody constructs were prepared using TCO and nanobody 2D3 N-terminal tags, TEV, and C-terminal azides according to Example 4 below.

[0371] 3.13 Nanobody-PEG 12 -TCO-MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 16 [(α-NH-Cy5)1(α-NHAc) 15 (ε-NH-COPEG 1100 ) 16 ], compound 43 DBCO-PEG 12 - Using TCO and nanobody 2D3 N-terminal tag, TEV, and C-terminal azide, nanobody constructs were prepared according to Example 4 below.

[0372] 3.14 Nanobody-PEG 12 -TCO-MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 32 [(α-NH-Cy5)1(α-NHAc) 31 (ε-NH-COPEG 1100 ) 32 ], Compound 44 DBCO-PEG 12 - Using TCO and nanobody 2D3 N-terminal tag, TEV, and C-terminal azide, nanobody constructs were prepared according to Example 4 below.

[0373] 3.15 Nanobody-PEG 12 -TCO-MeTzPh-PEG4PEG 24 -CO[N(PN)2][3H-Lys]4[Lys(α-NH-Glu-VC-PAB-MMAE)8(ε-NH-COPEG1100)8], Compound 45 DBCO-PEG 12 - Using TCO and nanobody 2D3 N-terminal tag, TEV, and C-terminal azide, nanobody constructs were prepared according to Example 4 below.

[0374] 3.16 Nanobody-N3 / DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO / (MeTzPh)PEG4CO-NHPEG 24CO-[N(PN)2][Lys]8[((α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)6)(ε-NH-COPEG 1100 )8], compound 74 DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazin dendrimer (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]4[Lys]8[((α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)6)(ε-NH-COPEG 1100 )8], compound 66 (1 equivalent), and nanobody-N3 ("nanobody-N3-C-terminal tag") (1 equivalent) were prepared according to general procedure H. SDS-PAGE analysis showed a band corresponding to approximately 37 kDa of the nanobody-dendrimer complex (Figure 1).

[0375] 3.17 Nanobody-N3 / BCN-NHPEG2-Glu-NHPEG 24 CO-NHPEG3-TCO / (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)6)(ε-NH-COPEG 1100 )8], compound 75 BCN-NHPEG2-Glu-NHPEG 24 CONHPEG3-TCO linker compound 32 (1 equivalent), tetrazin dendrimer (MeTzPh)PEG4CO-NHPEG 24 The compounds were prepared using the general procedure H with CO-[N(PN)2][Lys]4[Lys]8[((α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)6)(ε-NH-COPEG1100)8], compound 66 (1 equivalent), and nanobody-N3 ("nanobody-N3-C-terminal tag") (1 equivalent). SDS-PAGE analysis showed a band corresponding to approximately 37 kDa of the nanobody-dendrimer complex (Figure 1).

[0376] 3.18 BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)1(α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)5)(ε-NH-COPEG1 100 )8],G3, compound 76 DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazin dendrimer BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)5)(ε-NH-COPEG 1100 The compounds were prepared using the general procedure H with compound 67 (1 equivalent) and nanobody-N3 ("nanobody-N3-C-terminal tag") (1 equivalent). SDS-PAGE analysis showed a band corresponding to approximately 37 kDa of the nanobody-dendrimer complex (Figure 1).

[0377] 3.19 Nanobody-N3 / DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO / (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH-Cy5)1(α-NH-Glu-10-O-SN38)7)(ε-NH-COPEG 1100 )8], Compound 77 DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazin dendrimer (MeTzPh)PEG4CO-NHPEG 24The compound was prepared using CO-[N(PN)2][Lys]8[((α-NH-Cy5)1(α-NH-Glu-10-O-SN38)7)(ε-NH-COPEG1100)8], compound 68 (1 equivalent), and nanobody-N3 ("nanobody-N3-C-terminal tag") (1 equivalent) according to the general procedure H. SDS-PAGE analysis showed a band corresponding to approximately 37 kDa of the nanobody-dendrimer complex (Figure 2).

[0378] 3.20 BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody) 1-4 (α-NHCy5)1(α-NH-COPEG9-Val-Ala-PAB-P-Trigger-NMeCO-CTX) 22 )(α-NH2) 5-8 (ε-NH-COPEG 1100 ) 32 ],G5,Compound 78 DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazine dendrimer BHA[Lys]2[Lys]4[Lys]8[Lys] 16 [Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 (α-NHCy5)1(α-NH-COPEG9-Val-Ala-PAB-P-Trigger-NMeCO-CTX) 22 (NH2) 5-8 (ε-NH-COPEG 1100 ) 32 The compound was prepared using the general procedure H with compound 70 (1 equivalent) and nanobody-N3 ("nanobody-N3-C-terminal tag") (1 equivalent). SDS-PAGE analysis showed a band corresponding to approximately 95 kDa of the nanobody-dendrimer complex (Figure 2).

[0379] 3.21 BHA[Lys]4[((α-NH-COPEG 24NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)2)(ε-NH-COPEG 1000 )4],G2, Compound 86 and BHA[Lys]4[((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody) 2-3 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)) 0-1 (ε-NHCOPEG 1000 )4], G2, compound 87 BHA[Lys]4[((α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)1)(ε-NH-COPEG 1000 Using compound 99, [4], G2, the preparation was carried out according to general procedure H, except for the following:

[0380] Step 1: TCO linker solution 80 was prepared with neat DMSO. Step 2: Dissolve the dendrimer in Tris buffer at pH 8. When the TCO linker and dendrimer have completely reacted (UPLC or LCMS), dilute the reaction mixture in Tris buffer so that the final concentration of DMSO is 5% (v / v) or less, and purify by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO).

[0381] The purification method is the same as that used for the purification of compounds 74 and 76, and nanobody-dendrimer complex compound 86 (232 μg; 0.45 μg / μL / HEPES buffer, pH 8) and nanobody-dendrimer complex compound 87 (60 μg; 0.30 μg / μL / HEPES buffer, pH 8) can be obtained.

[0382] 3.22 BHA[Lys]8[((α-NH-COPEG24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)6)(ε-NH-COPEG 412 )8], G3, compound 88 and BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody) 2-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 3-5 (ε-NH-COPEG412)8], G3, compound 88a BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 3-6 )(ε-NH-COPEG 412 Using compound 100 (8), G3, the following was prepared according to general procedure H, excluding the following:

[0383] Step 1: A solution of TCO linker compound 80 was prepared with neat DMSO. Step 2: Dissolve the dendrimer in Tris buffer at pH 8. When the TCO linker and dendrimer have completely reacted (UPLC or LCMS), dilute the reaction mixture in Tris buffer so that the final concentration of DMSO is 5% (v / v) or less, and purify by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO).

[0384] The purification method was the same as that used for the purification of compounds 74 and 76 to obtain nanobody-dendrimer complex compound 88 (140 μg; 1.48 μg / μL in HEPES buffer at pH 8) and compound 88a (not pure).

[0385] 3.23 BHA[Lys]8[(( α -NH-COPEG24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)6)(ε-NH-COPEG 1000 )8], G3, compound 89 and BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody) 2-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)) 3-5 (ε-NH-COPEG 1000 )8], G3, compound 89a BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 3-6 )(ε-NH-COPEG 1000 )8]G3 and compound 101 were used to prepare the following according to general procedure H, excluding the following:

[0386] Step 1: A solution of TCO linker (compound 80) was prepared with neat DMSO. Step 2: Dissolve the dendrimer in Tris buffer at pH 8. When the TCO linker and dendrimer have completely reacted (UPLC or LCMS), dilute the reaction mixture in Tris buffer so that the final concentration of DMSO is 5% (v / v) or less, and purify by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO).

[0387] The purification method was the same as that used for the purification of compounds 74 and 76 to obtain nanobody-dendrimer complex compound 89 (249 μg; 2.68 μg / μL in HEPES buffer at pH 8) and compound 89a (not pure).

[0388] 3.24 BHA[Lys] 16[((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 14 (ε-NH-COPEG 1000 ) 16 ]G4, compound 90 and BHA[Lys] 16 [((a-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody) 2-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 11-13 (ε-NHCOPEG 1000 ) 16 ],G4,Compound 91 BHA[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) 1( α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 14 (ε-NH-COPEG 1000 ) 16 Using ], G4, and compound 102, preparations were made according to general procedure H, excluding the following:

[0389] Step 1: A solution of TCO linker (compound 80) was prepared with neat DMSO. Step 2: Dissolve the dendrimer in Tris buffer at pH 8. When the TCO linker and dendrimer have completely reacted (UPLC or LCMS), dilute the reaction mixture in Tris buffer so that the final concentration of DMSO is 5% (v / v) or less, and purify by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO).

[0390] The purification method was the same as that used for the purification of compounds 74 and 76 to obtain nanobody-dendrimer complex compound 90 (203 μg; 0.975 μg / μL and 0.556 μg / μL / HEPES buffer, pH 8) and compound 91 (101 μg; 0.34 μg / μL / HEPES buffer, pH 8).

[0391] 3.25 BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 30 (ε-NH-COPEG 1000 ) 32 ], G5, compound 92 and BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody) 2-4 [α-Lys(α-NHCy5)(ε-NHDFO)]1(α-NH2) 27-29 (ε-NH-COPEG 1000 ) 32 ], G5, compound 93 BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz)) 1-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 27-30 )(ε-NH-COPEG 1000 ) 32 Using compound 103, the following was prepared according to general procedure H, with the exception of:

[0392] Step 1: A solution of TCO linker compound 80 was prepared with neat DMSO. Step 2: Dissolve the dendrimer in Tris buffer at pH 8. When the TCO linker and dendrimer have completely reacted (UPLC or LCMS), dilute the reaction mixture in Tris buffer so that the final concentration of DMSO is 5% (v / v) or less, and purify by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO).

[0393] The purification method was the same as that used for the purification of compounds 74 and 76, to obtain nanobody-dendrimer complex compound 92 (370 μg; 3.66 μg / μL / HEPES buffer, pH 8) and product compound 93 (177 μg; 1.72 μg / μL / HEPES buffer, pH 8).

[0394] 3.26 BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhMeTz))2)(ε-NH-COPEG 24 NH-COPEG4(PhMeTz)) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody2],G2,Compound 94a,BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhMeTz))1(ε-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)3], G2, compound 94, and BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody)4], G2, compound 95 BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24Using NH-COPEG4(PhMeTz)4], G2 dendrimer, and compound 24, preparations were made according to general procedure H, excluding the following:

[0395] Step 1: A solution of TCO linker compound 51 was prepared with water. Step 2: Dissolve the dendrimer in MQ water.

[0396] The purification method was the same as that used for the purification of compounds 74 and 76, to obtain nanobody-dendrimer complex compounds 95 and 94 as an inseparable mixture (final concentration of 294 μg in HEPES buffer, pH 8).

[0397] Also, DBCO-Glu-NHPEG 24 A solution of CO-NHPEG3-TCO compound 80 (10 mg / mL solution / 57 μL MQ water, 0.322 μmol) was added to a solution of nanobody-N3 ("nanobody-N3-C terminal tag") (2.5 mg, 0.161 μmol) / 900 μL Tris buffer at pH 8. After the reaction mixture was left to stand in RT for 2 days, the reaction mixture was purified by centrifugal ultrafiltration using Tris buffer pH 8 (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO; 10 x 450 μL). To this solution, a solution of compound 105 (230.0 μg / 500 mL MQ water; 0.026 μmol) was added, and the reaction mixture was left to stand overnight in RT. The purification method was the same as that used for the purification of compounds 74 and 76, to obtain nanobody-dendrimer compounds 95, 94a, and 94 as an inseparable mixture (final concentration 124 μg in HEPES buffer, pH 8).

[0398] 3.27 BHA[Lys]4[((α-NH-COPEG 24 NH-COPEG4(PhTzMe) / TCO-PEG8-Nanobody)1(α-Lys(α-NHCy5)1(ε-NHDFO)1)1(α-NH2)2)(ε-NH-COPEG 1000 )4], G2, compound 97

[0399] A solution of dibromomaleimide linker compound 106 was prepared by dissolving 2.0 mg in 1 mL of DMSO (1.0 mL). To a solution of nanobody ("nanobody-N3-C-terminal tag") (compound 79) (1.0 mg, 0.058 μmol / 715 μL Tris buffer, pH 8), a solution of TCEP (0.5 M / PBS, pH 7) (2.3 μL, 1.174 μmol) was added. The reaction mixture was heated at 37°C for 1 hour, then DMSO (673 μL) was added, followed by the addition of linker compound 57 (93.0 μg, 0.117 μmol) / DMSO solution (47.0 μL). After 1.5 hours, the reaction mixture was cooled to RT and centrifuged. To the precipitate, a solution of compound 99 (100 μg, 17 μL, 1.78 mg / 300 μL MQ water) was added, and the solution was left overnight at 4°C. Compound 97 (29 μg) was obtained in solution / Tris buffer, pH 8 (final concentration = 1.78 mg / mL) using the same method as that used for the purification of compounds 74 and 76.

[0400] 3.28 BHA[Lys]4[((α-NH-COPEG 24 -NH-COPEG4(PhTzMe) / TCO-PEG3-nanobody) 1-3 (α-Lys(α-NHCy5)1(ε-NHDFO)1)1(α-NH2) 0-2 )(ε-NH-COPEG 1000 )4], G2, compound 98 A solution of TCO-PEG3-aldehyde (conjugate probe) linker (0.7 mg, 1.46 μmol) / DMSO (50 μL) was added to a solution of nanobody ("Nanobody-N3-C-") (compound 79) (1.07 mg / mL stock solution / PBS buffer, pH 6.5, 931 μL), followed by the addition of NaBH3CN (0.09 mg, 1.5 μmol) / water (19 μL). The reaction mixture was cooled to 4°C, and the reaction was monitored by UPLC analysis. After 16 hours, the reaction mixture was diluted with PBS buffer (pH 6.5) to a total volume of 2.0 mL and purified by centrifugal ultrafiltration using PBS buffer pH 6.5 (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO; 14 x 450 μL). UPLC: 5-20-30ACN%, 15 min, 0.01% TFA buffer used; nanobody (compound 79) Rt = 8.46 min, m / z 13802; product: 9.99 min, m / z 14263. To a nanobody-PEG3-TCO solution (500 μL PBS pH 6.5), a solution of G2 dendrimer compound 107 (179 μg, 0.020 μmol) / MQ water (15 μL) was added. After standing overnight at 4°C, the reaction mixture was purified using the same method as used for the purification of compounds 74 and 764 to obtain compound 99 (12 μg; shown in Figure 1i, lane 1) as solution / Tris buffer, pH 8 (final concentration = 575 μg / mL). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0401] Example 4: Synthesis of HER2-targeted dendrimer complexes a) Nanobody arrangement information Nanobody 2D3 arrangement compound 79 EVQLESGSLVQPAGSLLSCAASGFTFDDYAMSWVLVPGKLEWVSSINWSGTHTDYADSVKGRFTISRNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSAGGTQTVSS Nanobody 2D3N-terminal tag, TEV, C-terminal azide ("N-terminal tag-nanobody-N3") GGSHHHHHHGMASMTGGQQMGRDLYENLYFQGEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS# Nanobody 2D3, C-terminal tag, TEV, azide ("Nanobody-N3-C-terminal tag") EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS#ENLYFQGHHHHHH #=Non-natural amino acids.

[0402] B) Nanobody-N3 expression plasmid The coding sequence of anti-HER2 nanobody clone 2D3 (US Patent No. 20110028695(A1) Sequence ID: 1986) was inserted into the expression plasmid pET-His6-TEV-1B (Addgene plasmid 29653) using standard molecular biological techniques. A codon-optimized DNA sequence of E. coli K12 was synthesized and cloned into plasmid pET-His6-TEV-1B.

[0403] To incorporate non-natural amino acids into recombinant proteins, an amber stop codon (TAG) was inserted in-frame at the end of the nanobody coding sequence, followed by an ochre (TAA) or opal (TGA) stop codon to terminate translation. When using a his6 tag at the C-terminus, an amber stop codon (TAG) was inserted in-frame at the end of the nanobody coding sequence, followed by a sequence encoding the TEV protease cleavage site and the 6his tag, and then an ochre (TAA) or opal (TGA) stop codon for translation termination.

[0404] c) Expression and purification of nanobody-N3 Anti-HER2 nanobody 2D3 expression was performed using nanobody expression plasmids and orthogonal pair expression plasmids pEVOL-pAcFRS.2.t1 (Amiram et al., Nat. Biotechnol (2015), 33(12)) to transform *E. coli* strain B95(DE3) (Mukai et al., Scientific Reports (2015), 5,9699). Cells were incubated in Terrific broth (25 g / L tryptone, 30 g / L yeast, and 5 g / L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) in baffled shake flasks at 37°C. 600Cells were cultured until a cell density of 0.7–1.0 was reached. Recombinant protein expression was induced by adding 1.5 mM IPTG and 0.05% w / v L-(+)-arabinose, followed by 1.5 mM p-azidophenylalanine, and carried out at 25°C for 20 hours. Cells were collected by centrifugation, homogenized under high pressure, and then treated with a protease inhibitor cocktail, lysozyme, and DNAse to produce a cell lysate. Insoluble materials could be dissolved in refolding buffer (6 M guanidine HCl, 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8). After clarification by high-speed centrifugation and filtration through a 0.45 μM membrane filter, his6-tagged nanobodies were purified by immobilized metal affinity chromatography (IMAC) using nickel-charged nitrilotriacetate-agarose. On-column refolding was achieved by washing the initially bound protein with a column volume of 3M guanidine HCl, 50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8, followed by two column volumes of 50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8. The bound protein was eluted with two column volumes of 50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8. After IMAC, -nanobody-N3 was further purified by anion exchange chromatography using a HiTrap Q HP column (GE Healthcare, catalog 17115301). The binding and washing steps were performed using Tris buffer (20mM, pH 8), and elution was performed using a 0% to 50% gradient of 1M NaCl buffer (1M NaCl, 20mM Tris, pH 8). The obtained related nanobody fractions were buffered and exchanged with 20 mM Tris, pH 8, using an Amicon 10k MWCO filter unit (Merck, catalog UFC901008).

[0405] d) Conjugation of nanobody-N3-C terminal tags to dendrimers Except for using the linker DBCO-Glu-NHPEG24CO-NHPEG3-TCO (compound 51, Click Chemistry Tools), the nanobody-N3-C terminal tags were conjugated to dendrimer compounds 81-85 using bio-orthogonal click chemistry, following the procedure described for compounds 74 and 76, to generate compounds 86-95.

[0406] When compounds 41-45 were run on the gel, bands were observed in the appropriate microwave mass (MW) of the nanobody-dendrimer complexes. The purity of each nanobody-dendrimer complex was confirmed using non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 3). After electrophoretic separation on 4-15% polyacrylamide gel (Bio-rad, catalog 4561086), imaging with a Typhoon Biomolecular Imager (GE Healthcare) revealed that the nanobody-dendrimer complexes appeared as fluorescent bands approximately corresponding to the size of the dendrimer plus the additional mass of the nanobodies. Subsequent staining with Coomassie Brilliant Blue (CBB) confirmed the presence of nanobodies in the same locations. No other bands were detected by CBB, indicating the absence of unreacted nanobodies and high purity of the preparation.

[0407] Example 5: SPR binding test of affibody-MMAE dendrimer and Erb2 ECD Direct fixation of ErbB2-ECD Using a ProteOn XPR36 instrument, ErbB2-ECD was immobilized on the surface of a GLC sensor chip (Bio-Rad) at 25°C using HBS-P+ electrophoresis buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween20). This immobilization was performed using the standard amine coupling method described in the amine coupling kit (Bio-Rad). Lane 1 was activated with a 50:50 mixture of EDC (0.5 mM) and sulfo-NHS (0.125 mM). ErbB2 protein was diluted to 2 mg / mL, pH 5.0, with 10 mM sodium acetate and injected onto the activated surface channel. The remaining activated site was blocked with 1 M ethanolamine-HCl (pH 8.5). Protein binding was observed with an average response level of approximately 590 RU (1 RU = 1 pg protein / mm2).

[0408] SPR experimental analysis All SPR binding experiments were performed at 25°C using HBS-EP+ / BSA (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, 0.1 mg / ml BSA) as the electrophoresis buffer (RB) of the instrument. After immobilization, various concentrations of anti-ErbB2 affibodies and their MMAE-dendrimer complexes were injected onto the immobilized ErbB2 protein at 60 μl / min, and their association was monitored for 90 seconds. Subsequently, electrophoresis buffer was injected into the bound Ab-ErbB2 complex, and dissociation was observed for up to 60 minutes. Herceptin and affibo controls did not dissociate from the tip surface in electrophoresis buffer within a reasonable time frame (within 60 minutes). All binding measurements were performed three times.

[0409] Processing and analysis of SPR data The collected experimental data were processed using Scrubber-Pro software (www.biologic.com.au). Each set of experimental data was fitted to a Langmuir 1:1 coupling model to derive kinetic parameters (ka = association rate constant and kd = dissociation rate constant) and equilibrium dissociation constant (KD = kd / ka). All coupling parameters are reported as mean + / - standard deviation.

[0410] SPR results The affibody-MMAE-dendrimer complex specifically bound to the ErbB2 protein through a certain process, yielding a classical kinetic binding profile. Subsequently, kinetic binding analysis of the affibody-MMAE-dendrimer complex was performed using a dose-response method. The kinetic and affinity parameters obtained from the SPR sensorgram are summarized below. Note that, since this affibody is a dimer, its binding constant is lower than that observed with monomeric affibody used in dendrimer complexes. No binding was observed with compounds 20 and 21. This result clearly demonstrates nanomolar specific binding to the affibody-dendrimer target, even at smaller amounts compared to native affibody. [Table 4]

[0411] Example 6: Cell growth inhibition (SRB assay) by affibody-MMAE dendrimers HER2 - (ES2) and HER2 + Inhibition of cell growth in the (SKOV3) cell line was measured using a sulforhodamine B (SRB) assay [Voigt W. "Sulforhodamine B assay and chemosensitivity," Methods Mol. Med. 2005, 110, 39-48.], with each experiment performed twice on various cancer cell lines after 72 hours. GI50 is the concentration required to inhibit the growth of all cells by 50%, according to the NCI standard protocol.

[0412] All compounds were tested based on equivalent drug load. The results indicate that PEG1100-targeted dendrimer complexes are more effective in inhibiting cell growth than dendrimers with a PEG2000 surface. Affibodies alone were not effective in this assay in either HER2+ or HER2- cell lines. [Table 5]

[0413] Example 7: In vivo tolerability of dendrimer-MMAE dendrimers Mice were intravenously injected with a dendrimer (0.1-0.3 ml of PBS solution) once a week for three weeks (days 1, 8, and 15). Mice were weighed daily, and signs of toxicity were observed. Animals were monitored for up to 10 days after the final drug administration. Mice exceeding ethical endpoints (more than 20% weight loss, poor general condition) were immediately euthanized, and observations were recorded.

[0414] Administration of compound 26 at a dose of 1 mg / kg was well-tolerated in both nude SCID mice and balb / c mice, and no animals required slaughter due to poor health.

[0415] Example 8: In vitro efficiency of the complex [Table 6]

[0416] GI 50 result The cytotoxicity of Herceptin®, Kadcyla®, Lapatinib, compound 36 (control), and 2D3 nanobody-targeted dendrimers (compound 41) against MDA-MB-231, SKOV-3, SK-Br-3, NCI-N87, and OE-19 cells was evaluated using an MTT assay. Cells were seeded at a density of 5 x 10³ in 96-well plates and incubated overnight. Subsequently, cells were treated with 1 log units of serial dilutions of the test composition for 72 hours (6 days for Herceptin). During the last 2 hours of incubation, 10% medium volume of MTT (thiazolyl blue tetrazolium bromide (Merck, Cat#M5655, 5 mg / ml sterile solution)) was added. Reduction of MTT in living cells generates a purple, insoluble formazan metabolite. After 2 hours of incubation, all culture medium was removed from the assay plate, 100 μl of DMSO was added, and the absorbance at 570 nm was immediately read. GI 50 This is defined as a concentration that inhibits cell growth / proliferation by 50%. Cell viability and subsequent GI 50 The values ​​were obtained from blank-corrected dose-response curves using 4-parameter nonlinear curve fitting in GraphPad Prism 7.02. As a result, it was revealed that the exemplary complexes in this disclosure have potent cytotoxic effects, particularly against HER2-overexpressing cell lines such as SK-BR-3, NCI-N87, and OE19. [Table 7]

[0417] Generation of HER2+MDA-MB-231 breast cancer cell lines for paired hi / lo HER2 cell lines. MDA-MB-231 breast cancer cells were transfected with plasmid HER2 WT (Addgene, 16257) using Lipofectamine 3000, according to the manufacturer's instructions. After passage in the presence of 500 μg / ml Geneticin (Thermo Fisher, catalog 10131035), cells stably overexpressing HER2 WT were isolated by fluorescence-activated single-cell sorting using MoFlo Astrios (Beckman Coulter). The clonal cell population isolated in this process was further passaged with Geneticin, followed by a second cell sorting and screening for transgene expression. Subsequently, selected clones were further grown, and a stock of this cell line, called MDA-MB-231 / HER2, was cryopreserved to create a master stock of stably transfected cells.

[0418] IC50 results for the lo / hi expression MDA-MB-231 model. The cytotoxicity of compound 36 (control) and compound 41 (target) against MDA-MB-231 and MDA-MB-231 / HER2 transfected cells was evaluated using the alamarBlue assay. 5 x 10⁶ samples were taken in a 96-well plate. 3 Cells were seeded at a density and incubated overnight. The cells were then treated with the indicated concentration of the test composition for 48 hours. AlamarBlue (Thermo Fisher, DAL1025) was added during the last 4 hours of incubation. Reduction of alamarBlue within the viable cells generates a red fluorescent metabolite, which can be read with a plate reader (excitation 560 nm / emission 610 nm). Cell viability and subsequent IC (Implantation) were then assessed. 50 The ICs were obtained from blank-corrected dose-response curves using 4-parameter nonlinear curve fitting and variable slope (4 parameters) in GraphPad Prism 7.02. The table below shows the ICs between compound 36 (control) and compound 41 (target) and MDA-MB-231 cells and MDA-MB-231 / HER2 cells. 50The values ​​are shown. IC values ​​for compound 36 (control) and compound 41 (target) against MDA-MB-231 / HER2. 50 The values ​​were 2.842 μM and 13.55 nM, respectively. Compound 41 (target) showed approximately 140 times greater inhibitory effect on MDA-MB-231 / HER2 cell growth compared to compound 36 (control).

[0419] Dose-response curves and IC2 for MDA-MB-231 and MDA-MB-231 / HER2 at different MMAE concentrations of compound 36 (control). 50 The values ​​are shown below. The values ​​represent the mean ± standard deviation (sd; n=4). [Table 8]

[0420] Dose-response curves and IC2 for compound 41 (target) at different MMAE concentrations for MDA-MB-231 and MDA-MB-231 / HER2. 50 The value is the mean ± standard deviation (sd; n=4). [Table 9]

[0421] Example 9: Binding of 2D3-dendrimer complex to HER2+ cells Using nanobody-dendrimer complexes of different sizes labeled with cyanine 5 (Cy5), compounds 42, 43, and 44, we demonstrated the binding of non-target compounds 37, 38, and 39 (G3, G4, and G5) to HER2+ human cell lines. HER2-expressing human metastatic cancer cell line MDA-MB-453 (ATCC HTB-131) and HER2-negative epithelial adenocarcinoma cell line MDA-MB-231 (ATCC HTB-26) were maintained at 37°C under a 5% CO2 humidified atmosphere in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS and penicillin-streptomycin (100 U / mL), and subcultured with trypsin until confluence. Human adenocarcinoma ovarian cell line SKOV-3 (ATCC® HTB-77) cells were subcultured in RPMI medium supplemented with 10% (v / v) FBS and penicillin-streptomycin (100 U / mL) under a 5% CO2 humidified atmosphere at 37°C until confluence.

[0422] For measuring cell associations using fluorescence microscopy, use an 8-well chamber slide with 1x10⁶ cells. 5 Cells were seeded at a rate of 1 / well and allowed to adhere overnight. The following day, non-conjugate dendrimer compounds 37, 38, and 39 (control) or nanobody dendrimer compounds 42, 43, and 44 were added to the medium until a final concentration of 0.5 μg / ml was reached, and the cells were incubated on ice for 1 hour. Cells were stained, and the cell membrane (wheat germ agglutinin-alexa fluor 488) and nucleus (Hoechst 33342) were visualized. Cells were washed three times with 400 μl of cold Fluorobrite medium supplemented with 10% FBS. Cells were imaged using a standard "Pinkel" DAPI / FITC / CY5 filter set with an Olympus IX83 microscope fitted with a 40x0.9NA air objective lens.

[0423] For measuring cell association by flow cytometry, a cell suspension was added to a 96-well assay plate at a rate of 1 × 10⁵ cells / well. After incubation with nanobody dendrimers or non-conjugate dendrimers on ice for 1 hour, the cells were washed three times with 200 μl of DMEM containing 10% FBS to remove unbound dendrimers. The cells were then resuspended in cold Fluorobrite medium supplemented with 10% FBS, and Cy5 fluorescence was measured using excitation light at 642 nm and emission focused at 661–691 nm. To measure the internalization rate, nanobody dendrimers were incubated with cells at 37°C for 24 hours, 4 hours, 1 hour, 0.5 hours, and 0.1 hours, after which unbound nanobody dendrimers were removed by washing and analyzed by flow cytometry.

[0424] In vitro meeting trial MDA-MB-231, MDA-MB-231 / HER2 (HER2 knock-in) (as described above), or SKOV-3 cells were seeded in 500 μL of suitable growth medium supplemented with 10% (v / v) fetal bovine serum (FBS) in 24-well plates (1 x 10⁵ cells per well). Compound 36 or 41 was incubated at 3.33 nM, 37°C, and under a 5% CO₂ humidified atmosphere for incubation times ranging from 1 to 24 hours. After incubation, unbound / unassociated particles were removed from adherent cells by gently washing three times with DPBS (300 μL / well). Cells were removed from the plates by treatment with TrypLE® Express Enzyme (1X), no phenol red (150 μL / well) at room temperature for 5–10 minutes. The plates were then placed on ice. Subsequently, cell binding and association of the sample were determined by flow cytometry by obtaining signals from Cy5.

[0425] Flow cytometry: These results indicate that nanobody-dendrimer compounds 42, 43, and 44 bind to HER2-positive MDA-MB-453 cells. Non-conjugate dendrimer compounds 37, 38, and 39 did not bind to MDA-MB-453 cells. The data shown are the mean MFI of cells treated in three wells, shown with the standard deviation. Statistical analysis was performed using ANOVA with Dunnett's multiple comparison test. [Table 10]

[0426] Flow cytometry results These results clearly demonstrate minimal binding of compound 36 (control) to all three cell lines over 24 hours. Compound 41 (target) showed increased binding depending on the HER2 receptor expression level of the cell lines. Flow cytometry revealed that after 24 hours, MDA-MB-231 / HER2 cells treated with compound 41 (146.1±9.6) showed approximately 9 times stronger fluorescence compared to cells treated with compound 36 (16.05±1.89). Furthermore, SKOV-3 cells treated with compound 41 (277.5±5.9) showed approximately 16 times stronger fluorescence compared to cells treated with compound 36 (16.4±6.86). The results are shown in Figure 4.

[0427] Dendrimer sizes G3-G5: Additional dendrimer generation studies were conducted: compounds 37 and 42 (G3), compounds 38 and 43 (G4), and compounds 39 and 44 (G5), both unconjugated and conjugated to 2D3, respectively. The unconjugated control dendrimer compounds 37, 38, and 39 showed significantly less association with MDA-MB-231 (HER2-negative) and MDA-MB-231 / HER2 (HER2-positive). The 2D3-conjugated dendrimers 42, 43, and 44 significantly associated with MDA-MB-231 / HER2 over 24 hours, with compound 44 (G5) having the highest cell association rate (73.47% ± 0.92), followed by compound 42 (G3, 60.73 ± 0.21) and compound 43 (G4, 56.27 ± 1.02). Regardless of dendrimer formation, 2D3 HER2-nanobodies conjugated with dendrimers exhibit significantly improved binding to cells overexpressing the HER2 receptor.

[0428] Percentage of 24-hour cell association values ​​between compounds 42 (G3), 43 (G4), and 44 (G5) and MDA-MB-231 and MDA-MB-231 / HER2 cells. Values ​​are mean ± standard deviation (SD; n=3). [Table 11] [Table 12] [Table 13] [Table 14]

[0429] Percentage of 24-hour cell association values ​​between compounds 37 (G3), 38 (G4), and 39 (G5) and MDA-MB-231 and MDA-MB-231 / HER2 cells. Values ​​are mean ± standard deviation (SD; n=3). [Table 15] [Table 16]

[0430] Mean fluorescence intensity values ​​over 24 hours for compounds 42 (G3), 43 (G4), and 44 (G5) in MDA-MB-231 cells and MDA-MB-231 / HER2 cells. Values ​​are mean ± standard deviation (SD; n=3). [Table 17] [Table 18] Mean fluorescence intensity values ​​over 24 hours for compounds 37 (G3), 38 (G4), and 39 (G5) in MDA-MB-231 cells and MDA-MB-231 / HER2 cells. Values ​​are mean ± standard deviation (SD; n=3). [Table 19] [Table 20]

[0431] The flow cytometry results for these complexes are shown in Figure 5.

[0432] Example 10: Test demonstrating the internalization of the complex into HER2-overexpressing cells Confocal cell uptake MDA-MB-231, MDA-MB-231 / HER2, and SKOV-3 cells were plated at 1.0 x 10⁴ cells / well onto μ-slide 8-well chambered coverslips (ibidi) and incubated overnight at 37°C with 5% CO₂. Compounds 36 and 41 (3.33 nM) were then added, and the cells were incubated for 1, 3, 6, and 24 hours. After washing, the cells were fixed with 1% paraformaldehyde for 20 minutes at room temperature. The cell membranes were treated with Alexa Fluor488-wheat germ agglutinin (AF488-WGA, 5 μg mL). -1The cells were stained with 4',6-diamidino-2-phenylindole (DAPI, 2 μg mL) at room temperature. -1 Counterstaining was performed using DPBS at room temperature for 10 minutes. Fluorescence images and optical sections were collected using a confocal microscope (Leica SP8). Images were processed with Fiji (Image J 1.52n).

[0433] Confocal microscopy images are shown in Figures 6-8. In the confocal images, even after 24 hours of incubation, compound 36 (Figure 6a) and compound 41 (Figure 6b) showed almost no association with MDA-MB-231. On the other hand, target compound 41 was internalized by MDA-MB-231 / HER2 and SKOV-3 cells after 24 hours of incubation (Figures 7a and 8a), while compound 36 was not internalized (Figures 7b and 8b).

[0434] Example 11 Tumor distribution of tritium-labeled MMAE conjugate dendrimers (compounds 40 and 45) MDA-MB-231 / HER2 cells (5 x 10⁶ cells in 50 μL of PBS: Matrigel) were subcutaneously transplanted into the fourth mammary gland adipose pad of female NOD / SCID mice (5-7 weeks old). The solid tumor was 100 mm 3 The mice were allowed to grow until (approximately 3-4 weeks). The mice were divided into two distinct groups of 6 mice each, and either compound 40 (control group) or compound 45 (targeted group) (0.5 μCi per 100 μL, PBS pH 7) was injected into the tail vein under isoflurane sedation. After 48 hours, the mice were anesthetized with isoflurane, and blood was collected by cardiac puncture immediately before cervical vertebral dislocation. Subsequently, selected organs (tumors, liver, spleen, kidney, pancreas, lung, heart, and brain, etc.) were removed, weighed, and tritium was measured. 3 H) was processed for in vivo distribution.

[0435] The results are shown in Figure 9. Targeted dendrimer compound 45 accumulated in HER2-positive tumors (3.53% dose / g ± 0.43), representing approximately 80% increased tumor uptake over a wider range than the non-conjugate control compound 40 (1.88% dose / g ± 0.28) (p<0.05). The difference in blood concentrations between the targeted dendrimer and the control dendrimer was not significant, at 4.52% dose / g ± 0.24 and 3.69% dose / g ± 0.31, respectively. Since the difference in pharmacokinetics is unlikely to affect tumor retention, dendrimer retention was improved by 2D3HER2-nanobody targeting.

[0436] Example 12: Efficacy of tumor uptake and confocal imaging of Cy5-labeled MMAE conjugate dendrimers (compounds 36 and 41) in the SKOV3 tumor model. In the flank of a female NOD SCID mouse (8 weeks old), 3 x 10 6 Individual SKOV3 cells / PBS:Matrigel (1:1) were subcutaneously inoculated. The solid tumor was 200 mm 3 The mice were allowed to grow until approximately 3 weeks. The mice were divided into five different groups and administered either compound 36 (control group) or compound 41 (target group) (0.5 mg / kg in MMAE equivalent in 250 μL of PBS, pH 7), Kadcyla® 40 mg / kg, and Herceptin® (40 mg / kg) by tail vein injection under isoflurane sedation.

[0437] (a) Confocal imaging Mice were anesthetized with isoflurane 48 hours later, and blood was collected by cardiac puncture immediately before cervical dislocation (n=2 / group). The tumors were then excised, fixed overnight with 4% paraformaldehyde, washed with PBS, and embedded in agarose. The tumors were then incised to 100 μm using a vibratome, and the sections were stained for nuclei (DAPI, simultaneous, time, supplier) and blood vessels (CD31, simultaneous, time, supplier). Fluorescence images and optical sections were collected using a confocal microscope (Leica SP8). The results are shown in Figures 10 and 11. Targeted dendrimer (compound 41) showed uptake in the central and peripheral parts of the tumor, but compound 36 did not.

[0438] (b) Effectiveness of the complex Tumor measurements were performed at regular intervals until the ethical endpoint was met (n=6 / group). The results are shown in Figure 12 (plot of mean tumor volume over time), Figure 13 (plot of survival rate over time), and Figure 14 (plot of mean weight change over time). Targeted dendrimer compound 41 showed complete tumor regression compared to compound 36, Herceptin®, and Kadcyla®.

[0439] Example 13: Kinetics of internalization of fourth-generation multiple nanobody dendrimers in MDA-MB-231 / HER2+ cell lines In the fourth generation, we compared the binding affinity of Cy5-labeled nanobody-dendrimer complexes containing one (single nanobody) or two to four (multiple nanobodies) anti-HER2 2D3 nanobodies separated as described above, and desferrioxamine (DFO), a sideropho-derived chelator conjugated to either compound 90 or 91, to HER2+ human cell lines with different numbers of nanobodies attached per dendrimer.

[0440] HER2 knock-in epithelial adenocarcinoma cell line MDA-MB-231 / HER2 (described above) was maintained at 37°C in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS and penicillin-streptomycin (100 U mL-1) under a 5% CO2 humidified atmosphere.

[0441] For cell binding measurements by flow cytometry, 20,000 cells / well were plated overnight in DMEM medium supplemented with 10% FBS in a 96-well culture plate. Single or multiple nanobody dendrimers were added to the cells at concentrations of 3 nM, 6 nM, 12 nM, and 30 nM, and incubated at 37°C for 0.5 hours, 1 hour, 2 hours, 4 hours, and 6 hours. Control cells prepared similarly were pre-cooled and incubated with 30 nM single and multiple nanobody dendrimers on ice for 6 hours.

[0442] After incubation with nanobody conjugated dendrimers, cells were washed three times with ice-cold PBS supplemented with 1% bovine serum albumin to remove unbound dendrimers, and then released from the plate using TrypLE® Express Enzyme (1X), no phenol red (Gibco, 12604013). Cells were resuspended in cold DPBS for analysis by flow cytometry (Stratedigm S1000EON, California). The presence of dendrimers was measured by obtaining a signal from Cy5. To estimate the amount of internalized dendrimers at each concentration and time point, the Cy5 signal of cells incubated with dendrimers on ice was taken as the largest surface-bound dendrimer assuming no internalization. This signal was subtracted from the signal measured in cells incubated at 37°C (this represents surface-bound, internalized dendrimers), and only the signals from internalized dendrimers were isolated (see Figure 15).

[0443] Multiple (compound 91) conjugated anti-HER2 nanobody dendrimers are internalized more rapidly in the body at lower concentrations compared to the single (compound 90) compound. At higher concentrations, compound 90 shows higher levels of internalization than compound 91 after approximately 2 hours.

[0444] Example 14: Confocal imaging of single and multiple nanobody complexes in HER2 Hi cell lines. HER2 hi,SKOV-3 cells were placed on a μ-slide 8-well chamber coverslip (ibidi) at a size of 1.0 x 10⁶. 4 Cells were plated in wells and incubated overnight at 37°C with 5% CO2. Compound 91 (multiple 2D3-dendrimer complexes) and Compound 90 (single 2D3-dendrimer complex) (3.33 nM) were then added and incubated for 1, 3, 6, and 24 hours. Cells were washed with DPBS and then fixed with 1% paraformaldehyde for 20 minutes at room temperature. The cell membrane was treated with Alexa Fluor488® complex (AF488-WGA, 5 μg mL) of wheat germ agglutinin. -1 The cells were stained with DPBS at room temperature for 10 minutes. The cell nuclei were stained with Hoechst 33342 (2 μg mL). -1 Counterstaining was performed using PBS at room temperature for 10 minutes. Fluorescence images and optical sections were collected using a confocal microscope (Leica SP8). Images were processed with Fiji (ImageJ 1.52p).

[0445] Confocal microscopy images are shown in Figures 16 and 17. Both compounds 90 and 91 bind to and internalize SKOV-3 cells after 24 hours of incubation. Notably, compound 91 shows a greater degree of binding and internalization compared to compound 90 at 3 and 6 hours.

[0446] Multiple (compound 91) conjugated anti-HER2 nanobody G4 dendrimers are internalized more rapidly than a single nanobody G4 dendrimer (compound 90) at this concentration, and the difference is visible up to 6 hours, but the difference disappears after 24 hours.

[0447] Example 15: Imaging study using targeted Zr radionuclide-containing dendrimer-SKOV3 breast cancer xenograft. The accumulation of 89Zr-labeled HER2-targeted and non-targeted dendrimer constructs was investigated in a SKOV3 mouse xenograft model of ovarian cancer. In vivo distribution up to day 9 post-injection was measured by PET-CT and validated (where possible) by ex vivo gamma-scintillation imaging of organs excised on days 2 and 9. The study was conducted in three parts.

[0448] Tumor development and growth 5x10 6 SKOV3 cells (50:50 Matrigel: 50 μL of PBS) were injected via SC into the right flank of healthy female NOD-SCID (approximately 20 g) 8-week-old mice. The tumors were allowed to grow for 4 weeks before injection of the imaging compound. All tumors were palpable during imaging, and their size during the imaging experiment was approximately 3-5 mm.

[0449] Test compound The compounds in the table below were labeled as follows. Compounds for Table Tests [Table 21]

[0450] Radiolabeling of test compounds with Zr-89 and RadioTLC analysis All constructs (pre-treated for iron removal as necessary: ​​http: / / jnm.snmjournals.org / content / 44 / 8 / 1271.long) were subjected to an excess of dendrimers (see Table 1 for excess amounts) in 0.1M pH 7.4 HEPES buffer. 89Zr was incubated with the solution at 37°C for 45 minutes. Samples were taken from each solution and mixed with 50 mM DTPA in a 1:1 ratio. 5 μL of each solution was spotted onto TLC paper (silica gel-impregnated Agilent iTLC-SG glass microfiber chromatography paper) and chromatographed in 50:50 H2O:ethanol. The plates were then imaged with an Eckert & Ziegler Mini-Scan and Flow-Count iTLC Reader. Unbound zirconium was removed by purification according to the manufacturer's protocol using 7K MWCO Zeba Spin Columns (Thermo Scientific) as needed. All samples showed labeling of over 95%. For quality control, free zirconium was removed. 89 Zr and DTPA bound 89 We conducted control experiments to monitor the elution behavior of Zr, and also, 89 To identify any unbound chelating agents labeled with Zr, each sample was performed with or without DTPA. Representative RadioTLC images are shown in Figure 18, which are: 89 All Zr is bound to the dendrimer and is free. 89 This indicates that Zr was not present. RadioTLC includes: [Table 22]

[0451] Test injection details In in vivo imaging experiments, 100 μL of the test compound was injected into the tail vein of two mice (29G needle, approximately 1.5–3.5 MBq), and tumor accumulation and in vivo distribution were observed at various time points. Ex vivo quantification was performed using gamma counting to determine organ distribution 9 days after injection. In in vivo distribution experiments, the construct was injected into two mice, and ex vivo tumor accumulation and in vivo distribution 48 hours after injection were monitored using gamma counting.

[0452] result PET-CT imaging Still images were taken for 30-90 minutes at 4 hours, 24 hours, 48 ​​hours, 5 days, 7 days, and 9 days after injection. PET images were reconstructed using the order-subset expectation maximization (OSEM2D) algorithm and analyzed using Inveon Research Workplace software (IRW 4.1) (Siemens), which allows for the fusion of CT and PET images and the definition of regions of interest (ROIs). CT and PET datasets from each animal were aligned using IRW software (Siemens) to ensure overlap of target organs. Activity per voxel was converted to nci / cc using a conversion factor obtained by scanning a cylindrical phantom filled with 89Zr of known activity, taking into account the efficiency of the PET scanner. Activity concentration was measured per 1 cm³ of tissue. 3 The results are expressed as a percentage of decay-corrected injectable activity per unit and can be approximated as injectable dose / g percent (ID / g%). The results are shown in Figure 19 as graphs showing the zirconium injectable dose rate (percentage) per gram in (a) the kidney, (b) the liver, and (c) the tumor over 9 days for compounds 89, 90, and 92. Representative PET images are shown in Figure 20. Representative maximum intensity projections of the radioisotope-labeled complexes. All PET data are expressed in becquerels / voxels (cm³) and thresholds have been set to highlight tumor uptake.

[0453] Organs were removed on days 2 and 9 after injection, and signal intensity was quantified and imaged by ex vivo gamma analysis. Furthermore, n=2 were collected from each cohort 48 hours later, and the in vivo distribution was evaluated by gamma analysis. The results of the ex vivo in vivo distribution are shown in the table in Figure 21, showing the tumor:organ ratio of ex vivo signal at injection dose / g on days 2 and 9.

[0454] As a result, particularly from the day 2 results for compound 88 (low molecular weight G3 dendrimer), a higher tumor-to-blood ratio was observed in the targeted samples compared to the non-targeted samples. On day 9, the tumor-to-blood ratio was even better in the targeted samples compared to the non-targeted samples, especially for compounds 90 and 92 (larger G4 and G5 dendrimers).

[0455] Furthermore, the table in Figure 22 also shows the percentage of Zr dose / gram injected into ex vivo tumors and organs.

[0456] The results show a high signal in the targeted tumor compared to the untargeted area, particularly for compounds 90 and 92 (larger G4 and G5 dendrimers) on day 2, and for compound 89 (G3 dendrimer) on day 9.

[0457] conclusion 1. Larger dendrimers resulted in better tumor accumulation. 2D3 showed rapid clearance and low accumulation. 2. Targeted therapy results in higher accumulation in tumors compared to untargeted dendrimers. 3. Compound 88 (G3 1K-nanobody) shows a significant enhancement of signaling in tumors compared to other small molecular weight dendrimers. 4. Compounds 90 and 92 (G4 and G5 nanobodies) showed ID / g of over 12% in tumors at 48 hours, and at 9 days they showed ID / g of over 4% and over 8%, respectively. 5. Low molecular weight nanobodies containing dendrimers, as well as nanobodies alone, exhibit high retention in the kidneys. No abnormal accumulation was observed in clearance organs, and liver and spleen signals showed expected concentration ranges typical of similar systems.

[0458] Example 16: G5 and PEG in combination with or without cabazitaxel 1000 In vitro association studies of dendrimers MDA-MB-231, MDA-MB-231 / HER2 (HER2 knock-in), or SKOV-3 cells were seeded (1 x 10⁵ cells per well) in 500 μL of appropriate growth medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin in 24-well plates. Incubation times ranged from 1 to 24 hours, cells were incubated with compound 70 (G5, PEG1000, CTX) or 78 (2D3, G5, PEG1000, CTX) at 3.33 nM, 37°C, and under a 5% CO₂ humidified atmosphere. After incubation, unbound / unassociated particles were removed from adherent cells by gently washing three times with DPBS (300 μL / well). Cells were removed from the plates by treatment with TrypLE® Express Enzyme (1X), no phenol red (150 μL / well) at room temperature for 5–10 minutes. The plates were then placed on ice. Cell binding and association of the samples were then determined by obtaining signals from Cy5 by flow cytometry.

[0459] Flow cytometry results These results clearly demonstrate minimal binding of compound 70 (control) to all three cell lines over 24 hours. Compound 78 (target) showed increased binding depending on the HER2 receptor expression level of the cell lines. Flow cytometry revealed that after 24 hours, MDA-MB-231 / HER2 cells (4,269±322) treated with compound 78 (target) showed approximately 340 times stronger fluorescence compared to cells treated with compound 70 (control) (12.4±0.64). Furthermore, SKOV-3 cells (1,817±60.8) treated with compound 78 (target) showed approximately 160 times stronger fluorescence compared to cells treated with compound 70 (control) (11.4±0.42). The results are shown in Figures 23-25.

[0460] Mean fluorescence intensity (MFI) values ​​over 24 hours for compound 70 (control) and compound 78 (target) with MDA-MB-231, MDA-MB-231 / HER2, and SKOV-3 cells. Values ​​are mean ± standard deviation (SD; n=2). [Table 23] [Table 24] [Table 25]

[0461] Those skilled in the art will understand that numerous variations and / or modifications can be made to the embodiments described above without departing from the broad general scope of this disclosure. Therefore, these embodiments are considered illustrative and not limiting in all respects.

[0462] References Abdollahpour-Alitappeh et al (2017) Novelty in Biomedicine 4: 145-151. Al-Lazikani et al. (1997) J Mol Biol 273: 927-948. Arezumand et al. (2017) Front Immunol 8:1746. Bethesda, Md., 1987 and 1991. Chetterjee et al. (2013) Biochemistry 52(10). Chothia and Lesk (1987) J Mol Biol 196: 901 -917. Chothia et al. (1989) Nature 342: 877-883. Giudicelli et al. (1997) Nucleic Acids Res 25: 206-211. Honnegher and Plukthun (2001) J Mol Biol 309: 657-670. Hussack et al. (2018) BMC Res notes 11(1):866. Kabat Sequences of Proteins of Immunological Interest, National Institutes of He alth, Messerer et al. (2004) Clin. Cancer Res 10(19): 6638-6649. Milla et al. (2012) Current Drug Metabolism 13(1): 105-119. Mukai et al. (2015) Scientific Reports 5: 9699. Nord et al. (1995) Protein Eng 8:601-608. Oroudjev et al. (2010) Mol Can Thera 9:2700-2713. Owen et al. (2011) WO2012167309. Padlan et al. (1995) FASEB J Off Publ Fed Am Soc Exp Biol 9: 133-9. Pohlmann et al. (2009) Clin Cancer Research 15(24): 7479-7491. Pruszynski et al. (2013) Nucl Med Biol 40(1): 52-59. Revets et al. (2011) US Application No. 20110028695 Vaneycken et al. (2011) FASEB J 25(7): 2433-2446. Verel et al. (2003) J Nucl Med 44(8): 1271-1281. Voigt (2015) Methods Mol Med 110: 39-48. Wu et al. (2018) Translational Oncology 11(2): 366-373. The inventions described in the original claims of this application are listed below. [Invention 1] A dendrimer-targeting agent conjugate, a) i) Core unit (C), and ii) A dendrimer comprising a constituent unit (BU), wherein each constituent unit is a lysine residue or an analog thereof, The core unit is covalently attached to at least two constituent units via amide linkages, and each amide linkage is formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit, forming a dendrimer; b) A HER2 targeting agent comprising a peptide portion having a maximum molecular weight of approximately 80 kDa and containing an antigen-binding site, which is covalently linked to the dendrimer by a spacer group; c) A therapeutic agent covalently bonded to the surface constituent unit of the dendrimer; d) Hydrophilic polymer groups covalently bonded to the surface constituent units of the dendrimer and The composite, including the above. [Invention 2] The complex according to Invention 1, wherein the peptide portion is selected from a heavy chain antibody, Fab, Fv, scFv, or a single-domain antibody. [Invention 3] The complex according to invention 1 or 2, wherein the peptide portion includes or consists of a heavy chain variable (VH) domain. [Invention 4] The complex according to any one of Inventions 1 to 3, wherein the peptide portion includes a light chain variable (VL) domain or consists of a light chain variable (VL) domain. [Invention 5] The composite according to any one of Inventions 1 to 4, wherein the targeting agent has a molecular weight of approximately 5 kDa to approximately 30 kDa. [Invention 6] The composite according to Invention 5, wherein the targeting agent has a molecular weight of approximately 5 kDa to approximately 15 kDa. [Invention 7] The composite according to Invention 6, wherein the targeting agent has a molecular weight of approximately 10 kDa to approximately 16 kDa. [Invention 8] The complex according to any one of Inventions 1 to 7, wherein the targeting agent comprises less than 120 amino acid residues. [Invention 9] The complex according to any one of Inventions 1 to 8, wherein the targeting agent comprises or consists of any of the amino acid sequences described herein. [Invention 10] A complex according to any one of Inventions 1 to 9, wherein one targeting agent is covalently linked to the dendrimer. [Invention 11] A composite according to any one of Inventions 1 to 9, wherein two or more targeting agents are covalently linked to the dendrimer. [Invention 12] The composite according to any one of Inventions 1 to 11, wherein the covalent bond between the targeting agent and the spacer group is formed by a reaction between complementary reactive functional groups present on the targeting agent precursor and the spacer group precursor. [Invention 13] The complex according to Invention 12, wherein the targeting agent precursor comprises a non-natural amino acid residue, and the non-natural amino acid residue has a side chain containing a reactive functional group. [Invention 14] The aforementioned non-natural amino acid residues

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Claims

1. A dendrimer-targeting agent conjugate, a) i) Structure: 【Chemistry 1】 Including the core unit (C), and ii) A dendrimer including a third or fourth generation component unit (BU), Each constituent unit is a lysine residue, The core unit is covalently attached to at least two constituent units via amide links, and each amide link is formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the constituent unit. The aforementioned dendrimer and; b) A HER2 targeting agent that is a single-domain antibody, wherein the single-domain antibody has a molecular weight in the range of 10 kDa to 16 kDa and is a single-domain antibody that includes an antigen-binding site, and the HER2 targeting agent includes the amino acid sequences of complementarity-determining regions (CDRs) 1, 2 and 3 derived from the amino acid sequence of SEQ ID NO: 1, and is covalently linked to the core unit by spacer groups containing polyethylene glycol (PEG) groups; c) A therapeutic agent covalently linked to the surface constituent units of the dendrimer via an enzymatically cleavable linker containing a peptide group, wherein the therapeutic agent is auristatin or a topoisomerase inhibitor; d) A hydrophilic polymer group covalently linked to the surface constituent unit of the dendrimer, wherein the hydrophilic polymer group is a PEG group with an average molecular weight of 900 to 2300 Da, and The composite, including the above.

2. The complex according to claim 1, wherein the targeting agent includes or consists of the amino acid sequence described in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

3. The composite according to claim 1 or 2, wherein the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting agent.

4. The complex according to any one of claims 1 to 3, wherein the therapeutic agent is monomethyl auristatin E.

5. The composite according to any one of claims 1 to 4, wherein the therapeutic agent is covalently linked to the surface constituent units of the dendrimer via a cleavable linker, and the cleavable linker comprises any one of a Val-Cit-PAB group, a Val-Ala-PAB group, a Val-Arg-PAB group, a Val-Ala-PAB-P-trigger group, or a Val-Arg-PAB-P-trigger group.

6. i) The composite according to any one of claims 1 to 5; ii) Pharmacopoeia-acceptable excipients and A pharmaceutical composition containing the following:

7. The pharmaceutical composition according to claim 6, for the treatment of cancer.

8. The pharmaceutical composition according to claim 7, wherein the cancer is ovarian cancer, breast cancer, gastric cancer, uterine cancer, or another cancer characterized by abnormal expression of the ERBB2 gene.

9. Use of the complex according to any one of claims 1 to 8 in the manufacture of a pharmaceutical product for the treatment of cancer.

10. The use according to claim 9, wherein the cancer is ovarian cancer, breast cancer, gastric cancer, uterine cancer, or another cancer characterized by abnormal expression of the ERBB2 gene.

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