Targeted dendrimer conjugates

Abstract

To provide conjugates useful for targeting cancers in which HER2 is overexpressed.SOLUTION: Provided are dendrimer-targeting agent conjugates comprising a dendrimer, a HER2 targeting agent, and a therapeutic agent, wherein the dendrimer comprises a core unit and a lysine or lysine analog building unit, the HER2 targeting agent is a peptide moiety having a molecular weight of up to about 80kDa and comprising an antigen binding site, covalently linked by a spacer group, and the therapeutic agent is covalently linked to surface building units of the dendrimer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates generally to therapeutic methods useful for targeting HER2-overexpressing cancers. More specifically, the present disclosure relates to targeted delivery of a therapeutic moiety using a dendrimer-targeting moiety conjugate comprising a HER2 antibody fragment or mimetic and a therapeutic moiety, such as a residue of a supercytotoxic agent. [Background technology]

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

[0003] Much effort has been expended to develop effective cancer treatments, but the problem of finding new treatments that have sufficient efficacy at the target, suitable pharmacokinetic properties to enable delivery to the site of action, sufficient duration of action, and a good safety profile is extremely difficult, time-consuming, and expensive.

[0004] The development of monoclonal antibody therapies, such as trastuzumab (Herceptin®) and rituximab (Mabthera®), is one approach that has achieved notable success. For example, Herceptin® was approved in 1998 and 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 forms of cancer, and blocking signals that stimulate cancer cell growth. However, Herceptin® also has drawbacks. For example, known side effects include myocardial damage and heart failure. Herceptin® is also not fully 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 active against rapidly dividing cells. Cisplatin, used for the treatment of various cancers, including ovarian, bladder, testicular, and squamous cell carcinoma, is a long-established example. Another group of cytotoxic agents are ultra-cytotoxic agents, such as auristatins, such as monomethyl auristatin E (MMAE), which are highly potent but can also cause severe toxicity and adverse side effects in some patients. As a result, such ultra-cytotoxic agents cannot be safely administered to patients as chemotherapy agents themselves. Research to identify means to harness such ultra-cytotoxic agents has led to the development of antibody-drug conjugates, such as brentuximab vedotin (Adcetris®), which contains MMAE conjugated to brentuximab (a CD30 antibody) via a linear linker and has been approved for the treatment of Hodgkin's 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 causes of this failure include lack of efficacy, lack of specificity, poor drug absorption / bioavailability, uncontrolled biodistribution, rapid metabolism and clearance, and instability during storage. Indeed, a balance must be struck between preventing the achievement of therapeutic cytotoxicity necessary to kill cancer cells and reducing adverse toxicity and side effects to a level that can be tolerated by patients.

[0007] Various approaches have been investigated to test and improve the therapeutic efficacy of compounds with suboptimal properties. For example, sustained-release formulations can be developed for compounds that are rapidly eliminated. One example in the field of chemotherapy is the product Onivyde®, a liposomal formulation of the active ingredient irinotecan, which significantly reduces its plasma excretion rate compared to conventional formulations (Messerer et al., 2004). Other technologies include the use of depot injectable formulations (e.g., Lupron Depot®). Dendrimer technology has also been investigated to improve the properties of pharmaceutically active agents and avoid formulation problems. For example, WO 2012 / 167309 describes the provision of drug-dendrimer conjugates, particularly those containing the poorly soluble pharmaceutically active agent docetaxel.

[0008] Despite these and other advances in technology, there is a need for further development of safe and effective therapeutics in the treatment of diseases such as cancer.

[0009] Any discussion of documents, acts, materials, devices, articles or the like in this specification is not intended to be an admission that any or all of such matters form part of the prior art or were common general knowledge in the art to which this disclosure pertains. Summary of the Invention

[0010] In a first aspect, a dendrimer-targeting agent conjugate is provided, the conjugate comprising: a) i) Core Unit (C), and ii) a dendrimer comprising building blocks (BU), each building block being a lysine residue or analog thereof; a dendrimer in which a core unit is covalently attached to at least two building blocks via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block; b) a HER2 targeting agent, which is a peptide moiety having a molecular weight of up to about 80 kDa and containing an antigen-binding site, covalently linked to the dendrimer by a spacer group; c) a therapeutic agent covalently linked to a surface building unit of the dendrimer; d) hydrophilic polymeric groups covalently linked to the surface building units of the dendrimer.

[0011] In some embodiments, the peptide moiety is selected from a heavy chain antibody, a Fab, an Fv, an scFv, or a single domain antibody. In some embodiments, the peptide moiety is a heavy chain variable (V H ) domain or heavy chain variable (V H In some embodiments, the peptide moiety consists of a light chain variable (V L ) domain or light chain variable (V L ) 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 is composed 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, wherein the non-natural amino acid residue has a side chain that comprises a reactive functional group.

[0017] In some embodiments, the unnatural amino acid residue is: [ka] is a residue of

[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, hi 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 the 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 reaction of a spacer group precursor with a complementary reactive functional group present on the targeting agent precursor. In some embodiments, the spacer group comprises 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 an 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 a surface building unit of the dendrimer via a linker. In some embodiments, the therapeutic agent is covalently linked to a surface building unit of the dendrimer via a cleavable linker. In some embodiments, the cleavable linker comprises a Val-Cit-PAB group.

[0024] In some embodiments, the conjugate comprises a hydrophilic polymer group covalently linked to a surface-building unit of the dendrimer. In some embodiments, the hydrophilic polymer group is a PEG group, which is covalently linked to a surface-building unit of the dendrimer. In some embodiments, the PEG group has an average molecular weight in the range of about 500 to about 2500 g / mol.

[0025] In some embodiments, the spacer group comprises 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 building blocks. In some embodiments, the dendrimer has 3 to 5 generations of building blocks. In some embodiments, the dendrimer has 3 generations of building blocks. In some embodiments, the dendrimer has 4 generations of building blocks. In some embodiments, the dendrimer has 5 generations of building blocks.

[0030] In some embodiments, each of the constituent units comprises: [ka] Includes.

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

[0032] In some embodiments, the complex 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 the free therapeutic agent.

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

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

[0036] In a third aspect, i) a conjugate described herein; ii) a pharmaceutically acceptable excipient.

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

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

[0039] In a fourth aspect, there is provided a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition described herein.

[0040] In a fifth aspect, there is provided the use of a conjugate or composition as described herein in the manufacture of a medicament 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 aberrant expression of the ERBB2 gene.

[0042] In a sixth aspect, there is provided a method of killing HER2-expressing cells, comprising: A method is provided which includes contacting a conjugate as defined herein with a HER2-expressing cell, thereby allowing the conjugate to be internalized into the cell, whereby the therapeutic agent kills the HER2-expressing cell.

[0043] It will be understood that further aspects, embodiments, and examples are described herein, which may include one or more of the above-described embodiments or features. [Brief explanation of the drawings]

[0044] [Figure 1] FIG. 1 shows the results of SDS-PAGE analysis of compounds 74, 75, and 76 stained with Coomassie blue. [Figure 2] FIG. 1 shows SDS-page and fluorescent imaging analysis of compounds 77 and 78. [Figure 3]Figure 1 shows SDS-PAGE and fluorescence imaging analysis of compounds 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] Figure 1 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 per measurement. Values ​​are mean ± standard deviation (SD; n=3). [Figure 5-1] This figure shows the results of flow cytometry analysis of HER2-positive cells (MDA-MB-231 / HER2) and HER2-negative cells (MDA-MB-231) incubated with dendrimer at 3.33 nM for 24 hours at 37°C. At least 10,000 cells were counted per measurement. Values ​​are means ± standard deviation (SD; n = 3). [Figure 5-2] (Same as above.) [Figure 6] Confocal microscopy images of MDA-MB-231 cells treated with a) 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] Confocal microscopy images of MDA-MB-231 / HER2 cells treated with a) 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]Confocal microscopy images of SKOV-3 cells treated with a) 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] Figure 1 shows tumor and blood distribution data after 3H-labeled Compound 40 and Compound 45 dendrimers were administered 48 hours after sacrifice. All data were normalized by tissue content. All data represent mean ± SEM (n=5); (NS=not significant; *=p-value<0.05). [Figure 10] Figure 1 shows representative ex-vivo tumor distribution of compounds 36 and 41 after sacrifice at 48 hours. Data show typical fields of view for (a) untargeted dendrimer (compound 36) and (b) targeted dendrimer (compound 41). [Figure 11] FIG. 1 shows images demonstrating that the targeted dendrimer (compound 41) was taken up in the central and peripheral regions of the tumor, whereas the control compound 36 was not. [Figure 12] FIG. 1 shows a plot of mean tumor volume over time following treatment with vehicle, control compound 36, targeted complex 41, Kadcyla®, or Herceptin® in mice inoculated with SKOV3 cells. [Figure 13] FIG. 1 shows survival over time following treatment with vehicle, control compound 36, targeted complex 41, Kadcyla® or Herceptin® in mice inoculated with SKOV3 cells. [Figure 14] FIG. 1 shows the mean percent weight change over time following administration of vehicle, control compound 36, targeted complex 41, Kadcyla® or Herceptin® to mice inoculated with SKOV3 cells. [Figure 15] FIG. 1 shows the internalization kinetics of fourth generation dendrimers and single (compound 90, MFI single) or multiple (compound 91, MFI multiple) conjugated anti-HER2 nanobodies. [Figure 16] Confocal microscopy images show SKOV-3 cells after incubation with compound 91 (multiple 2D3-dendrimer conjugates) at 37 °C for a) 1 h, b) 3 h, c) 6 h, or d) 24 h. 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] Confocal microscopy images show SKOV-3 cells after incubation with compound 90 (single 2D3-dendrimer complex) at 37 °C for a) 1 h, b) 3 h, c) 6 h, or d) 24 h. Compound 91 is stained with Cy5 (magenta), cell membranes with AF488-WGA (green), and nuclei with Hoechst 33342 (blue). Scale bar = 30 μM. [Figure 18] 1 is a radio-TLC image of a compound of the present disclosure showing that 89Zr is bound to the dendrimer. [Figure 19-1] 1 is a graph showing the percentage of injected zirconium dose per gram in (a) kidney, (b) liver, and (c) tumor over a 9-day period for compounds 89, 90, and 92. [Figure 19-2] (Same as above.) [Figure 20] Representative maximum intensity projections of PET images of the radioconjugate from animals from 4 hours to 9 days post-treatment. Data are expressed in becquerels / voxel (cm) and thresholded to highlight tumor uptake. [Figure 21] 1 is a table showing tumor:organ ratios of ex vivo signal of injected zirconium (dose / gram) for compounds of the present disclosure on days 2 and 9. [Figure 22] 1 is a table showing the percentage of injected zirconium (dose / gram) in ex vivo tumors and organs for compounds of the present disclosure on days 2 and 9. [Figure 23] FIG. 1 shows flow cytometry analysis and mean fluorescence intensity values ​​of Compound 70 (control) and Compound 78 (target) with MDA-MB-231 / HER2 cells over 24 hours. [Figure 24] FIG. 1 shows flow cytometry analysis and mean fluorescence intensity values ​​of compound 70 (control) and compound 78 (target) with MDA-MB-231 cells over 24 hours. [Figure 25] Figure 1 shows flow cytometry analysis and mean fluorescence intensity values ​​of Compound 70 (control) and Compound 78 (target) with SKOV-3 cells over 24 hours. Keys indicating sequence listing: SEQ ID NO: 1: 2D3 nanobody. SEQ ID NO: 2: 2D3 nanobody with N-terminal tag, TEV, C-terminal azide. SEQ ID NO: 3: 2D3 nanobody with C-terminal tag, TEV, azide. DETAILED DESCRIPTION OF THE INVENTION

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

[0046] As used herein, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, and not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. As used herein, the term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is considered to explicitly provide support for both meanings or either meaning.

[0047] As used herein, unless stated to the contrary, the term "about" refers to ±10%, more preferably ±5% of the specified value.

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

[0049] As used herein, the term "subject" refers to any organism susceptible to a disease or condition associated with HER2 (human epidermal growth factor receptor 2). In one embodiment, the subject can 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] As used herein, 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 aggressiveness of cancer. In one embodiment, the term "treating cancer" refers to extending progression-free survival. As used herein, the term "progression-free survival" refers to the period during and after cancer treatment in which a patient remains with the disease, i.e., cancer, without a recurrence of the disease or an increase in symptoms of the disease. This refers to the length of time.

[0051] As used herein, the term "prevention" includes preventing a particular disorder or condition. For example, as used herein, the term "preventing cancer" refers to preventing the onset or persistence of symptoms associated with cancer. In one embodiment, the term "preventing cancer" refers to slowing or stopping the progression of cancer. In one embodiment, the term "preventing cancer" refers to slowing or preventing metastasis.

[0052] As used herein, a "therapeutically effective amount" refers to a conjugate containing a therapeutic agent administered in an amount sufficient to alleviate or prevent to some extent one or more of the symptoms of the disorder or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. In one embodiment, the term "therapeutically effective amount" refers to a conjugate administered in an amount sufficient to result in a reduction in the size of a cancerous tumor. In one embodiment, the term "therapeutically effective amount" refers to a conjugate administered in an amount sufficient to result in an extension of progression-free survival. As used herein, an "effective amount" refers to an amount of conjugate effective to achieve a desired pharmacological effect or therapeutic improvement without undue side effects, or with a reduced side effect profile. A therapeutically effective amount can be determined by routine experimentation, including, but not limited to, a dose-escalation clinical trial. A "therapeutically effective amount" includes, for example, a prophylactically effective amount. In one embodiment, a prophylactically effective amount is an amount sufficient to prevent metastasis. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject, depending on variations in the compound's metabolism, the age, weight, and general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. An appropriate "effective amount" in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0053] As used herein, the term "attached" refers to a connection between chemical moieties by a covalent bond. The term "covalent bond" is used interchangeably with the term "covalently attached."

[0054] dendrimer In a first aspect, a dendrimer-targeting agent conjugate is provided, the conjugate comprising: a) i) Core Unit (C), and ii) a dendrimer comprising building blocks (BU), each building block being a lysine residue or analog thereof; a dendrimer in which a core unit is covalently attached to at least two building blocks via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block; b) a HER2 targeting agent, which is a peptide moiety having a molecular weight of up to about 80 kDa and containing an antigen-binding site, covalently linked to the dendrimer by a spacer group; c) a therapeutic agent covalently linked to a surface building unit of the dendrimer; d) hydrophilic polymeric groups covalently linked to the surface building units of the dendrimer.

[0055] The dendrimer conjugate has been demonstrated to be a highly effective anticancer agent in in vitro and in vivo studies, and experimental studies have demonstrated improved efficacy over Herceptin® and Kadcycla® (a HER2-emtansine antibody-drug conjugate). In contrast to Herceptin® itself, the conjugate is rapidly internalized into HER2-overexpressing cancer cells. The encapsulation properties are expected to aid in the delivery of a high percentage of therapeutic agents administered via the conjugate to the desired site of action. The ability to specifically deliver therapeutic moieties to target cells, through rapid internalization, helps reduce unwanted side effects (i.e., toxicity) associated with exposure of otherwise non-cancerous cells to cytotoxic agents. Such an approach is particularly advantageous when delivering cytotoxic therapeutic moieties (e.g., ultracytotoxic therapeutic moieties), which may have toxic effects if circulated freely in plasma.

[0056] As used herein, the term "dendrimer" refers to a molecule comprising a core and dendrons attached to the core. Each dendron is composed of generations of branched building blocks, with each generation of building blocks resulting in a branched structure with increasing branching. "Conjugates" may include pharmaceutically acceptable salts or solvates as defined herein.

[0057] As used herein, the term "building block" refers to a branched molecule that is a lysine residue or analog thereof that has three functional groups: one functional group (e.g., derived from a carboxylic acid group) for attachment to the core or previous generation building block, and at least two functional groups (e.g., derived from amines) for attachment to the next generation building block or to form the surface of the dendrimer molecule.

[0058] Those skilled in the art will appreciate that the conjugates may be produced in various forms, such as, for example, salt forms (e.g., if there are ionizable groups present in the conjugate), different solvates, etc. It will be understood that the present disclosure relates to all such forms of dendrimer-targeting moiety conjugates.

[0059] Suitable salts of the complex include those formed with organic or inorganic acids or bases.In this specification, the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts.Exemplary acid addition salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts such as potassium and sodium salts, alkaline earth metal salts such as calcium and magnesium salts, and salts with organic bases such as dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di-, and tri-lower alkylamines such as ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl, or dimethylpropylamine, or mono-, di-, and tri-hydroxy lower alkylamines such as mono-, di-, and triethanolamine. Pharmaceutically acceptable salts may contain another molecule, such as an acetate ion, a succinate ion, or another counterion. A counterion can be any organic or inorganic moiety that stabilizes the charge of the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, it can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ion.It will also be understood that non-pharmaceutically acceptable salts are within the scope of the disclosure, as they may be useful as intermediates in preparing pharmaceutically acceptable salts, or may be useful during storage or transport.

[0060] Those skilled in the art of organic chemistry and medicinal chemistry will understand that many organic compounds can form complexes with solvents in which they are reacted, precipitated, or crystallized. Such complexes are known as "solvates." For example, complexes with water are known as "hydrates." As used herein, the phrase "pharmaceutically acceptable solvates" or "solvates" refers to the association of one or more solvent molecules with a compound of the present 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 core units (C) of the dendrimer are covalently attached to the building blocks via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the building block. Thus, the core units may be formed, for example, from core unit precursors containing amino groups. Any suitable amino-containing molecule may be used as the core unit precursor.

[0062] Typically, the core unit provides additional functionality to the nitrogen atom used for covalent attachment of the building blocks, and may contain a functional group, for example, to attach a targeting moiety via a spacer group. In other words, the HER2 targeting agent is typically covalently linked to the dendrimer core unit via a spacer group. In some embodiments, the core unit can be derived from a precursor having three reactive nitrogen atoms, two of which can be used to attach building blocks and one of which can be used to attach a spacer group.

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

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

[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 building blocks. For example, in some embodiments, the core unit can be derived from ethylenediamine, 1,4-diaminobutane, or 1,6-diaminohexane. In some embodiments, the core unit has the following structure: [ka] wherein the core unit comprises a lysine residue (BHA-Lys) in which the acid moiety is capped with a benzhydrylamine to form the corresponding amide, e.g., a core unit precursor having two reactive (amino)nitrogens: [ka] It can be formed from

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

[0067] Configuration Unit The building blocks (BU) are lysine residues or analogs thereof and may be formed from suitable building block precursors, such as lysine or lysine analogs containing suitable protecting groups. The lysine analogs have two amino nitrogen atoms for binding to subsequent building blocks and an acyl group for binding to previous building blocks or core units. Examples of suitable building blocks include: [ka] are listed, The acyl group of each building block provides a covalent attachment point for attachment to the core or previous generation building block, and each nitrogen atom is attached to a subsequent generation building block or linked bond. The hydroxybenzoate provides a covalent attachment point for covalently attaching an end group such as a therapeutic agent.

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

[0069] In some preferred embodiments, the constituent units are each [ka] and; The acyl group of each building block provides a covalent attachment point for attachment to the core or a previous generation building block, and each nitrogen atom provides a covalent attachment point for covalent attachment to a subsequent generation building block or to a terminal group such as a linked therapeutic agent.

[0070] The outermost generation unit (BU outer ) may be formed by lysine or lysine analogue building blocks of those used in other generation building blocks (BU), as described above. The outermost generation building block (BU outer ) is the outermost generation structural unit from the core unit of the dendrimer, that is, the outermost generation structural unit (BU outer ) does not have any subsequent generation components attached to it.

[0071] It will be understood that the dendrons of a dendrimer can be synthesized to any desired number of generations, for example, by attaching building blocks (BUs) as appropriate. In some embodiments, the building blocks (BUs) of each generation may be formed from the same building block, e.g., all generations of building blocks may be lysine building blocks. In other embodiments, the building blocks of one or more generations may be formed from different building blocks relative to the building blocks of other generations.

[0072] The dendrimer portion typically has 1 to 5 generations of building blocks. In some embodiments, the dendrimer is a 1 generation building block dendrimer. In some embodiments, the dendrimer is a 2 generation building block dendrimer. In some embodiments, the dendrimer is a 3 generation building block dendrimer. In some embodiments, the dendrimer is a 4 generation building block dendrimer. In some embodiments, the dendrimer is a 5 generation building block dendrimer. For example, a 3 generation building block dendrimer is a dendrimer having a structure comprising three building blocks covalently linked to each other, such as when the building block is lysine, including the structure: [ka] may include:

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

[0074] In some embodiments, each generation of constitutional units in each dendron (X) has the formula [BU]2 (b-1) where b is the generation number. A dendron (X) having three generations of complete building blocks is represented as [BU]1-[BU]2-[BU]4.

[0075] Targeting Agents 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 comprises a peptide moiety having a molecular weight of up to about 80 kDa and an antigen-binding site that specifically binds to or has affinity for the target molecule (i.e., HER2). This interaction can occur by any type of bond or association, such as covalent, ionic, hydrogen, or van der Waals forces.

[0077] In one embodiment, the HER2 targeting agents described herein have 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. The antibody has a molecular weight of 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 about 3 kDa to about 80 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 3 kDa to about 60 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 3 kDa to about 50 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 3 kDa to about 40 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 3 kDa to about 30 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 3 kDa to about 20 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 3 kDa to about 15 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 3 kDa to about 13 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 5 kDa to about 15 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of about 5 kDa to about 12 kDa. In one embodiment, the HER2 targeting agent has a molecular weight of 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" refers to a portion or fragment of an antibody that is capable of specifically binding to an antigen, such as an F as defined herein. V , V H , V L or variable regions. This term will be understood to encompass fragments derived directly from antibodies, as well as proteins produced using recombinant means. In one embodiment, the antibody fragment is selected from a Fab, Fv, scFv, heavy chain antibody, domain antibody, heavy chain antibody, diabody, or triabody.

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

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

[0083] As used herein, a "single domain antibody (sdAb)", also known as a "domain antibody (dAb)" or "nanobody", is an antibody that contains a single variable region heavy chain V H or light chain V L In one embodiment, the variable region is derived from the family Camelidae. In one embodiment, the variable region is derived from a shark. In one embodiment, the variable region is derived from V H V, derived from the Camelidae family H is.

[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 the amino acid sequence of a single domain described in, 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 the amino acid sequence of a single domain described in U.S. Patent No. 20110028695. In some embodiments, the single domain antibody is a nanobody described in 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 comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology to that set forth in U.S. Patent No. 20110028695.

[0086] In one embodiment, the single domain antibody comprises the amino acid sequence of the single domain disclosed in Example 7. In one embodiment, the single domain antibody is 2D3, comprising the amino acid sequence set forth in SEQ ID NO: 1986 of US20110028695.

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

[0088] In some embodiments, the targeting agent does not compete with trastuzumab and / or pertuzumab for HER2 binding. In some embodiments, the targeting agent competes with trastuzumab and / or pertuzumab for HER2 binding. In one embodiment, the targeting agent is 2D3, and the targeting agent competes with trastuzumab and / or pertuzumab for HER2 binding. In some embodiments, the targeting agent binds to the extracellular domain of HER2. In some embodiments, the targeting agent binds to extracellular domain I, II, III, and / or IV of HER2. In one embodiment, the targeting agent binds to extracellular domain III of HER2. In some embodiments, the targeting agent binds to the dimerization arm of ErbB2. In an example where the targeting agent binds to the dimerization arm of ErbB2, In some embodiments, the targeting agent can compete with pertuzumab for HER2 binding. 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 and has a molecular weight of about 4 kDa to about 80 kDa, or about 5 kDa to about 80 kDa, or about 5 kDa to about 60 kDa, or about 5 kDa to about 50 kDa, or about 5 kDa to about 40 kDa, or about 5 kDa to about 30 kDa, or about 5 kDa to about 20 kDa, or about 5 kDa to about 15 kDa, or about 5 kDa to about 12 kDa, or about 10 kDa to about 16 kDa.

[0090] In some embodiments, the HER2 targeting agent contains fewer than about 500, fewer than about 400, fewer than about 300, fewer than about 200, fewer than about 150, fewer than about 140, fewer than about 130, fewer than about 120, fewer than about 110, or fewer than about 100 amino acid residues. In some embodiments, the HER2 targeting agent contains about 50 or more, about 75 or more, about 100 or more, or about 120 or more amino acid residues. In some embodiments, the HER2 targeting agent contains fewer than 120 amino acid residues. In some embodiments, the HER2 targeting agent contains between about 100 and about 120 amino acid residues.

[0091] In some embodiments, the HER2 targeting agent is a mimetic of an antibody or antibody fragment. As used herein, the term "mimetic" or "mimetics" refers to a compound that can bind an antigen like an antibody or antibody fragment, but is not structurally related to an antibody. This term is understood to not include the antibodies or antibody fragments described herein. This term is understood to include synthetic (in vitro produced) mimetics and mimetics produced using recombinant means. This term is understood to include protein mimetics.

[0092] In one embodiment, the mimetic is selected from an affibody, an aptamer, an affilin, an affimer, an affitin, an anticalin, an avimer, an alphabody, a monobody, a DARPin, a Fyomer, a fibronectin type III-derived protein scaffold, a phytocystatin-derived protein scaffold, and a paratope mimetic peptide. Similar to antibodies, mimetics can also be used as targeting moieties.

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

[0094] In one embodiment, the mimetic is about 3 kDa to about 20 kDa, or about 4 kDa to about 18 kDa, or about 6 kDa to about 16 kDa, or about 6 kDa to about 14 kDa, or about 6 kDa to about 12 kDa, or about 6 kDa to about 10 kDa, or about 6 kDa to about 8 kDa. In one embodiment, the mimetic is about 3 kDa to about 20 kDa. In one embodiment, the mimetic is about 3 kDa to about 20 kDa. In one embodiment, the mimetic is about 4 kDa to about 18 kDa. In one embodiment, the mimetic is about 6 kDa to about 16 kDa. In one embodiment, the mimetic is about 6 kDa to about 14 kDa. In one embodiment, the mimetic is about 6 kDa to about 12 kDa. In embodiments, the mimetic is between about 6 kDa and about 10 kDa. In one embodiment, the mimetic is between about 6 kDa and about 8 kDa.

[0095] In some embodiments, the targeting agent is an affibody. As used herein, the term "affibody" refers to any of a class of very small (approximately 6 kDa) polypeptide antibody mimetics based on a three-alpha helix bundle domain, approximately 58 amino acids in length, known as the "Z domain." Typically, the affibody scaffold is based on a modified version of the B domain of Protein A. Affibodies are characterized by extremely high stability (resistant to temperatures as high as 90°C and having target affinities in the nanomolar to picomolar range). See, e.g., 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 and has a molecular weight ranging from about 3 kDa to about 10 kDa, or from about 3 kDa to about 8 kDa, or from about 4 kDa to about 8 kDa, or from about 4 kDa to about 7 kDa, or from about 5 kDa to about 7 kDa, or about 6 kDa.

[0097] In some embodiments, the targeting agent is an affibody and has 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 is made up of 40-80 amino acid residues, or 50-70 amino acid residues, or 55-65 amino acid residues, or 56-60 amino acid residues, or about 58 amino acid residues.

[0098] For purposes of this disclosure, the term "antibody" includes four-chain proteins, e.g., two light chains and two heavy chains, such as recombinant or engineered antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies, and half antibodies, bispecific antibodies) capable of specific binding to one or a few closely related antigens via Fv. Antibodies generally contain a constant domain, which may be arranged into a constant region and a constant fragment, a fragment crystallizable (Fc). Exemplary forms of antibodies contain a four-chain structure as a basic unit. Full-length antibodies contain two covalently linked heavy chains (approximately 50-70 kDa) and two light chains (approximately 23 kDa each). The light chains generally contain a variable region and a constant domain, and in mammals are either kappa or lambda light chains. The heavy chains generally contain a variable region and one or two constant domains connected to additional constant domain(s) by a hinge region. Mammalian heavy chains are any one of α, δ, ε, γ, and μ types. 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 noncovalent interactions. The number of interchain disulfide bonds varies depending on the type of antibody. Each chain has an N-terminal variable region (VH or VL, each approximately 110 amino acids long) and one or more constant domains at the C-terminus. The light chain constant domain (CL: approximately 110 amino acids long) is aligned with and disulfide-bonded to the first heavy chain constant domain (CH: approximately 330-440 amino acids long). The light chain variable region is aligned with the heavy chain variable region. An antibody heavy chain can contain two or more additional CH domains (e.g., CH2, CH3, etc.) and can include a hinge region between the CH1 and CH2 constant domains. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass. In one example, the antibody is a human antibody, a deimmunized form thereof, a germline form, or an affinity matured form thereof.

[0099] The terms "full-length antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment thereof. Specifically, a whole antibody includes an antibody having heavy and light chains, including constant regions. The constant region may be a wild-type sequence constant region (e.g., a human wild-type sequence constant region) or an amino acid sequence variant thereof.

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

[0101] As used herein, the term "complementarity-determining region" (synonym: CDR, i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the variable region of an antibody, the presence of which is a major contributor to specific antigen binding. Each variable region typically has three CDR regions, designated CDR1, CDR2, and CDR3. Each complementarity-determining region may contain the amino acid residues of the "complementarity-determining region" 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 is located at residues 50-65, and CDRH3 is located at residues 95-102. In the light chain, CDRL1 is located at residues 24-34, CDRL2 is located at residues 50-56, and CDRL3 is located at residues 89-97. These CDRs may also contain multiple insertions, for example, as described in Kabat (1987 and / or 1991). The present disclosure relates to all numbering systems, including, but not limited to, the Kabat numbering system, the canonical numbering system, 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 in Giudicelli et al. (1997); or the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html). The CDRs are FRs and CDRs defined by the Kabat numbering system. In one example, the CDRs and / or FRs are defined according to the Kabat numbering system, e.g., as depicted in bold in Figures 9A-9D. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include any of the five C-terminal amino acids described herein, or any one or more of those amino acids are substituted with another naturally occurring amino acid. Additionally or alternatively, optionally, the light chain CDR1 does not include any of the four N-terminal amino acids described herein, or any one or more of those amino acids are substituted with another naturally occurring amino acid. In this regard, Padlan et al. (1995) established that the five C-terminal amino acids of heavy chain CDR2 and / or the four N-terminal amino acids of light chain CDR1 are generally not involved in antigen binding. In one example, the CDRs and / or FRs are defined according to the Chothia numbering system, e.g., as depicted in underlined text in Figures 9A-9D.

[0102] As used herein, the term "Kabat numbering system" refers to the scheme for numbering the variable regions of antibodies and identifying CDRs (hypervariable regions) as described in Kabat (Kabat et al., 1987 and / or 1991).

[0103] As used herein, the term "Chothia numbering system" refers to the numbering system for the variable regions of an antibody as described by Chothia and Lesk (Chothia and Lesk, 1987) or Al-Lazikani (Al-Lazikani et al., 1997). refers to the scheme for numbering and identifying CDRs (structural loops).

[0104] As used herein, the term "antigen-binding domain" is intended to mean a region of a compound that is capable of specifically binding 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 an F-binding domain as described herein. V , V H, or V L Contains one or more of the following:

[0105] As used herein, the terms "bind" or "binding," in reference to the interaction of a protein or its antigen-binding domain with an antigen, means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures, rather than proteins in general. If an antibody is binding to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") and the antibody in a reaction containing labeled "A" will reduce the amount of labeled "A" that binds to the antibody.

[0106] As used herein, "specifically bind," "bind specifically," or similar phrases mean that a protein of the present disclosure reacts or associates with a particular antigen (e.g., HER2) or cells expressing it more frequently, more rapidly, more persistently, and / or with higher affinity than with alternative antigens or cells. For example, a protein that specifically binds to an antigen binds to that antigen with higher affinity (e.g., 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 150-fold, 200-fold or more affinity), avidity, ease, and / or longer persistence than it binds to other antigens. It is also understood by reading this definition that, for example, a protein that specifically binds to a first antigen may or may not specifically bind to a second antigen. Such "specific binding" does not necessarily require exclusive or undetectable binding to another antigen, as 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 conjugate via a spacer. In some embodiments, the covalent attachment or linkage between the targeting agent and the spacer group is formed by reaction between a complementary reactive functional group present on the targeting agent precursor and a spacer group precursor, which may or may not be attached to the remainder of the conjugate upon reaction with the HER2 targeting moiety precursor, depending on the process used to prepare the conjugate.

[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 site of the targeting agent precursor can be, for example, a cysteine, a lysine, an N-terminal amine, a tyrosine, a carbohydrate, an unnatural amino acid, or a transaminase (e.g., transglutaminase or sortase A) or recognition sequence. Covalent attachment sites are known in the art (e.g., Milla P. et al., 2012). In silico crystal structure predictions can be used to identify positions in the polypeptide sequence of a targeting agent that are structurally suitable for covalent attachment of the conjugate (e.g., away from the paratope, CDRs, functions important for correct folding and conformation, and / or residues with highly charged, polar, or bulky side groups). From these candidate positions, empirical screening can be performed to determine substitution sites that maximize retention of function while enabling robust conjugates. 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 comprises a non-natural amino acid residue for attachment to a spacer. The non-natural amino acid residue may have a side chain with a reactive functional group complementary to the reactive functional group of the spacer group precursor. In some embodiments, the unnatural amino acid residue comprises an azido group, for example, a 4-azidophenylalanine residue, e.g., [ka] The azide group can undergo a cycloaddition reaction with an alkyne group that may be present in the spacer precursor group. In some embodiments, the unnatural amino acid is a diene-containing amino acid, such as [ka] Spirocyclopentadiene-containing amino acids such as: The diene group can undergo a cycloaddition reaction (e.g., a Diels-Alder reaction) with an alkene group, such as present in maleimides. Further examples of unnatural amino acids that can be used to attach to the spacer include those containing a carbonyl group, such as a ketone, and those containing a methylcyclopropylene group. Additional examples of unnatural amino acids include: [ka] Examples include:

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

[0112] Spacer group As used herein, the term "spacer group" refers to a chemical entity that serves to attach a targeting agent to a dendrimer. That is, the spacer group binds the targeting agent more tightly (e.g., via covalent attachment) to the dendrimer. The purpose of the spacer group is to position the targeting agent so that it can bind to the HER2 receptor without undue adverse influence from other components of the dendrimer.

[0113] As described above, the targeting agent can be covalently attached to the dendrimer, for example, via the dendrimer core. In some embodiments, a spacer group is covalently attached to the core unit. In some embodiments, the targeting agent is covalently attached to the dendrimer via the dendrimer core. That is, the targeting agent, such as an antibody fragment, is covalently attached to the dendrimer core by a spacer group. Covalently attaching the targeting agent to the dendrimer via a spacer attached to the dendrimer core can be beneficial when the dendrimer is sterically congested, and the spacer group can be long enough to protrude beyond the surface of the dendrimer, thereby allowing the targeting agent to bind to its receptor, etc., in vivo.

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

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

[0116] In some embodiments, the spacer group comprises multiple PEG groups flanked by other functional groups, e.g., the spacer group may comprise PEG groups linked via, for example, amide groups or other functional groups useful for connecting moieties of the spacer group.

[0117] To attach the spacer group to a 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 groups such as hydroxy, carboxy, activated esters such as NHS or pentafluorophenol esters, amino, azide, maleimides (such as sulfomaleimides), dienes (cyclopentadiene, e.g., spiro[2.4]hepta-4,6-diene groups), tetrazines, alkyne-containing groups such as citraconimides, BCN (bicycle[6.1.0]non-4-yn-9-yl), and DBCO (dibenzocyclocyne-amine), activated alkenes such as methyl-cyclopropylene-containing moieties, carbonyl groups such as thiols, aldehydes, and ketones, alkoxyamines, haloacetates, biotin, tetrazines, alkene-containing groups such as TCO (trans-cyclooctene), methyl-cyclopropylene groups, and PTAD or other tyrosine-reactive groups.

[0118] For example, in some embodiments, the spacer group intermediate may include two reactive groups (e.g., one at each orthogonal terminus). That is, at least one of the reactive groups can react with a complementary group present on either the targeting agent-containing intermediate or the dendrimer-containing intermediate to attach the spacer group to a component of the conjugate under conditions under which the other reactive group is stable and substantially non-reactive. This allows the spacer to be attached (e.g., covalently attached) to either the dendrimer or the targeting agent, after which the other reactive group can react with a complementary group present on the remaining component to link the targeting agent to the dendrimer.

[0119] In some embodiments, the spacer group is attached (e.g., covalently attached) to the targeting agent by reaction of precursors containing an alkyne group and an azide group, respectively (e.g., the intermediate containing the targeting group may contain an azide group, and the intermediate containing the spacer group may contain an alkyne group). Such reactions can result in the formation of triazole-containing groups, e.g., [ka] leading to the formation of The following structure: [ka] It can be formed by reacting a precursor having

[0120] As another example, the spacer group may be a triazole-containing group, e.g., [ka] or may be attached via formation of the following structure: [ka] It can be formed by reacting a precursor having

[0121] In some embodiments, the spacer group is attached (e.g., covalently attached) to the targeting agent by reaction of precursors containing an alkene (e.g., a strained alkene such as trans-cyclooctene) and a tetrazine group, respectively. Such reactions can result in pyridazine-containing groups with nitrogen protrusions, e.g., [ka] and also results in the formation of the following structure: [ka] It can be formed by reaction of a precursor having

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

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

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

[0125] As described above, the targeting agent precursor may include an unnatural amino acid residue. In some embodiments, the targeting agent includes an unnatural amino acid residue. The unnatural amino acid residue may be, for example, any unnatural amino acid capable of exhibiting a reactive side chain, where the reactive side chain carries a functional group complementary to a functional group present on the spacer group precursor. In this manner, the complementary functional groups can react, resulting in attachment of the targeting moiety precursor to the spacer group precursor. In some embodiments, the unnatural amino acid residue is a 4-azidophenylalanine residue. In some embodiments, the spacer group precursor includes an alkyne group for conjugating to a targeting moiety precursor comprising an unnatural amino acid residue. In some embodiments, the spacer group precursor includes an alkyne group for conjugating to a targeting agent precursor comprising a 4-azidophenylalanine residue. In some embodiments, the spacer group precursor includes an alkyne group that is a dibenzylcyclooctane group for conjugating to a targeting moiety precursor comprising a 4-azidophenylalanine residue. In some embodiments, one end of the spacer group forms part of the targeting moiety with a DBCO group (e.g., [ka] or by a cycloaddition reaction with an azide moiety on a 4-phenylalanine residue to form a triazole-containing group such as [ka] The targeting moiety is attached by reaction with an azide moiety on the 4-phenylalanine residue (forming a triazole-containing group such as

[0126] In some embodiments, one end of the spacer group is attached (e.g., covalently attached) to the dendrimer by an amidation reaction (e.g., by reaction of activated esters) between an amino group present on the core or on a surface unit and a carboxyl group present on the spacer group. In some embodiments, the spacer group precursor comprises a tetrazine group. In some embodiments, the spacer group precursor comprises a maleimide group, for example, for conjugation to a diene (such as a cyclopentadiene, e.g., a 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 at the dendrimer core and an azide group for conjugating to a targeting agent precursor comprising 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 at the dendrimer core and an azide group for conjugating to a targeting agent precursor comprising 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 at the dendrimer core and a maleimide group for conjugating to a targeting agent precursor comprising 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 at the dendrimer core and a thiol or masked thiol group for conjugating to a targeting agent precursor comprising a reactive maleimide moiety. In some embodiments, the spacer group precursor comprises a PEG chain with a reactive carboxyl group for coupling to an amine at the dendrimer core and a tetrazine group for conjugating to a targeting agent precursor comprising a reactive alkene moiety. In some embodiments, the spacer group precursor comprises a PEG chain with a reactive carboxyl group for coupling to an amine at the dendrimer core and a maleimide group for conjugating to a targeting agent precursor comprising a reactive diene (such as a cyclopentadiene, e.g., a spiro[2.4]hepta-4,6-diene group).

[0128] In some embodiments, linking a targeting agent to a dendrimer can be achieved by attaching (e.g., covalently attaching) a first spacer group to a targeting agent intermediate and a second spacer group to a dendrimer (e.g., to the dendrimer core), 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 can provide for easy connection between the dendrimer and the targeting agent. For example, the first spacer group intermediate may include a first reactive group at one end that is complementary to the reactive group on the targeting agent (e.g., an alkyne group complementary to an azide group, which can react together to form a triazole group), and a second reactive group complementary to the reactive group on the second spacer group (e.g., a tetrazine-containing group complementary to a trans-cyclooctene-containing group). The second spacer group intermediate may, for example, contain a third reactive group at one end that is complementary to a reactive group on the dendrimer (e.g., a carboxylic acid group that is complementary to a reactive group on an amine group) and a fourth reactive group at the other end that is complementary to a reactive group on the first spacer group intermediate (e.g., a trans-cyclooctene-containing group that is complementary to a reactive group on a tetrazine group). For example, the first and spacer groups may be attached via a group produced by reacting a trans-cyclooctene group with a tetrazine group, resulting in the structure: [ka] may include:

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

[0130] The targeting agent precursor is reacted with a spacer group precursor to form an 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, covalent attachment sites 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. Attachment 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, such that the spacer group is attached 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 or linked to the spacer group via the C-terminus of the targeting agent.

[0131] therapeutic agent As used herein, the term "therapeutic agent" refers to an agent that exerts a therapeutic effect in vivo, for example, against HER-2-expressing cancer cells. Examples of therapeutic agents include, but are not limited to, anti-cancer agents, also known as chemotherapeutic agents, anti-neoplastic agents, and cytotoxic agents. In some embodiments, a therapeutic agent is a chemotherapeutic agent. In some embodiments, a therapeutic agent is a cytotoxic agent. As used herein, the term "cytotoxic agent" refers to an agent that exhibits chemotherapeutic properties. Examples of anti-cancer agents include, but are not limited to, DNA alkylating agents and cross-linking agents (e.g., platinum-containing agents such as cisplatin and oxaliplatin), DNA damaging agents, e.g., PARP (poly ADP-ribose polymerase) inhibitors (e.g., olaparib, rucaparib, niraparib, talazoparib), anti-microtubule agents (e.g., paclitaxel, docetaxel, cabazitaxel), topoisomerase inhibitors (e.g., irinotecan, topotecan, SN-38, Anticancer drugs include anti-cancer drugs such as 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 antimitotic agents. Such anticancer drugs can be used in the treatment of cancer with the aim of curing (i.e., eliminating the cancer patient) or alleviating / alleviating symptoms, which will improve the quality of life and prolong the life of cancer patients. Anticancer drugs can also be used in place of other non-chemotherapeutic cancer treatments (i.e., radiation therapy, surgery / surgical intervention). It may be used in combination with

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

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

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

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

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

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

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

[0139] In some embodiments, the therapeutic agent is a hyper-cytotoxic agent. As used herein, "hyper-cytotoxic agent" refers to an agent that exhibits very potent chemotherapeutic properties, but is itself so toxic that it cannot be administered alone as an anti-cancer agent. That is, hyper-cytotoxic agents exhibit chemotherapeutic properties, but generally cannot be safely administered to subjects because the harmful and toxic side effects outweigh the benefits of chemotherapy.

[0140] Examples of super-cytotoxic agents include nemorubicin, dolastatins (e.g., dolastatin-10, dolastatin-15), auristatins (e.g., monomethylauristatin-E, monomethylauristatin-F), maytansinoids (e.g., maytansine, mertansine / emtansine (DM1, ravtansine (DM4))), calicheamicins (e.g., calicheamicin γ1), esperamicins (e.g., esperamicin A1), and pyrrolobenzodiazepines (PDB).

[0141] In some embodiments, the therapeutic agent is an 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: [ka] is.

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

[0144] In some embodiments, the super cytotoxic agent is a maytansinoid. In one embodiment, the super cytotoxic agent is maytansine. In one embodiment, the super cytotoxic agent is ansamitocin. In one embodiment, the super cytotoxic agent is emtansine / mertansine (DM1). In one embodiment, the super cytotoxic agent is ravtansine (DM4). Maytansinoids are understood to inhibit microtubule assembly by binding to tubulin.

[0145] The chemical structure of maytansine is: [ka] is.

[0146] The chemical structure of emtansine / mertansine is: [ka] is.

[0147] In some embodiments, the super cytotoxic agent exhibits in vitro IC50 activity against cancer cell lines (e.g., SKBR3 cells and / or HEK293 cells and / or MCF7 cells). 50 or 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 activities of MMAE and emtansine have been reviewed in Abdollahpour-Alitappeh et al. (2017) and Oroudjev et al. (2010), respectively.

[0148] It will be understood that the dendrimer-targeting agent conjugates can have a distribution of therapeutic agents per targeting agent, for example, it is possible that the dendrimer-targeting agent conjugates can have up to about eight or more therapeutic agents per targeting agent.

[0149] It will be appreciated that the therapeutic agent-to-targeting agent ratio (DTR) of each dendrimer-targeting agent conjugate species may vary among populations. Typically, it is desirable to produce dendrimer-targeting agent complexes with a high DTR. This generally results in a more effective therapy. However, if the DTR is too high, for example, if the therapeutic agent is a highly cytotoxic agent, undesirable toxicity may result.

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

[0151] In some embodiments, the dendrimer-targeting agent conjugate has a DTR of about 1 to about 32. In some embodiments, the dendrimer-targeting agent conjugate has a DTR of about 7 to about 32. In some embodiments, the dendrimer-targeting agent conjugate is a G5 dendrimer and has a DTR of about 26 to about 32. In some embodiments, the dendrimer-targeting agent conjugate is a G4 dendrimer and has a DTR of about 14 to about 16. In some embodiments, the dendrimer-targeting agent conjugate 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 reduced compared to a dendrimer-targeting agent conjugate with only the targeting agent. In some embodiments, the dendrimer-targeting agent conjugate has a DTR of about 2 to about 16. In some embodiments, the dendrimer-targeting agent conjugate has a DTR of about 8 to about 16. In some embodiments, the dendrimer-targeting agent conjugate is a G5 dendrimer and has a DTR of about 4 to about 16. In some embodiments, the dendrimer-targeting agent conjugate is a G5 dendrimer and has a DTR of about 6 to about 16. In some embodiments, the dendrimer-targeting agent conjugate is a G4 dendrimer and has a DTR of about 4 to about 8. In some embodiments, the dendrimer-targeting agent conjugate is a G4 dendrimer and has a DTR of about 4 to about 8. In some embodiments, the dendrimer-targeting agent conjugate is a G3 dendrimer and has a DTR of about 2 to about 4.

[0153] The ability of dendrimer therapeutics to carry more than one therapeutic agent obviates the need for ultra-cytotoxic agents. For example, the ability to target the delivery of multiple cytotoxic agents, such as docetaxel, onto a single dendrimer may result in comparable or even greater therapeutic efficacy when compared to the delivery of a single super-cytotoxic agent, such as MMAE. Eliminating the use of super-cytotoxic agents may also be an advantage.

[0154] Linker In some embodiments, a therapeutic agent is attached to the dendrimer via a linker. The linker preferably preserves the favorable properties of the therapeutic agent and remains substantially intact and non-toxic in the systemic circulation. Linker groups can be used to provide a suitable group for attaching a pharmaceutically active agent to a dendrimer, for example, when the available functionality of the pharmaceutically active agent is not suitable for direct attachment to a building block. Alternatively, or additionally, linker groups can be used to facilitate controlled release of the pharmaceutically active agent from the dendrimer scaffold, thereby providing therapeutically effective concentrations and a desirable pharmacokinetic profile of the pharmaceutically active agent for a suitable period of time (e.g., an extended period of time).

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

[0156] Those skilled in the art will appreciate that any one of a variety of suitable linkers may be used, which should provide sufficient stability during systemic circulation while simultaneously allowing for rapid and efficient release of the active form of the cytotoxic drug at its site of action upon internalization, for example, by a cancer cell.

[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. A cleavable linker comprises, by itself or in conjunction with a linkage to a pharmaceutically active agent, 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 the tumor environment, acid labile, reducing environment labile, hydrolytically unstable, or protease labile.

[0158] Chemically labile linkers include, but are not limited to, acid-labile 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 blood, peptide linkers and their peptide bonds are expected to advantageously have good serum stability. Val-Ala, Val-Cit, Phe-Lys, Phe-Arg, Phe-Cit, Val-Arg, Val-Cit, Ala-Arg, and Ala-Cit linkers are preferred. The linker is rapidly hydrolyzed by cathepsin B. In some embodiments, the linker is an enzymatically cleavable linker. For example, in some embodiments, the linker comprises amino acid residues that can be recognized and cleaved by an enzyme.

[0159] In some embodiments, the linker comprises 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 formula:

[0160] For example, the PAB group may be covalently attached to an amine group present on the therapeutic moiety through a carbonyl group to form a carbamate linkage, or may be attached to an amine group present on an outer building block through a diacyl linker that forms an amide bond with a valine amino group and an amine group present on the outer building block.

[0161] In some embodiments, the linker has, for example, the structure: [ka] The amino acid sequence of the present invention comprises or consists of a glutaric acid-valine-citrulline-para-aminobenzyl alcohol group having the formula:

[0162] In some embodiments, the linker has, for example, the structure: [ka] The amino acid sequence of the amino acid sequence of the present invention comprises or consists of a valine-alanine-para-aminobenzyl alcohol group having the formula:

[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, attachment to the linker can be via an ester group, which has been found to be cleavable in vivo to release the pharmaceutically active agent at a desirable rate.

[0164] In some embodiments, the core unit is formed from a core unit precursor that includes an amino group. In some embodiments, the building block is a lysine residue or analog thereof, the pharmaceutically active agent comprises a hydroxyl group, the residue of the pharmaceutically active agent is attached through the oxygen atom of the hydroxyl group, and the cleavable linker is a diacyl linker such that there is an ester linkage between the residue of the pharmaceutically active agent and the linker and an amide linkage between the linker and a nitrogen atom present on the outermost building block. In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, the residue of the pharmaceutically active agent is attached through the oxygen atom of the hydroxyl group, and the cleavable linker has the formula: [ka] wherein A is a C2-C alkyl group interrupted by O, S, SS, NH, or N(Me). 10 A is 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 straight-chain (i.e., linear) or branched saturated hydrocarbon group. By way of example, an alkyl group can be an alkyl group containing 1 to 10 carbon atoms (i.e., C 1~10 In one example, an alkyl group may contain 1 to 6 carbon atoms (i.e., C 1~6 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 straight-chain (i.e., linear) or branched saturated hydrocarbon group. By way of example, an alkylene group can be an alkylene group having 2 to 10 carbon atoms (i.e., C 2~10 In one example, an alkylene group can be a group containing 2 to 6 carbon atoms (i.e., C2~6 Examples of alkylene groups include, for example, -CH2CH2-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2-, and the like.

[0167] Such linkers are particularly suitable for linking to therapeutic agents via hydroxyl groups present in the therapeutic agent, in which case the conjugate contains an ester linkage between the linker and the therapeutic agent moiety and an amide linkage between the linker and the dendrimer moiety.

[0168] In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, the residue of the pharmaceutically active agent is attached through the oxygen atom of the hydroxyl group, and the cleavable linker has the formula: [ka] A is a diacyl linker group of the formula: 10 It is an alkylene group.

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

[0170] A particular type of cleavable linker is one that contains 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 a disulfide moiety.

[0171] In some embodiments, the linker comprises an alkyl chain interrupted by a disulfide moiety, wherein one or both of the 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 may comprise the group: [ka] may include:

[0172] Another particular type of cleavable linker is one that contains a diphosphate moiety. Such linkers are susceptible to hydrolysis by lysosomal acid pyrophosphatase and acid phosphatase enzymes. In some embodiments, the linker contains a pyrophosphate moiety: [ka] It contains an alkyl chain interrupted by a diphosphate moiety, also called a diphosphate group.

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

[0174] Another particular type of cleavable linker is an orthoester moiety: [ka] It includes:

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

[0176] For example, an alkylene group or a cycloalkylene group (e.g., C 1-10 Alkylene group or C 3-10 Examples of non-cleavable linkers include diacyl linkers bridged by a cycloalkylene group. Further examples of non-cleavable linkers include thioether linkers. Specific examples of non-cleavable linkers include those formed by using SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). SMCC can be used to react the maleimide functionality with a thiol group present on or attached to the therapeutic moiety to form a thioether linkage. The carboxylic acid functionality can be used to react with amino groups present on the outer building blocks.

[0177] In some embodiments, the linker comprises a Val-Cit-PAB group. In some embodiments, the linker comprises a Val-Arg-PAB group. In some embodiments, the linker comprises 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 comprises a Val-Cit-PAB group, the therapeutic moiety is MMAE, and the Val-Cit-PAB group is attached to the therapeutic moiety as follows (i.e., Val-Cit-PAB-MMAE): [ka] .

[0179] In one embodiment, the linker is a Val-Cit-PAB group, the therapeutic moiety is MMAF, and the Val-Cit-PAB group is attached to the therapeutic moiety 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 agent moiety is MMAE, and the glutaric acid-Val-Cit-PAB group is Attached to the agent portion (i.e., Glutarate-Val-Cit-PAB-MMAE): [ka] .

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

[0182] In some embodiments, the therapeutic agent is covalently linked to a surface building unit of the dendrimer via a connector group. For example, the therapeutic agent may be covalently linked to a surface building unit of the dendrimer via a connector group in addition to a linker group. The connector group allows the therapeutic agent to be tethered at different lengths from the surface of the dendrimer. For example, a connector group may be utilized to extend the distance of the therapeutic agent from the surface of the dendrimer. This may avoid any potential problems associated with steric hindrance, which may hinder access to the cleavable linker and reduce the ability of the therapeutic agent to interact with the target. In some embodiments, the dendrimer-targeting agent complex comprises a therapeutic agent covalently linked to the surface of the dendrimer via a linker group and a connector group. 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 comprise any chemical moiety that serves to increase the distance of the therapeutic agent from the surface of the dendrimer. In some embodiments, the connector group comprises one or more hydrophilic polymer groups. In some embodiments, the connector group comprises from about 1 to about 50 hydrophilic polymer groups. In some embodiments, the connector group comprises a PEG group. In some embodiments, the connector group comprises a PEOX group. In some embodiments, the connector group comprises a polysarcosine group. In some embodiments, the connector group comprises 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 comprises from about 1 to about 50 PEG moieties. In some embodiments, the connector group comprises 4 PEG moieties. In some embodiments, the connector group comprises 12 PEG moieties. In some embodiments, the connector group comprises 24 PEG moieties. In some embodiments, the connector group comprises 48 PEG moieties.

[0184] In some embodiments, the linker comprises a connector group, (PEG) X -Val-Arg-PAB-P-Trigger- moiety, where X can be a number from about 1 to about 50, as described above.

[0185] In one embodiment, the linker comprises a connector group and a (PEG)9-Val-Arg-PAB-P-Trigger-NHCO moiety: [ka] is.

[0186] In one embodiment, the linker comprises a connector group and the moiety: (PEG)9-Val-Arg-PAB-P-Trigger-N(CH3)CO: [ka] is.

[0187] In some embodiments, the linker comprises a connector group, (PEG) x The moiety is —OP(O)OH—OP(O)OH—O—, where again, X can be a number from about 1 to about 50, as described above.

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

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

[0190] For example, (PEG)X -Val-Arg-PAB-P-Trigger- or (PEG) X The use of certain linkers such as -OP(O)OH-OP(O)OH-O- can result in release of the therapeutic agent.

[0191] (PEG) X -Val-Arg-PAB-P-Trig-NHCO, (PEG) X In an example such as -Val-Arg-PAB-P-Trigger-N(CH3)CO, the linker is first activated by cleavage of the dipeptide moiety by a cellular enzyme such as cathepsin B, revealing a self-immolating 2-(aminomethyl)pyrrolidine connector. After intramolecular cyclization, loss of the hydroxyl-containing therapeutic agent occurs. Exemplary hydroxyl-containing therapeutic agents include cabazitaxel, docetaxel, and SN-38.

[0192] (PEG) x In the example of the -OP(O)OH-OP(O)OH-O- moiety, the linker not only cleaves specifically and cleanly to yield hydroxyl-containing drugs such as cabazitaxel, docetaxel, and SN-38, but also has the added advantage of providing the drug monophosphate, which is the active species of nucleotide inhibitors, e.g., gemcitabine.

[0193] Pharmacokinetic Modification Group The conjugates contain multiple hydrophilic polymeric groups covalently linked to the surface building blocks of the dendrimer, which can modify the pharmacokinetic properties of the conjugates, e.g., provide improved pharmacokinetic properties.

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

[0195] In some embodiments, the hydrophilic polymer group comprises repeating units of an amino acid, alkyloxy, or alkyl(acyl)amino group. In some embodiments, the hydrophilic polymer group comprises repeating units of an amino acid, such as sarcosine. In some embodiments, the hydrophilic polymer group comprises repeating units of an alkyloxy group (e.g., the hydrophilic polymer is a PEG group). In some embodiments, the hydrophilic polymer comprises repeating units of an alkyl(acyl)amino group (e.g., the hydrophilic polymer is a PEOX group).

[0196] In some embodiments, the hydrophilic polymeric group comprises at least 10 monomeric units. In some embodiments, the hydrophilic polymeric group comprises up to 100 monomeric units. In some embodiments, the hydrophilic polymeric group comprises 10 to 100, or 10 to 50 monomeric 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 the present disclosure typically has a molecular weight of It includes mixtures of PEGs with some variability (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 "polyethylene glycol terpolymer" refers to a polymer having an average molecular weight of about 2100 daltons, i.e., ± about 10% (PEG 1890 ~PEG 2310 ) refers to polyethylene glycol. ~2300 " refers to an average molecular weight of about 2300 daltons, or ±10% (PEG 2070 ~PEG 2530) refers to polyethylene glycol having a molecular weight of 1.3 or less. Three commonly used methods for calculating average MW are number average, weight average, and Z-average molecular weight. As used herein, the phrase "molecular weight" is intended to refer to a weight average molecular weight that can be measured using techniques well known in the art, including, but not limited to, 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 viscometry.

[0198] In some embodiments, the PEG groups have an average molecular weight in the range of 500 to 2500 daltons. In some embodiments, the PEG groups have an average molecular weight in the range of 800 to 1200 daltons. In some embodiments, the PEG groups have an average molecular weight in the range of 900 to 1100 daltons. In some embodiments, the PEG groups have an average molecular weight in the range of 1500 to 2500 daltons. In some embodiments, the PEG groups have an average molecular weight in the range of 1900 to 2300 daltons. In some embodiments, the PEG groups have 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 a measure of the distribution of molecular weights in a polymer sample. The polydispersity index (PDI) is a measure of the distribution of molecular weights in a polymer sample. w ) to the number average molecular weight (M n ) and indicates the distribution of individual molecular weights within a batch of polymer. The polydispersity index (PDI) can have a value greater than or equal to 1, but as a polymer approaches uniform variation in length and average molecular weight, the PDI approaches 1.

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

[0202] In some embodiments, the hydrophilic polymeric group is a PEOX group. In some embodiments, the dendrimer comprises a plurality of PEOX groups covalently linked to the surface building units of the dendrimer moiety. A PEOX group is a polyethyloxazoline group, i.e., a group having the formula: [ka] It is a group containing repeating units of the formula:

[0203] PEOX groups are so named because they can be produced by polymerization of ethyloxazoline. The PEOX materials used to produce the dendrimers of the present disclosure typically contain mixtures of PEOX with some variation in molecular weight (i.e., ±10%); therefore, when a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEOX composition. In some embodiments, the second end 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 end group comprises a PEOX group having an average molecular weight ranging from 750 daltons to 2500 daltons, or from 1000 daltons to 2000 daltons. End-capped PEOX groups can be used if desired. In some embodiments, the PEOX group is a methoxy-terminated PEOX.

[0204] In some embodiments, the hydrophilic polymeric group is comprised of polysarcosine groups. In some embodiments, the polysarcosine groups have an average molecular weight of at least 750 daltons, at least 1000 daltons, or at least 1500 daltons. In some embodiments, the hydrophilic polymeric group comprises polysarcosine groups having an average molecular weight ranging from 750 daltons to 2500 daltons, or from 1000 daltons to 2000 daltons.

[0205] The hydrophilic polymeric groups may be attached to the outer building units via any suitable means, hi some embodiments, a linking group is used to attach the hydrophilic polymeric groups to the outer building units.

[0206] Hydrophilic polymeric groups are typically attached through the use of precursors that contain reactive groups reactive with amine groups, such as reactive acyl groups (capable of forming amide bonds) or aldehydes (capable of forming amine groups under reductive amination conditions).

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

[0208] In some embodiments, the PEOX groups are covalently attached to the PEOX linking group (L1') through a linkage formed between a nitrogen atom present in the PEOX group and a carbon atom present in the PEOX linking group, and each PEOX group is covalently attached to a building block through an amide linkage formed between a nitrogen atom present in the building block and a carbon atom of an acyl group present in the PEOX linking group. In some embodiments, each L1'-PEOX group is [ka] is.

[0209] In some embodiments, the hydrophilic polymeric group is a polysarcosine group, i.e., a group having the formula: [ka] This includes groups containing repeating units of the formula:

[0210] In some embodiments, the polysarcosine group is attached to the constitutional unit via an amide linkage formed between a nitrogen atom present in the constitutional unit and a carbon atom of an acyl group present in the polysarcosine group. In some embodiments, the hydrophilic polymeric 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 daltons to 2500 daltons, or 1000 daltons to 2000 daltons.

[0211] In many cases, a population of dendrimers that are surface functionalized contains random stoichiometry and topology of functional groups. For example, reacting a population of dendrimer-targeting agent conjugates containing, for example, 64 reactive surface groups with one or more reactive functional groups can result in a diverse population of functionalized dendrimers, with some dendrimers containing more functional groups than others. When multiple different surface groups are available for reaction with reactive functional groups, a wide distribution of dendrimers with different surface topologies can also be obtained.

[0212] The dendrimer-targeting agent conjugates, intermediates, and processes of the present invention may enable high loading of therapeutic agents by covalently attaching them to surface building blocks. Such dendrimer-targeting agent conjugates may facilitate the release of therapeutically effective levels of the therapeutic agent over an extended period of time after administration, thereby reducing the frequency and / or number of doses required. The dendrimer-targeting agent conjugates of the present invention may also enable targeted delivery of the therapeutic agent to its site of action. As a result, such dendrimer targeting moieties may reduce off-target activity, such as cytotoxic activity.

[0213] Internalization of dendrimer-targeting agent conjugates The dendrimer-targeting agent conjugates provide targeted anti-cancer drug therapy. It is understood that the beneficial properties of the conjugates are at least partially related to the observed rapid internalization of exemplary conjugates containing MMAE therapeutic agents into target cells. In some embodiments, the 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 conjugate into a cell. The ability of a dendrimer-targeting agent conjugate to specifically seek out, bind to, and rapidly internalize the HER2 receptor on target HER2-expressing cancer cells means that a reduced amount of the conjugate circulates in the plasma. In this way, the chances of a therapeutic agent, such as a supercytotoxic moiety like MMAE, being undesirably cleaved from the dendrimer-targeting agent conjugate outside of the site where HER2 is overexpressed (e.g., tumor cells) are reduced, and the potential for side effects or toxicity associated with non-specific release of the therapeutic agent in the subject is reduced. In contrast, administering an equivalent dose of a therapeutic agent, such as a supercytotoxic moiety, can cause extreme toxicity to the point where the therapeutic agent cannot be safely administered alone. For example, it is understood that MMAE and MMAF are too toxic to be safely administered alone to a subject. Therefore, dendrimer-targeting agents Agent conjugates are believed to provide a safer means for delivering therapeutic agents, such as ultracytotoxic agents such as MMAE and MMAF.

[0215] In some embodiments, administration of the conjugate results in reduced side effects compared to administration of an equivalent dose of the free therapeutic agent, hi some embodiments, administration of the conjugate results in at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, or at least a 90% decrease in the plasma concentration of the therapeutic agent (e.g., Cmax or the concentration at a particular time point 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 ultracytotoxic (e.g., MMAE, MMAF) and administration of the dendrimer-targeting agent conjugate results in at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, or at least a 90% decrease in the plasma concentration of the therapeutic agent (e.g., Cmax or the concentration at a particular time point 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] Thus, provided are methods for killing HER2-expressing cells, the methods comprising contacting a conjugate as defined herein with a HER2-expressing cell, thereby causing the conjugate to be internalized within the cell, and thereby causing the therapeutic agent to kill the HER2-expressing cell. In some embodiments, the therapeutic agent is a supercytotoxic agent (e.g., MMAE, MMAF). In some embodiments, the conjugate (e.g., the supercytotoxic agent-containing conjugate) is internalized within the cell by endocytosis.

[0218] The conjugates of the present disclosure have been shown to be effective in in vivo cancer studies, i.e., xenograft studies. It is believed that the relatively small size (e.g., low overall molecular weight of the conjugate) aids in tumor tissue penetration and the effectiveness of the present treatment. Thus, in some embodiments, the molecular weight of the conjugate is in the range of at most about 120 kDa, at most about 100 kDa, at most about 80 kDa, at most about 60 kDa, at most about 50 kDa, at most about 45 kDa, or at most about 40 kDa.

[0219] composition In some embodiments, the dendrimer-targeting agent conjugate 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 molecular composition among the dendrimer-targeting agent conjugates present in a given composition. For example, as noted above, one or more of the synthetic steps used to produce a dendrimer-targeting agent conjugate may not proceed completely to completion, resulting in the presence of dendrimer-targeting agent conjugates that all contain the same number of therapeutic agent moieties, no targeting agent, or incomplete generation building blocks.

[0221] In one example, the composition comprises a plurality of dendrimers that do not all comprise the same number of therapeutic agent moieties. In one example, the composition comprises a plurality of dendrimers that do not all comprise the same number of targeting agents. In some embodiments, the composition comprises a plurality of dendrimer-targeting agent conjugates, wherein the average number of targeting agents covalently linked to the dendrimers is 1. In some embodiments, the composition comprises a plurality of dendrimer-targeting agent conjugates, wherein the average number of targeting agents covalently linked to the dendrimers is 1.2 ... The composition comprises a plurality of dendrimer-targeting agent conjugates, wherein the average number of targeting agents covalently attached to the dendrimer is between 1 and 1.5. In some embodiments, the composition comprises a plurality of dendrimer-targeting agent conjugates, wherein the average number of targeting agents covalently attached to the dendrimer is between 0.5 and 5. In some embodiments, the composition comprises a plurality of dendrimer-targeting agent conjugates, wherein the average number of targeting agents covalently attached to the dendrimer is between 1 and 4. In some embodiments, the composition comprises a plurality of dendrimer-targeting agent conjugates, wherein the average number of targeting agents covalently attached to the dendrimer is between 1 and 1.5. Thus, a composition is provided comprising a plurality of dendrimer-targeting agent conjugates, or pharmaceutically acceptable salts thereof, wherein the dendrimer-targeting agent conjugate is as defined herein. In some embodiments, a composition for therapeutic use is provided, comprising a dendrimer-targeting agent conjugate and a therapeutically acceptable excipient. 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 present disclosure also provides pharmaceutical formulations or compositions for both veterinary and human medical use, which comprise a conjugate of the present disclosure, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilizers, etc.

[0223] Accordingly, there is also provided a composition for pharmaceutical use comprising i) a conjugate as defined herein, and ii) a pharmaceutically acceptable excipient.

[0224] The excipient(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation / composition and not unduly adversely affecting the recipient thereof.

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

[0226] Compositions of the present disclosure can include, for example, polymeric excipients / additives or carriers, such as polyvinylpyrrolidone, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylmethylcellulose, ficoll (a polymeric sugar), hydroxyethyl starch (HES), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin), polyethylene glycol, and pectin.

[0227] The compositions may further include diluents, buffers, citrate salts, 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 phosphatidylcholines, phosphatidylethanolamines, 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 compositions according to the present disclosure are described in "Remington: The Science & Practice of Pharmacy," 19th ed., Williams & Williams, (1995), and "Physician's Desk Reference," 52nd ed., Medical Economics, Montvale, NJ (1998), and "Handbook of Pharmaceutical Excipients" (3rd ed.), ed. A.H. Kibber, Pharmac This is described in European Journal of Clinical Nutrition, Vol. 1, No. 1, 2000.

[0228] The dendrimer-targeting agent conjugates of the present disclosure can be formulated into compositions, including those suitable for administration by, for example, pulmonary inhalation, aerosol administration, or parenteral administration (e.g., intraperitoneal, intravenous, subcutaneous, or intramuscular injection). The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the dendrimer-targeting agent conjugate into association with a carrier, which constitutes one or more accessory ingredients. Generally, compositions are prepared by bringing the dendrimer-targeting agent conjugate into association with a liquid carrier to form a solution or suspension, or by bringing the dendrimer-targeting agent conjugate into association with formulation ingredients suitable for forming a solid, optionally particulate, product, and, if applicable, shaping the product into the desired delivery form. Solid formulations of the present disclosure, when micronized, typically contain particles ranging in size from about 1 nanometer to about 500 microns. Generally, for solid formulations intended for intravenous administration, the particles range in size from about 1 nm to about 10 microns in diameter. The composition may comprise dendrimer-targeting moiety conjugates of the present disclosure that are nanoparticles having a particle size of 1000 nm or less, e.g., 5-1000 nm, particularly 5-500 nm, more particularly 5-400 nm, e.g., 5-50 nm, particularly 5-20 nm. In one example, the composition comprises dendrimer-targeting moiety conjugates with an average diameter of 5-20 nm. In some embodiments, the dendrimer-targeting moiety conjugates are polydispersed in the composition, with a PDI of 1.01-1.8, particularly 1.01-1.5, more particularly 1.01-1.2. In one example, the dendrimer-targeting moiety conjugates are monodispersed 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 suitable for reconstitution with an aqueous vehicle prior to injection.

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

[0231] In some embodiments, the composition is formulated for parenteral injection 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, it may be delivered by lavage or aerosol. In one embodiment, the composition is formulated for intraperitoneal delivery and is used to treat intraperitoneal cancers, such as malignant epithelial tumors (e.g., ovarian cancer) and peritoneal carcinomatosis (e.g., gastrointestinal, particularly colon cancer, gastric cancer, gynecological cancer, and primary peritoneal neoplasms).

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

[0234] As described below, the dendrimer-targeting agent conjugates of the present disclosure can be administered in combination with, for example, one or more additional pharmaceutically active agents. For example, the dendrimer-targeting agent conjugates can be administered in a composition together with the additional pharmaceutically active agents. Examples of further 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 the present disclosure can be administered not only together with other chemotherapeutic agents, but also, if appropriate, in a composition with other pharmaceutical agents.

[0236] How to use Also provided herein is a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate as defined herein, or a pharmaceutical composition comprising a conjugate as defined herein.

[0237] Also provided herein is the use of a conjugate as defined herein, or a composition comprising a conjugate as defined herein, in the manufacture of a medicament for the treatment of cancer.

[0238] Also provided herein is a conjugate as defined herein, or a pharmaceutical composition comprising a conjugate as defined herein, for use in therapy. Also provided herein is a conjugate as defined herein, or a pharmaceutical composition comprising a conjugate as defined herein, for use in the treatment of cancer.

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

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

[0241] In some embodiments, the cancer is characterized by aberrant or overexpression of HER2 (also known as ERBB2). Such aberrant or overexpression of HER2 is known to occur in, for example, 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 endometrial cancer.

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

[0243] combination Drugs are often administered in combination with other drugs, particularly during chemotherapy. Thus, in some embodiments, the dendrimer-targeting agent conjugate is administered in combination with one or more additional pharmaceutically active agents, e.g., one or more additional anti-cancer agents / drugs. The dendrimer-targeting agent conjugate and one or more additional pharmaceutically active agents can be administered simultaneously, sequentially, or separately. For example, they may be administered as part of the same composition or by administering separate compositions. The one or more additional pharmaceutically active agents may be, for example, anti-cancer agents for the treatment of colon 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 therapy, taxanes, and aromatase inhibitors.

[0244] dose It will be understood that the term "therapeutically effective amount" refers to a dendrimer-targeting agent conjugate administered in an amount sufficient to alleviate or prevent to some extent one or more symptoms of the disorder or condition being treated. A therapeutically effective amount of a dendrimer-targeting agent conjugate can be referred to, for example, based on the amount of dendrimer-targeting agent conjugate administered. Alternatively, it can be determined based on the amount of therapeutic agent that the administered dendrimer-targeting agent conjugate can theoretically deliver, for example, the loading of a therapeutic agent to the dendrimer-targeting agent conjugate.

[0245] The dendrimer-targeting agent complex may be administered by any suitable route. For example, the dendrimer-targeting agent complex may be administered 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 ranging from 0.5 to 60 minutes, or from 0.5 to 15 minutes, or from 0.5 to 5 minutes. In another example, the dendrimer-targeting agent complex may be administered intraperitoneally. The route of administration may be targeted, for example, to the disease or disorder the subject has. For example, in some embodiments, the disease or disorder may be an intraperitoneal malignancy, such as a 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 cancer of the peritoneal cavity, such as malignant epithelial tumors (e.g., ovarian cancer) or peritoneal carcinomatosis (e.g., gastrointestinal, especially colorectal, gastric, gynecological cancers, and primary peritoneal neoplasms), and the dendrimer-targeting moiety conjugate is administered intraperitoneally.

[0246] In some embodiments, the dose of the dendrimer-targeting agent conjugate is between 2 and 100 mg of active agent / m 2 , 2 to 50 mg of active agent / m 2 , 2 to 40 mg of active agent / m 2 , 2-30 mg of active agent / m 2 , 2 to 25 mg of active agent / m 2 , 2 to 20 mg of active agent / m 2 , 5 to 50 mg of active agent / m 2 , 10-40 mg of active agent / m 2 , 15-35 mg of active agent / m 2 , 10-20 mg / m 2 , 20-30 mg / m 2 , or 25-35 mg of active agent / m 2 A dose of 10 mg / kg of active agent in mice is sufficient to deliver 30 mg / m 2 (The mg / kg dose in humans is converted to mg / m 2To convert, multiply by 37 (FDA Guidance 2005).

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

[0248] The targeted complexes of the present disclosure provide a therapeutic agent in a controlled manner. By controlling the release of the therapeutic agent from the complex, the level of circulating therapeutic agent (e.g., hypercytotoxicity) can also be controlled. For example, the maximum plasma concentration of the therapeutic agent can be significantly lower than the results obtained from administration of an equivalent amount of free therapeutic agent. In some embodiments, administration of the complex results in a maximum plasma concentration of the released therapeutic agent that is significantly lower than that obtained from administration of an equivalent dose of free therapeutic agent. , 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% decrease.

[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 conjugate comprises: Produced by a process comprising reacting a first intermediate comprising a HER2 targeting agent with a second intermediate which is a dendrimer: the first intermediate has a molecular weight of up to about 80 kDa and is a peptide moiety comprising an antigen-binding site, the targeting agent being covalently attached to a spacer precursor group, the spacer precursor group comprising a first reactive group; The second intermediate is i) Core Unit (C), and ii) comprising building blocks (BU), each building block being a lysine residue or an analog thereof; a dendrimer in which a core unit is covalently attached to at least two building blocks via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block; a therapeutic agent covalently linked to a surface building unit of the dendrimer; and hydrophilic polymeric groups covalently linked to the surface building units of the dendrimer; the second intermediate comprises a second reactive group; The first and second reactive groups are complementary such that they are suitable to react with each other to covalently link the HER2 targeting agent to the dendrimer.

[0251] The present disclosure also provides intermediates useful for the synthesis of dendrimer-targeting agent conjugates. Thus, intermediates for preparing dendrimer-targeting agent conjugates comprising a HER2 targeting agent that is a peptide moiety having a molecular weight of up to about 80 kDa and comprising an antigen-binding site, wherein the targeting agent is covalently attached to a spacer precursor group, the spacer precursor group comprising a reactive group suitable for reaction with a complementary reactive group present on the dendrimer intermediate.

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

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

[0254] In some embodiments, the reactive group present on the spacer precursor group is an alkene group, e.g., a reactive alkene that is suitable for reaction with a tetrazine-containing group, hi 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 comprising: a) i) Core Unit (C), and ii) a dendrimer comprising building blocks (BU), each building block being a lysine residue or analog thereof; a dendrimer in which a core unit is covalently attached to at least two building blocks via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block; b) a HER2 targeting agent, which is a peptide moiety having a molecular weight of up to about 80 kDa and containing an antigen-binding site, covalently linked to the dendrimer by a spacer group; c) a therapeutic agent covalently linked to a surface building unit of the dendrimer; d) PEG or PEOX groups covalently linked to the surface building units of the dendrimer. 2. The conjugate of clause 1, wherein the peptide moiety is selected from a heavy chain antibody, a Fab, an Fv, an scFv, or a single domain antibody. 3. The peptide portion is a heavy chain variable (V H ) domain or heavy chain variable (V H 3. The complex of clause 1 or 2, comprising a .sup.3 domain. 4. The peptide portion is the light chain variable (V L ) domain or light chain variable (V L 4. The complex of any one of clauses 1 to 3, comprising a ) domain. 5. The conjugate of any one of clauses 1 to 4, wherein the targeting agent has a molecular weight of about 5 kDa to about 30 kDa. 6. The conjugate of clause 5, wherein the targeting agent has a molecular weight of about 5 kDa to about 15 kDa. 7. The conjugate of clause 6, wherein the targeting agent has a molecular weight of about 10 kDa to about 16 kDa. 8. The conjugate of any one of clauses 1-7, wherein the targeting agent comprises fewer than 120 amino acid residues. 9. The conjugate of any one of clauses 1 to 8, wherein the targeting agent comprises or consists of any of the amino acid sequences defined herein. 10. The conjugate of any one of clauses 1-9, wherein the covalent linkage between the targeting agent and the spacer group is formed by reaction between complementary reactive functional groups present on the targeting agent precursor and the spacer group precursor. 11. The conjugate of clause 10, wherein the targeting agent precursor comprises a non-natural amino acid residue, the non-natural amino acid residue having a side chain comprising a reactive functional group. 12. The conjugate of clause 11, wherein the unnatural amino acid residue is a 4-azidophenylalanine residue. 13. The conjugate of any one of clauses 1-12, wherein the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting agent. 14. The conjugate of any one of clauses 10 to 13, wherein the spacer group precursor comprises a reactive functional group that is an alkyne group. 15. The conjugate according to clause 14, wherein the alkyne group is a dibenzocyclooctyne group. 16. The conjugate of any one of clauses 1 to 15, wherein the therapeutic agent is an ultra-cytotoxic agent. 17. The conjugate of clause 16, wherein the therapeutic agent is an auristatin or a maytansinoid. 18. The conjugate of clause 17, wherein the therapeutic agent is monomethyl auristatin E. 19. The conjugate of clause 17, wherein the therapeutic agent is monomethyl auristatin F. 20. The conjugate of any one of clauses 1-19, wherein the therapeutic agent is covalently linked to a surface-building unit of the dendrimer via a linker. 21. The conjugate of any one of clauses 1 to 20, wherein the therapeutic agent is covalently linked to a surface-building unit of the dendrimer via a cleavable linker. 22. The conjugate of clause 21, wherein the cleavable linker comprises a Val-Cit-PAB group. 23. The conjugate of any one of clauses 1 to 22, comprising a PEG group covalently linked to a surface-building unit of the dendrimer. 24. The conjugate of clause 23, wherein the PEG group has an average molecular weight in the range of about 500 to about 2500 g / mol. 25. The conjugate of any one of clauses 1 to 24, wherein the spacer group comprises a PEG group. 26. The core unit is the structure [ka] 26. The conjugate of any one of clauses 1 to 25, comprising: 27. The conjugate of any one of clauses 1 to 26, wherein the dendrimer has constitutional units of generations 1 to 5. 28. The conjugate according to clause 27, wherein the dendrimer has three generations of building blocks. 29. The constituent units are, respectively: [ka] 29. The conjugate of any one of clauses 1 to 28, wherein 30. A conjugate according to any one of clauses 1 to 29 for administration in combination with a further active agent. 31. The conjugate of any one of clauses 1 to 30, wherein the conjugate is internalized into HER2-expressing cells. 32. The conjugate of any one of clauses 1 to 31, wherein administration of the conjugate results in reduced side effects compared to administration of an equivalent dose of the free therapeutic agent. 33. The conjugate of any one of clauses 1 to 32, wherein administration of the conjugate results in at least a 50% decrease in 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 a plurality of conjugates according to any one of clauses 1 to 33. 35. i) a complex according to any one of clauses 1 to 34; ii) a pharmaceutically acceptable excipient; and A pharmaceutical composition comprising: 36. The composition of clause 34 or 35, wherein the composition is formulated for parenteral delivery. 37. A conjugate 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 of treating cancer comprising administering a therapeutically effective amount of the conjugate of any one of clauses 1 to 33 or the composition of any one of clauses 34 to 37 to a subject in need thereof. 39. Use of a conjugate according to any one of clauses 1 to 33 or a composition according to any one of clauses 34 to 38 in the manufacture of a medicament for the treatment of cancer. 40. The method or use according to any one of 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. or a complex or composition for use. 41. A method for killing HER2-expressing cells, comprising: 34. A method comprising contacting the conjugate of any one of clauses 1 to 33 with a HER2-expressing cell, thereby causing the conjugate to be internalized into the cell, and thereby causing the therapeutic agent to kill the HER2-expressing cell.

[0257] The present disclosure is further illustrated by the following examples: It should be understood that the following descriptions are for the purpose of illustrating particular embodiments only and are not intended to be limiting with respect to the above descriptions.

[0258] Example The following nomenclature is used herein in connection with the synthesis of dendrimer conjugates: [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), inlet flow split to MS at approximately 50 μL / min.

[0260] Mass spectrometry data were acquired in positive or negative electrospray ionization mode as indicated. Raw data were analyzed using Maximum NMR spectroscopy (MS) as implemented in MassLynx software v4.0 provided by Waters Corporation. Deconvolution was performed using the Entropy algorithm (MaxEnt). 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 scaffolds has been previously described, inter alia, in WO 2007 / 082331, WO 2008 / 017125, WO 2012 / 167309 and WO 2015 / 184510. Those skilled in the art can use and adapt these methods to prepare the various dendrimers outlined herein.

[0262] In the following examples, [Lys] in the formulas refers to the lysine building blocks in the surface layer of the dendrimer.

[0263] Example 1 General Procedure A. Boc Deprotection To a stirred, ice-cooled suspension of the Boc compound (1.0 equiv.) in water was added TFA (40-200 equiv. / Boc group). After 5 min, the ice bath was removed and the reaction mixture was allowed to stir at room temperature overnight. The volatiles were removed in vacuo, and the remaining aqueous solution was further diluted with water and lyophilized to give the deprotected product in quantitative yield.

[0264] General Procedure B. Addition of a lysine layer to the dendrimer surface. To a stirred solution of TFA dendrimer (1.0 equiv.) in DMF under a nitrogen atmosphere, TEA (6.0 equiv. / NH2) was added, followed by DBL-oPNP (2.0 equiv. / NH2). The resulting reaction mixture was then allowed to stir at room temperature overnight. Volatiles were removed in vacuo, and the resulting crude material was purified using standard methods.

[0265] General Procedure C. PEGylation of dendrimer surfaces using HO-Lys-(α-NHBoc)(ε-NHPEG1100) wedges. To a stirred solution of TFA dendrimer (1.0 equiv.) / DMF under a nitrogen atmosphere, PyBOP (2.0 equiv. / NH2) and DIPEA (8.0 equiv. / NH2) were added. After 10 min, HO-Lys-(α-NHBoc)(ε-NHPEG) was added. 1100 A solution of 1.35 equiv. / NH2 in DMF was added, and the resulting reaction mixture was stirred at room temperature overnight. The volatiles were removed in vacuo, and the resulting crude material was purified using standard methods.

[0266] General Procedure D. Capping of the dendrimer surface with a HO-Lys-(α-NHBoc)(ε-NHFmoc) wedge followed by Fmoc deprotection. Step 1: To a stirred solution of HO-Lys-(α-NHBoc)(ε-NHFmoc) (1.5 equiv. / NH2) and NMM (2.5 equiv. / NH2) in DMF was added PyBOP (1.4 equiv. / NH2). The resulting reaction mixture was then stirred at room temperature for 15 min, and a solution of TFA-dendrimer (1.0 equiv.) and NMM (2.5 equiv. / NH2) in DMF was added. The resulting reaction mixture was then allowed to stir at room temperature for 1 h, then slowly added to ice-cold MeCN and stirred for 15 min. The resulting solid was collected by filtration, washed with MeCN (3 times), and lyophilized.

[0267] Step 2: To a DMF solution of Fmoc / Boc dendrimer (1.0 equiv.) was added piperidine (21 eq / Fmoc). After stirring at room temperature for 90 min, the solution was slowly added to ice-cold EtO. After 15 min, the precipitated solid was collected by filtration, washed with EtO, dissolved in HO, and stored frozen.

[0268] General Procedure E. Capping of Dendrimer Surfaces with DGA-doxorubicin or DGA-nemorubicin DGA-3'-NH-doxorubicin

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

[0270] Step 2: Add PyBOP (2.0 equiv. / dendrimer-NH) to the above reaction mixture, followed by 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 equiv.) and DIPEA (6.0 equiv. / dendrimer NH) / DMF solution were added. The resulting reaction mixture was then left stirring at room temperature overnight. Volatiles were removed in vacuo, and the resulting crude material was purified by SEC.

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

[0272] Step 2: Add PyBOP (2.0 equiv. / dendrimer-NH2) to the above reaction mixture. , followed by TFA-dendrimer (azide-PEG 24 -CO[N(PN)2[Lys]2[Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )]4 or azido- PEG 24 -CO[N(PN)2[Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NHPEG 1100 )]8) (1.0 equiv.) and DIPEA (6.0 An equivalent of dendrimer (NH) / DMF solution was added. The resulting reaction mixture was then left stirring at room temperature overnight. Volatiles were removed in vacuo and the resulting crude material was purified by SEC.

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

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

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

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

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

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

[0280] Example 1a Synthesis of intermediates 1.1 Azide-PEG 24 -CO[N(PNBoc)2] Compound 1 Azide-PEG under N2 atmosphere 24 To a stirred solution of 2.00 g (1.71 mmol) of methylpyridinium chloride and 1.33 g (2.56 mmol) of PyBOP in 20 mL of DMF was added 563 μL of NMM (5.12 mmol). After 10 min, 622 mg of N(PNBoc)2 was added. A solution of 1.88 mmol) in DMF (5 mL) was added, and the resulting reaction mixture was allowed to stir at room temperature overnight. Volatiles were removed in vacuo, and the resulting oil was dissolved in MeCN and purified by preparative HPLC (27-50-70% MeCN, Rt 47-50 min) to give a pale yellow oily solid (1.37 g, 54%). H-NMR (300 MHz, CD3OD) δ (ppm): 1.44 (m, 18H); 1.65-1.84 (m, 4H); 2.63 (t, J 6.3 Hz, 2H); 3.05 (dt, J 6.9 and 14.7 Hz, 4H); 3.36-3.41 (m, 6H); 3.60-3.78 (m, 98H). LCMS (hydrophilic method, formate buffer) R t =9.32 minutes ESI MS(+ ve)1486.3[M] + ;C 67 H 132 NO 29 [M] + Calculated m / z: 1486.8

[0281] 1.2 Azide-PEG 24 -CO[N(PNH2.TFA )2 ], compound 2 Azide-PEG 24 Prepared using —CO[N(PNBoc)2] (1.37 g, 922 μmol) according to general procedure A. The lyophilized product was obtained as a pale yellow oil (1.67 g, 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 NO 25 Calculated m / z for [M]+ = 1286.6.

[0282] 1.3 Azide-PEG 24 -CO[N(PN)2][Lys]2[Boc]4, G1, compound 3 Azide-PEG 24 Prepared according to general procedure B using —CO[N(PNH.TFA)] (186 mg, 145 μmol). The crude material was dissolved in MeCN and purified by preparative HPLC (30-80% MeCN, R t 33.5-36 min) to give 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] + Calculated m / z = 1942.4.

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

[0284] 1.5 Azide-PEG 24 -CO[N(PN)2][Lys]4[(α-Boc)(ε-NHPEG 1100 )]4, G2, compound 5 Azide-PEG 24 Prepared according to general procedure C using -CO[N(PN)2[Lys]2[NH2.TFA]4 (120 mg, 60.1 μmol). The crude material was dissolved in MeCN / HO (1:1) and purified by preparative HPLC (20–70% MeCN, Rt 31–32.5 min) to give 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+ / 7. Converts to 6852.

[0285] 1.6 Azide-PEG24 -CO[N(PN)2][Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )]4, G2, compound 6 Azide-PEG 24 -CO[N(PN)2[Lys]4[(α-Boc)(ε-NHPEG 1100 )]4 (244 mg, 35.6 μmol) according to general procedure A. The lyophilized crude product was redissolved in water and purified by preparative HPLC (22-70% MeCN, 0.01% TFA, R t 27 min) to give the product as a pale yellow sticky 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+ / 6. Converts to 6452.

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

[0287] 1.8 Azide-PEG 24 -CO[N(PN)2][Lys]4[NH2.TFA]8, G2, compound 8 Azide-PEG 24 Prepared according to general procedure A using —CO[N(PN)][Lys][Boc] (167 mg, 58.6 μmol). The crude aqueous solution was purified by preparative HPLC (10–60% MeCN, 0.1% TFA buffer; Rt 27–29 min) to give the product as a very pale yellow sticky solid (124 mg, 71% over two steps). 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 minutes, 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+ Calculated m / z for / 2: 1028.3, C 93 H 191 N 18 O 313+ [ M+3H ]3+ / Calculated m / z for 3: 685.9.

[0288] 1.9 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG1100)]8,G3,Compound 9 Azide-PEG 24Prepared according to general procedure C using —CO[N(PN)][Lys][NH.TFA] (123 mg, 41.5 μmol) to give the crude material as a brown oil. LCMS (hydrophilic method, formic acid buffer) 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+ / 12. Converts to 12 673.

[0289] 1.10 Azido-PEG24-CO[N(PN)2][Lys]8[(α-TFA)(ε-NHPEG 1100 )]8, G3, compound 10 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 1100 )]8 (526 mg, 41.5 μmol) 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 give the product as a pale yellow sticky solid (359 mg, 68% over two steps). LCMS (hydrophilic method, TFA buffer) Rt = 10.27 min, converted to 11880.

[0290] 1.11 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-Fmoc)]8,G3, Compound 11 Azide-PEG 24 Prepared using —CO[N(PN)][Lys][NH.TFA] (105 mg, 35.4 μmol) according to general procedure D. The product was obtained as a white solid (166 mg, 83%). 1H-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 Azide-PEG 24 Prepared according to general procedure D using —CO[N(PN)][Lys][(α-Boc)(ε-Fmoc)] (169 mg, 29.9 μmol) to give a fluffy solid (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 A solution of -COH (205 mg, 348 μmol), NMM (60 μL, 546 μmol) and PyBOP (171 mg, 329 μmol) in DMF (1.5 mL) was added to azide-PEG 24 -CO[N(PN)2[Lys]8[(α-Boc)(ε-NH2)]8 (0.5 mL) in DMF was added. The resulting reaction mixture was then heated at room temperature. The mixture was stirred at RT overnight, then concentrated in vacuo, dissolved in water, treated with TFA, and stirred at RT overnight. The mixture was concentrated, taken up in water, and purified using a Millipore Centrifugation filtration unit (3K MWCO regenerated cellulose) to give the lyophilized product (68% over two steps) as an off-white fluffy material. 1 H-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 solution 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 Azide-PEG 24 To a stirred solution of -CO[N(PN)2[Lys]8[(α-Boc)(ε-NH2)]8 (95.0 mg, 24.5 μmol) in DMF (4 mL) was added DIPEA (85 μL, 488 μmol), 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 (5K, Pall PES membrane). The retentate was collected and lyophilized to yield an off-white fluffy 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 Azide-PEG according to general procedure A 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 2000 )]8 (40.0 mg, 1.87 μmol) The product was obtained as an off-white fluffy 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 To a stirred solution of -COH (0.402 g, 0.305 mmol), PyBOP (0.205 g, 0.394 mmol), and NMM (130 μL, 1.18 mmol) in DMF (3 mL) was added NH(PNBoc) (0.147 g, 0.444 mmol) under a N atmosphere. The resulting reaction mixture was then allowed to stir at room temperature overnight. Volatiles were removed in vacuo, and the resulting oil was purified by silica chromatography (5%-10% MeOH / DCM) to afford the desired product, compound 106, as a red residue (0.474 g, 95%). 1HNMR(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] + Calculated m / z: 1631.0.

[0296] 1.17 (MeTzPh)-PEG 24 -CO[N(PNH2.HCl)2] Compound 47 (MeTzPh)-PEG in an ice / water bath 24 To —CO[N(PNBoc)2] compound 1 (0.420 g, 0.258 mmol) was slowly added 1.25 M HCl / MeOH solution (8 mL, 10.0 mmol). After 5 min, the ice bath was removed, and the resulting reaction mixture was allowed to stir at room temperature overnight. Volatiles were removed in vacuo to give the product, compound 107, 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,108 H);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] + Calculated m / z: 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] (0.418 g, 0.286 mmol) in DMF (2.0 mL) was added (MeTzPh)-PEG4-CO2H (0.15 g, 0.344 mmol), PyBOP (0.178 g, 0.342 mmol), and and NMM (80 μL, 0.727 mmol) were added. The resulting reaction mixture was then allowed to stir at room temperature overnight. The volatiles were removed in vacuo, and the resulting oil was cooled in an ice / water bath. 1.25 M HCl / MeOH solution (10.0 mL, 12.5 mmol) was then slowly added. After 5 min, the ice bath was removed, and the resulting reaction mixture was then allowed to stir at room temperature overnight. The volatiles were removed in vacuo, and the resulting oil was dissolved in MeCN / HO (8 mL, 1:1) and purified by preparative HPLC (10–50% MeCN, 0.1% formic acid buffer, RT 35 min) to give compound 108 (1.37 g, 54%) as a red solid. 1 HNMR(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] + Calculated m / z: 1678.0.

[0298] 1.19 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NHBoc]4G1 Compound 49 Under N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24To a stirred solution of —CO[N(PNH2.HCl)2] compound 48 (0.195 g, 0.111 mmol) and DBL-oPNP (0.146 g, 0.312 mmol) in DMF (3.0 mL) was added NMM (125 μL, 0.864 mmol). The resulting reaction mixture was then allowed to stir at room temperature overnight. The volatiles were removed in vacuo, and the resulting oily residue was purified by column chromatography on silica gel (5%-10%-15% MeOH / DCM) to give the desired product, compound 109, as a red oil (0.186 g, 72%). 1 HNMR(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] + Calculated m / z = 2335.4.

[0299] 1.20 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NH2.HCl]4G1 Compound 50 Ice-cold (MeTzPh)-PEG4CO-NHPEG 24 To compound 49 (0.215 g, 0.0921 mmol) was slowly added a solution of 1.25 M HCl / MeOH (6.0 mL, 7.50 mmol). After 5 min, the ice bath was removed, and the resulting reaction mixture was allowed to stir at room temperature overnight. Volatiles were removed in vacuo to give the product, compound 110, as a red oil (0.208 g, 108%). 1HNMR(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 O 33 [M] + Calculated m / z = 1935.2.

[0300] 1.21 (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]4[NHBoc]8G2 Compound 51 Under N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24 To a stirred solution of -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) in DMF (3.0 mL) was added NMM (215 μL, 0.196 mmol). The resulting reaction mixture was then allowed to stir at room temperature overnight. Volatiles were removed in vacuo, and the resulting oily residue was purified by silica chromatography (5%-10%-15% MeOH / DCM) to give 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,J 6.0Hz,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] + Calculated m / z = 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 To compound 51 (0.231 g, 0.0711 mmol) was slowly added a solution of 1.25 M HCl / MeOH (9.0 mL, 11.3 mmol). After 5 min, the ice bath was removed, and the resulting reaction mixture was allowed to stir at room temperature overnight. Volatiles were removed in vacuo to give the product, compound 51, 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 O37 [M+H] + Calculated m / z = 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 To a stirred solution of (MeTzPh)-PEG4CO-NHPEG (0.541 g, 0.402 mmol) and PyBOP (0.195 g, 0.375 mmol) in DMF (2 mL) was added NMM (225 μL, 2.96 mmol). After 10 min, the solution was cooled to room temperature and cooled to room temperature. 24 To the cooled residue was added 1.25M (8.0 mL, 10 mmol). After 10 min, 1.25M (8.0 mL, 10 mmol) was slowly added. The cold water bath was removed and the reaction was left stirring at room temperature overnight. Volatiles were removed in vacuo and dissolved in HO (16 mL). The solution was centrifuged using Millipore Amicon Ultra-15 Centrifugal Filter Units (4 units x 10 kDa MWCO units). * The retentate was lyophilized overnight to give 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 HO (5 mL) and spun at 4000 rpm for 5 minutes. This process was repeated. This crude solution was filtered through a 0.45 μm syringe filter and placed in a centrifuge. The unit was then spun at 4000 rpm for 15 minutes. The retentate was then diluted with HO (4 mL) and spun 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 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) in DMF (2.0 m To the stirred solution of 50 mL of HCl (190 μL, 1.973 mmol) was added NMM (190 μL, 1.973 mmol). The resulting reaction mixture was then allowed to stir at room temperature overnight. Volatiles were removed in vacuo, and the resulting oily residue was purified by silica chromatography (5%-10%-15% MeOH / DCM) to give the Boc-protected product as a red oil (0.128 g, 70%). The purified material was cooled in an ice / water bath, and then a solution of 1.25 M HCl / MeOH (6.5 mL, 8.5 mmol) was slowly added. After 5 min, the ice bath was removed, and the resulting reaction mixture was then allowed to stir at room temperature overnight. Volatiles were removed in vacuo to give the product, compound 54, as a red oil (0.107 g, 100%). LCMS (hydrophilic method, TFA buffer) RT = 7.48 min, ESI MS (+ve) 3471 [M+H] + ;[M+H] + Calculated m / z = 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 To a stirred solution of (MeTzPh)-PEG4-PEG (0.107 g, 0.132 mmol) and PyBOP (0.065 g, 0.125 mmol) in DMF (1.5 mL) was added NMM (50 μL, 0.455 mmol). After 10 min, the solution was cooled to room temperature and cooled to room temperature. 24 -CO[N(PN)2][Lys]8[NH2.HCl] 16 Compound 54 (0.0221 g, 0.00545 mmol) was added. The resulting reaction mixture was then allowed to stir at room temperature overnight. The volatiles were removed in vacuo, and the resulting crude product was cooled in an ice / water bath. To the cooled residue, 1.25 M HCl / MeOH (1.5 mL, 1.88 mmol) was slowly added. 10 min After this time, the cold water bath was removed and the reaction was left stirring at room temperature overnight. Volatiles were removed in vacuo and dissolved in HO (5 mL). This solution was purified by centrifugation using a Millipore Amicon Ultra-15 Centrifugal Filter Unit (10 kDa MWCO). The retentate was lyophilized overnight and further purified by Gilson (single gradient 60 min, 10 > 60% ACN (0.1% TFA buffer, RT 36 min)) to give 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,6 3H);3.52-4.01(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-NHPEG24 -CO[N(PN)2][Lys] 16 [NH2.TFA] 32 G4 Compound 56 Prepared similarly to compound 54 and deprotected using TFA / AcOH to give compound 56 (0.078 g, 78%) as a red solid. LCMS (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 To a stirred solution of (MeTzPh)-PEG4CO-NHPEG (0.476 g, 0.354 mmol) and PyBOP (0.170 g, 0.327 mmol) in DMF (3.0 mL) was added NMM (180 μL, 0.500 mmol). After 10 min, the solution was cooled to room temperature and cooled to room temperature. 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 allowed to stir at room temperature overnight. The volatiles were removed in vacuo, and the resulting crude product was dissolved in HO (1.0 mL) and TFA (1.0 mL) was added slowly. The reaction was allowed to stir at room temperature overnight. The mixture was diluted with 20 (12 mL) and purified by centrifugation using a Millipore Amicon Ultra-15 Centrifugal Filter Unit (10 kDa MWCO). The retentate was lyophilized overnight to give compound 57 (0.374 g, 91%) as a pink solid. 1HNMR(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)3.48-3.9 8(m,2960H);4.27-4.39(m,32H);7.30-7.33(m,2H);8.46-8.49(m,2H);HPLC analysis (hydrophilic method, using ammonium formate as a buffer) RT=8.60 min.

[0308] 1.28 (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NHCy5)1(α-NHAc)7)(ε-NH-COPEG1100)8], G3, Compound 37 A solution of Cy5-NHS ester (1.0 mL of a 1 mg / mL solution in DMF; 1.0 mg, 1.59 μmol) was added to (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[(α-NH2.HCl)8)(ε-NH-COPEG 1100 )8] was added to a vial containing compound 53 (20 mg, 1.59 μmol) in DMF (0.5 mL). To this solution, NMM (10 μL, 91.0 μmol) was added, and the resulting reaction mixture was then stirred at RT, protected from light. After 3.5 h, acetic anhydride (20 μL, 212 μmol) was added, and the reaction mixture was left stirring overnight. The reaction mixture was concentrated under reduced pressure, taken up in MQ water (5 mL), and split into two portions, which were then purified on two (pre-equilibrated) PD10 columns. Once the sample had entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The combined filtrates were lyophilized overnight. Lyophilization afforded the title product as a bright blue powder, 19.3 mg (93%). HPLC (C8XBridge, 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 a 1 mg / mL solution in L DMF; 0.52 mg, 844 nmol) was added to (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 16 [((α-NH2.HCl) 16 )(ε-NH-COPEG 1100 ) 16 ] was added to a vial containing compound 55 (20 mg, 844 nmol) in DMF (1.0 mL). To this solution, NMM (10 μL, 94.5 μmol) was added, and the resulting reaction mixture was then stirred at RT while protected from light. After 3.5 h, acetic anhydride (20 μL, 230 μmol) was added, and the reaction mixture was left stirring overnight. The reaction mixture was concentrated under reduced pressure, taken up in MQ water (5 mL), and split into two portions, which were then purified on two (pre-equilibrated) PD10 columns. Once the sample had entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The combined filtrate was lyophilized overnight. Lyophilization afforded the title product as a bright blue powder, 19.9 mg (98%). HPLC (C8XBridge, 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) = 8.40

[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 / mL solution in L DMF; 0.30 mg, 487 nmol) was added to (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 32 [((α-NH2.HCl) 32 )(ε-NH-COPEG 1100 ) 32 ] was added to a vial containing compound 57 (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 at RT. After 3.5 h, acetic anhydride (22 μL, 237 μmol) was added, and the reaction mixture was left stirring overnight. The reaction mixture was concentrated under reduced pressure, then taken up in MQ water (5 mL), split into two portions, and purified on two (pre-equilibrated) PD10 columns. Once the sample entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The combined filtrates were lyophilized overnight. Lyophilization afforded 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) was added triethylamine (37.0 μL, 0.264 mmol), DMAP (1.64 mg, 0.013 mmol), and p-nitrophenyl chloroformate (24.0 mg, 0.12 mmol). The mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure, and the resulting residue was purified by column chromatography on silica gel using a gradient of 0:100 to 50:50 (ethyl acetate:hexane) to give the title product as a white solid (28 mg, 21%). LCMS (hydrophilic method, TFA buffer) Rt = 6.55 min. ESI MS (+ve) 1001 [M] + ;C 52 H 60 N2O 18 Calculated m / z for [M]: 1001

[0312] 1.32 Fmoc-Val-Ala-PAB-P-Trigger-(NMeBoc), compound 59 This compound can be obtained by the method described in Dal Corso et al., Angew. Chem Int. eds. 2020, 59, 4176-4181. It can also be prepared as follows: Fmoc-Val-Ala-PAB-O-oPNP ester (Iris Biotech, 127.0 mg, 0.186 mmol) and (S)-tert-butylmethyl(pyrrolidin-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 under an inert atmosphere for 2 hours. LCMS analysis of the reaction mixture indicated the formation of the title compound. The solvent was removed under reduced pressure, and the resulting product was used in the next reaction without further purification. LCMS (hydrophilic method, TFA buffer), Rt = 6.60 min. ESI MS (+ve) 756 [M] +1; C 42 H 53 Calculated m / z for N5O8[M]+1: 756

[0313] 1.33 TFA.NH2-Val-Ala-PAB-P-Trig-(NMeBoc), Compound 60 To a stirred solution of compound 59 (141.0 mg, 0.186 mmol) in DMF (3 mL) was added piperidine (0.6 mL, 6.07 mmol) at 0 °C, and the reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure, and the resulting residue was dissolved in acetonitrile and purified using preparative HPLC (40-90% MeCN, 0.05% TFA buffer, RT approx. 45 min). After lyophilization, the title compound was obtained as a colorless liquid (63.0 mg, 52%). LCMS (hydrophilic method, TFA buffer) ESI MS (+ve) 556 [M] + Na; C 27 H 43 Calculated m / z for N5O6[M]+Na: 556; 1 H-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-TRIG-(NMeBo c), 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 h. LCMS analysis of the reaction mixture indicated product formation. LCMS (hydrophilic method, TFA buffer; 5–60% acetonitrile over 15 min), Rt = 11.32 min. ESI MS (+ve) 1048 [M] + HO; C 49 H 83 N5O 18 [M] + m / z calculated for H2O: 1048]. The solvent is removed under reduced pressure and the resulting product is used in the next reaction without further purification.

[0315] 1.35 COOH-PEG9-Val-Ala--PAB-P-TRIG-(NMe.TFA), compound 62 To a stirred solution of compound 61 (84.0 mg, 0.081 mmol) in dichloromethane (2 mL) at 0 °C, TFA (0.5 mL, 6 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the resulting residue was dissolved in acetonitrile and purified using preparative HPLC (5-70% acetonitrile, 0.05% TFA buffer, RT 32-33 min) to give the title compound as a thick, colorless liquid after lyophilization (87.0 mg, 100%). LCMS (hydrophilic method, FA buffer) ESI MS (+ve) 930 [M] +1; C 45 H 77 N5O 15 Calculated m / z for [M]+1: 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 To a solution of compound 46 (28.0 mg, 0.027 mmol) in DMF (3 mL), DIPEA (9.40 μL, 0.54 mmol) and compound 51 (27.3 mg, 0.027 mmol) were added. 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 the title product was obtained as a white solid (26 mg, 56%) after lyophilization. LCMS (hydrophilic method, TFA buffer) Rt = 5.57 min. ESI MS (+ve) 1792 [M]+; C 90 H 130 NO 31 Calculated m / z for [M]: 1792

[0317] Example 1b. Synthesis of BCN and DBCO Linkers 1.37 BCN-PEG2-Glu-CO-NHPEG 24 CO2H, compound 31 NH2-PEG 24 To a solution of -COOH (93.7 mg, 0.082 mmol) in a mixture of water / THF (1:1, 4 mL) was added sodium bicarbonate (15.2 mg, 0.181 mmol). After stirring the mixture at room temperature for 5 min, a solution of BCN-PEG2-NHS ester (50 mg, 0.093 mmol) in THF (2 mL) was added. The resulting reaction mixture was stirred at RT for 15 h. Volatiles were then 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) to give the title product as a white solid (42 mg, 33%) after lyophilization. 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 minutes; ESI MS(+ve)m / z 1566.7[M]+.

[0318] 1.38 BCN-PEG2-Glu-CO-NHPEG 24 CO-NHPEG3-T CO, compound 32 BCN-PEG2-Glu-CO-NHPEG 24 To a stirred solution of -COOH compound 31 (10.0 mg, 0.006 mmol) in 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 collected filtrate was purified by preparative HPLC (30–50% ACN, Rt 40–42 min), and the product-containing fractions were concentrated under reduced pressure to remove the ACN. The remaining aqueous solution was lyophilized overnight to give 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 Calculated m / z for [M]: 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) in 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 stirred at room temperature for 3 h and then concentrated under reduced pressure. The resulting residue was dissolved in ACN (2 mL) and filtered through a 0.45 μm filter. The filtrate was purified by preparative HPLC (40–70% ACN, Rt 27–29 min). The product-containing fractions were concentrated under reduced pressure to remove the ACN, and the remaining aqueous solution was lyophilized overnight to afford 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 Calculated m / z for [M]: 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 a 1 mg / mL solution in L DMF; 1.0 mg, 1.59 μmol) was added to MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys]8(α-NH2)8(ε-NHPEG 1100)8] (20 mg, 1.59 μmol) in DMF (0.5 mL) was added to a vial. To this solution, NMM (10 μL, 91.0 μmol) was added, and the resulting reaction mixture was then stirred at RT, protected from light. After 3.5 h, acetic anhydride (20 μL, 212 μmol) was added, and the reaction mixture was left stirring overnight. The reaction mixture was concentrated under reduced pressure, taken up in MQ water (5 mL), and split into two portions, which were then purified on two (pre-equilibrated) PD10 columns. Once the sample had entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The combined filtrates were lyophilized overnight. Lyophilization afforded the title product as a bright blue powder, 19.3 mg (93%). HPLC (C8XBridge, 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.

[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 a 1 mg / mL solution in L DMF; 0.52 mg, 844 nmol) was added to MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 16 [(α-NH2) 16 (ε-NHPEG 1100 ) 16] (20 mg, 844 nmol) in DMF (1.0 mL) was placed in a vial. To this solution, NMM (10 μL, 94.5 μmol) was added, and the resulting reaction mixture was then stirred at RT while protected from light. After 3.5 h, acetic anhydride (20 μL, 230 μmol) was added, and the reaction mixture was left stirring overnight. The reaction mixture was concentrated under reduced pressure, then taken up in MQ water (5 mL), split into two portions, and purified on two (pre-equilibrated) PD10 columns. Once the sample had entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The combined filtrates were lyophilized overnight. Lyophilization afforded the title product as a bright blue powder, 19.9 mg (98%). HPLC (C8XBridge, 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) = 8.40.

[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 / mL solution in L DMF; 0.30 mg, 487 nmol) was added to MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 32 [(α-NH2) 32 (ε-NHPEG 1100 ) 32] (20 mg, 435 nmol) in DMF (1.2 mL) was placed in a vial. To this solution, NMM (11 μL, 97.5 μmol) was added, and the resulting reaction mixture was then stirred at RT while protected from light. After 3.5 h, acetic anhydride (22 μL, 237 μmol) was added, and the reaction mixture was left stirring overnight. The reaction mixture was concentrated under reduced pressure, taken up in MQ water (5 mL), and split into two portions, which were then purified on two (pre-equilibrated) PD10 columns. Once the sample had entered the column bed, it was eluted with 3.5 mL of MQ water, and the filtrate was collected. The combined filtrates were lyophilized overnight. Lyophilization afforded 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 )4], G2, was prepared by general procedure C using compound 107 (3.0 mg, 0.414 μmol) and HO-Lys[(α-Cy5)(ε-DFO)] compound 108 (0.56 mg, 0.414 μmol) and purified by spin column (10 kDa MW cutoff, 10 x 450 μL MQ water washes) to give the desired 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 )8], G3, 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 by general procedure C and loaded onto spin columns (10 kDa MW cutoff 10 x 450 μL M The product, Compound 100, was purified by filtration (washing with MQ water) to give 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 )8], G3, 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 and purified on a spin column (10 kDa MW cutoff with 10 x 450 μL MQ water washes) to give the product, compound 27 (3.45 mg in 300 μL MQ water, final concentration).

[0326] 1.46 BHA[Lys]16 [((α-NH-COPEG 24 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 ], G4, compound 111 (11.8 mg, 0.454 μmol) and HO-Lys(α-NHCy5)( Prepared according to general procedure C using compound 108 (0.62 mg, 0.454 μmol) and loaded onto spin columns (10 x 450 μL MQ water with a 10 kDa MW cutoff) The product was purified by filtration (cleavage and washing) to give 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 ) 32Using [Chemical Formula 1], 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 and purified by spin column (10 x 450 μL MQ water washes, 10 kDa MW cutoff) to give 8.4 mg of product, compound 103, as a blue solid after lyophilization.

[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 NH-C To a stirred solution of [OPEG(PhTzMe)(G2)], compound 113 (11.0 mg, 0.0015 mmol) in DMF (3 mL) at RT, 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. After 18 h, the volatiles were 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 using a spin column (Amicon Ultra, 0.5 mL, 3 kDa MW cutoff), and the retentate was repeatedly washed with MQ water (10 × 450 μL) to give compound 24 (10 mg / mL in MQ water; 1.3 mL). LCMS (hydrophilic method, TFA buffer), gradient 20-90, Acetonitrile, 8 min; Rt = 5.64 min; 1HNMR (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 ] (50.0 mg, 0.007 mmol) and HOOCPEG 24 The residue was purified by SEC (Sephadex™ LH-20) using NH-COPEG4(PhTzMe) (Click Chemistry Tools, 13.39 mg, 0.010 mmol) and methanol as the eluent, except that the reaction vessel was wrapped in foil to protect from light, to give the product, Compound 107 (44.00 mg, 77%); 1HNMR(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(1-7 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) = 8.08-9.01.

[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 24 NH-COPEG Prepared according to general procedure C using 4 (PhTzMe) (Click Chemistry Tools; 11.2 mg, 0.008 mmol), except that the reaction vessel was wrapped in foil to protect from light, and the residue was purified by SEC (Sephadex™ LH-20) using methanol as the eluent to give the product, compound 109 (29 mg, 55%); 1HNMR(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 (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=8.16-8.93 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 ] (100.0 mg, 0.007 mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (Click Chemistry Tools; 15.97 mg, 0.010 mmol) was used, prepared according to general procedure C, except that the reaction vessel was wrapped in foil to protect from light. The residue was purified by SEC (Sephadex™ LH-20) using methanol as the eluent to give the product, compound 19 (SPL-9248) (69 mg, 66%). 1 HNMR(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 minutes), 80% ACN (7-12 minutes), 80-5% ACN (12-13 minutes), 5% ACN (13-15 minutes), 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)(100.0m g, 0.004 mmol) and HOOCPEG 24 Prepared according to general procedure C using NH-COPEG4(PhTzMe) (Click Chemistry Tools; 6.74 mg, 0.005 mmol), except that the reaction vessel was wrapped in foil to protect from light, and the residue was purified by SEC (Sephadex™ LH-20) using methanol as the eluent to give the product, Compound 111 (63 mg, 65%). 1 HNMR(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 NH-COPEG4(PhTzMe) (Click Chemistry Tools; 3.38 mg, 0.005 mmol) was used, prepared according to general procedure C, except that the reaction vessel was wrapped in foil to protect from light. The residue was purified by SEC (Sephadex™ LH-20) using methanol as the eluent to give the product, Compound 21 (68 mg, 72%); 1 HNMR(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 N H-COPEG4(PhTzMe)4],G2, compound 113 BHA[Lys]4[(α-NHBoc)4(ε-NH2)4](46.0mg,0. To a stirred solution of NMM (68.0 μL, 0.620 mmol) / DMF at 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 h, 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). The filtrate was purified by preparative HPLC. 30-80% ACN, 60 min, Mobile phase: MQ water and acetonitrile, R t 33.0-36.0 min, Compound 22 was obtained as a pink solid (52 mg, 22%). LCMS (hydrophilic method, TFA buffer) Rt = 5.66 min; 1 HNMR(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-COPEG2 4NH-COPEG4(PhTzMe)4], G2, compound 114 BHA[Lys]4[(α-NHBoc)4(ε-NH-COPEG 24 NH-COP To a stirred solution of EG4(PhTzMe)4, G2, and compound 113 (48.0 mg) in methanol (2 mL) was added 3 M hydrochloric acid / methanol (2 mL), and the reaction mixture was stirred at room temperature for 20 h. Volatiles were removed under reduced pressure to give BHALys[Lys]2[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe) 4], G2, Compound 23 was obtained as a pink solid, 41.0 mg (91%). LCMS (hydrophilic method, TFA buffer), gradient: 20 to 90 acetonitrile, 8 min; 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) was added DIPEA (32 μL, 0.224 mmol), 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 stream of nitrogen gas through the solution for several hours. The resulting residue was then purified by preparative HPLC. 30-60% ACN / MQ water + 0.1% TFA (60 min, Rt 39-42 min) afforded the product, compound 108, as a blue solid, 15 mg (34%). LCMS (hydrophilic method, TFA buffer) R t =5.93 minutes;ESI MS(+ve)1364[M]+;C 71 H 103 N 12 O 11Calculated 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 stirred solution of compound 116 (36 mg; 0.043 mmol) in DMF (4 mL) was added piperidine (1.5 mL), and the reaction mixture was stirred at room temperature for 1 h. 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 give the product, compound 59, as a blue solid, 12 mg (44%). LCMS (hydrophilic method, TFA buffer) R t = 7.65 min; ESI MS (+ve) 611 [M] + ;C 38 H 51 N4O3[M] + Calculated m / z = 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) in DMF (5 mL) was added NMM (52 μL, 0.472 mmol) and cyanine 5 NHS ester (Lumiprobes, 40.0 mg, 0.059 mmol). The reaction mixture was stirred overnight at room temperature, and then the volatiles were removed in vacuo. 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 purified by preparative HPLC; 20–90% ACN / MQ water + 0.1% formic acid (60 min, R t The resulting mixture was purified by LCMS (hydrophilic method, TFA buffer) to give 33 mg (67%) of compound 58 as a blue solid. t = 10.54 min; ESI MS (+ve) 833 [M] + ;C 53 H 61 Calculated m / z for N4O5[M]+ = 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 Azide-PEG 24 -CO[N(PN)2][Lys]4[(α-NH2.TFA)( ε-NHPEG1100 Prepared according to general procedure E using )]4 (50.0 mg, 7.24 μmol) to give 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 minutes.ESI M S(+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 Azide-PEG 24 CO-[N(PN)2][Lys]4[(α-NH2.TFA)( ε-NH-COPEG 1100 Prepared according to general procedure E using )]4 (50.0 mg, 7.24 μmol) to give 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) 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 Azide-PEG 24 -CO[N(PN)2][Lys]8[(α-NH2.TFA)( ε-NHPEG 1100 Prepared according to general procedure E using )]8 (50.0 mg, 3.91 μmol) to give 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 Azido-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG1100)]8,G3, Compound 65 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1100 )]8 (50.0 mg, 3.91 μmol) according to general procedure E to give 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.

[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 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 570 )8] (7.2 mg, 842 nmol) and HO-Gl Prepared according to general procedure F using u-vc-PAB-MMAE (10.0 mg, 8.08 μmol) to give a product concentration of 14.6 mg / 3.5 mL (240 μM).

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

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

[0346] 2.8 Azido-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 Azide-PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NH0-COPEG 1100 )]8(16.3mg,1. Prepared according to general procedure F using DGA-MMAF(OMe) (28 μmol) and DGA-MMAF(OMe) (10.6 mg, 12.3 μmol) to give a product concentration of 23.8 mg / 3.5 mL (365 μM).

[0347] 2.9 Azido-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-Pt(IV) acetate)(ε-NH-COPEG 1100 )]8,G3, compound 23 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)( ε-NH-COPEG 1100 To a stirred solution of )]8 (SPL19 and SPL32) (22.4 mg, 1.75 μmol) in DMF (0.5 mL) was added NMM (7.4 μL, 67.3 μmol). The resulting solution was then added to a stirred solution of diglycolic acid-1R,2R-cyclohexane-1,2-diamine-oxalatoplatinum(IV) acetate (10.4 mg, 17.7 μmol) and PyBOP (8.74 mg, 16.8 μmol) in DMF (0.5 mL). The resulting reaction mixture was then protected from light and stirred overnight at room temperature before being purified by SEC to give the product as an off-white solid (24 mg, 86%). 1H-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% of 9.0%, indicating that there are seven Pt-containing moieties in the macromolecule. 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 (100 mg, 0.00786 mmol, 1.0 eq) in DMF (300 μL) was prepared at RT. 24 COH (Click Chemistry Tools; 16 mg, 0.01 mmol, 1.3 equiv.), PyBOP (8 mg, 0.013 mmol, 1.6 equiv.), and DMF (200 μL) were added. The reaction mixture was stirred for 3 min, and then NMM (40 mg, 50 μL, 0.38 mmol) was added. l, 48 equiv.) was added. The contents were protected from light and stirred overnight at RT. The reaction mixture was diluted with MQ water and stored 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 fractions were confirmed by HPLC and collected in two separate fractions. Each fraction was concentrated under reduced pressure, and the resulting residue was taken up in MQ water, filtered (0.45 μm Acrodisc filter), and lyophilized to give the title product as a pink solid (69 mg, 66%). HPLC (C8 Xbridge, 3x100mm) gradient: 5% ACN / HO (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 24 A stirred solution of compound 31 (9.6 mg, 0.006 mmol, 1.3 equiv.) in DMF (200 μL) was prepared at RT. To this was added PyBOP (4 mg, 0.008 mmol, 1.6 equiv.) and NMM (23 mg, 25 μL, 0.226 mmol, 48 equiv.), and after 5 min, BHA[Lys][(α-NH2.TFA)(ε-NH-COPEG] 1000 )8] (60 mg, 0.006 mmol, 1.0 equiv.) was added, followed by DMF (200 μL). The contents were protected from light and stirred overnight at RT. The reaction mixture was diluted with ACN (10 mL) and then purified by SEC (400 drops / tube, ACN Sephadex LH20, 35 drops / min). Product-containing fractions were confirmed by HPLC, collected, filtered (0.45 μm Acrodisc filter), concentrated under reduced pressure, and lyophilized overnight to afford the title compound as a pale yellow solid (58 mg, 92% yield). HPLC (C8 Xbridge, 3x100mm) gradient: 5% ACN / HO (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-PA B-MMAE) 4-7 )(ε-NH-COPEG 1100 )8],G3, compound 36 A solution of Cy5-NHS (214 μL 4.2 mg / mL in DMF; 1.43 μmol, 1.0 equiv.) was dissolved in neat (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 )8] to give compound 8 (18 mg, 1.43 μmol). After complete dissolution, NMM (10 μL, 91.0 μmol) was added, and the resulting reaction mixture was stirred and protected from light. After 2 h, NMM (10 μL, 91.0 μmol) and PyBOP (7.3 mg, 14.0 μmol) were added to the dendrimer solution, followed by Glu-VC-PAB-MMAE / DMF solution (290 μL, 60 mg / mL solution, 14.0 μmol, 9.8 equiv.). The resulting reaction mixture was then stirred overnight at RT, protected from light.

[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 had entered the column bed, PBS (3.5 mL) was added to elute the product (appearing as a blue band). The final theoretical construct concentration was 8.7 mg / mL in PBS. The material was stored frozen at -80°C. HPLC (C8 Xbridge, 3x100 mm) gradient: 5% ACN / HO (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 / 83% peak associated with the MMAE-linker conjugate.

[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 To 53 (36.8 mg, 2.93 μmol) in NMM / DMF (7.8 μL / 0.5 mL) was added solid PyBOP (24.7 mg, 47.5 μmol). After complete dissolution, the dendrimer solution was added to HO-Glu-VC-PAB-MMAE (40 mg, 32.3 μmol) in NMM / DMF (7.8 μL / 0.5 mL). The resulting reaction mixture was then stirred overnight at RT, protected from light.

[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 per column). When all of 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 regenerated cellulose Amicon Ultra-0.5 mL centrifugation units (10K MWCO). The final theoretical concentration of the construct was 29–30 mg / mL in PBS. The material was stored frozen at -20°C. HPLC (C8 Xbridge, 3 x 100 mm) gradient. 5% ACN / HO (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 91.4% and MMAE / MMAE-linker-related peaks 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) in DMSO (100 μL) was prepared at room temperature. 24 CO[N(PN)2][Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100)8] Compound 53 (17.0 mg, 1.35 μmol) in DMF (200 μL) was added. The resulting reaction mixture was then stirred for 3 min, followed by the addition of NMM (10 μL, 91.0 μmol). The resulting solution was protected from light and stirred at RT for 4 h. PyBOP (7.0 mg, 13.5 μmol) was added, and 5 min later, 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 overnight. The reaction mixture was diluted with PBS buffer (4.5 mL) and divided into four Amicon Ultra centrifugal filters (10K MWCO). The filters were centrifuged (14K rcf, 15 min). The retentate was diafiltered against PBS (400 μL, 14K rcf, 15 min x 10). The retentates were combined to yield a pink solution. The concentration of compound 67 was approximately 16 mg in 2 mL. HPLC (C8XBridge, 3x100mm) gradient: 5% ACN / HO (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 stirred solution (100 μL) of p-SCN-deferoxamine (2.0 mg, 2.66 μmol) in DMSO was prepared at room temperature. 24 NH-COPEG4(PhMeTz))1(α-NH2)7)(ε-NHPEG 1100)8] Compound 34 (17.0 mg, 1.27 μmol) in DMF (200 μL) was added. The resulting reaction mixture was then stirred for 3 min, followed by the addition of NMM (10 μL, 91.0 μmol). The resulting solution was protected from light and stirred at RT for 4 h. PyBOP (7.0 mg, 13.5 μmol) was added, and after 5 min the reaction mixture was washed with neat HO-Gl u-Val-Cit-PAB-MMAE (9.17 mg, 7.41 μmol) was added to the mixture. The resulting reaction mixture was then left overnight. The reaction mixture was diluted with PBS buffer (4.5 mL) and divided into four Amicon Ultra centrifugal filters (10K MWCO). The filters were centrifuged (14K rcf, 15 min). The retentate was diafiltered against PBS (400 μL, 14K rcf, 15 min x 10). The retentates were combined to obtain a pink solution. The concentration of compound 68 was approximately 16 mg in 2 mL. HPLC (C8XBridge, 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) = 9.3-9.7 min (Blow 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] To a stirred solution of compound 53 (10 mg, 0.75 μmol) in DMF (2 mL) was added NMM (4.0 μL, 36.0 μmol) and sulfo-Cy5 NHS ester (0.58 mg, 0.75 μmol). The reaction mixture was stirred at room temperature for 2 h. 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 an additional 20 h. 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 give the title compound as a blue solid (6.0 mg). UPLC-TOF analysis of the reaction mixture confirmed the random attachment of sulfo-Cy5 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 NH-COPEG4(PhTzMe) (Click Chemistry Tools; 3.38 mg, 0.005 mmol) was prepared according to general procedure C (except that the reaction vessel was wrapped in foil to protect from light), and the residue was purified by SEC (Sephadex™ LH-20) using methanol as the eluent to give 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. 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) Graphite (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), 214nm, 0.4mL / min, Rt=8. 77 points.

[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) in DMF, NMM (8.0 μL, 72 μmol) was added, followed by Cy5-NHS ester (0.26 mg, 0.4 μmol), and the reaction mixture was stirred at room temperature overnight. Compound 63 (25.7 mg, 14.4 μmol) and PyBOP were added to the reaction mixture, which was stirred at room temperature for an additional 20 hours. The solvent was removed under reduced pressure, the crude residue was dissolved in acetonitrile (2 mL), and 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 give the title compound as a blue solid (27 mg; 75%). 1 HNMR (300 MHz, 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(0-1min),75-90%ACN(1-7min),90%ACN(7-12min),90-75%ACN(12-13min),75%ACN(13-15min),214min,0.4mL / min, Rt(min)=8.90-9.10.

[0359] Example 3 Synthesis of Targeted Dendrimer Conjugates 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 an 858 μM solution) was prepared according to General Procedure G (affibody-BCN:DGA-Dox dendrimer molar ratio; 1:3). SDS-PAGE analysis revealed a band corresponding to the affibody-dendrimer complex at approximately 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-COPEG1 100 )]4 (300 μL of an 858 μM solution) was prepared according to General Procedure G (affibody-BCN:DGA-Nemo dendrimer molar ratio; 1:3). SDS-PAGE analysis revealed a band corresponding to the affibody-dendrimer complex at approximately 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 )]8 (35 μL of a 580 μM solution) was prepared according to general procedure G (affibody-BCN:DGA-Dox dendrimer molar ratio; 1:1). Analysis showed a band corresponding to the affibody-dendrimer conjugate at around 27 kDa (700 nm). The conjugate yield was estimated to be 80% by adding Azide IR Dye 800CW and measuring the 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-COPEG1 100 )]8 (35 μL of a 580 μM solution) was prepared according to general procedure G (affibody-BCN:DGA-Nemo dendrimer molar ratio; 1:1). SDS-PAGE analysis showed a band corresponding to the affibody-dendrimer complex at approximately 27 kDa (700 nm). The conjugate yield was estimated to be 80% by adding Azide IR Dye 800CW and measuring the 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 Affibody 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)]8 (1.0 mL of a 240 μM solution) according to general procedure G. The purified material was lyophilized to yield a fluffy white powder (2.19 mg, 31%). SDS-PAGE analysis showed a band corresponding to the affibody-dendrimer complex at approximately 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-COPEG 2000 )8],G3, Compound 27 (Compound 78 / SPL40) Affibody BCN (1.0 mg, 143 nmol, 1.0 mg / mL in PBS) and azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-vc-PAB-MMAE)(ε-NH-COPEG 2000 )]8 (250 μL of a 233 μM solution) according to general procedure G. SDS-PAGE analysis showed a band corresponding to the affibody-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 )]8 (600 μL of a 903 μM solution) according to general procedure G. The purified material was lyophilized to yield a fluffy white powder (5.90 mg, 47%). SDS-PAGE analysis showed a band corresponding to the affibody-dendrimer complex at approximately 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 Azide-PEG 24 -CO[N(PN)2[Lys]4[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100 A solution of )]4 (10 μL of a 500 μM solution in PBS) was added to a solution of Fab-DBCO* (40 μL of a 13.4 μM solution in HEPES buffer). The resulting reaction mixture was then shaken (650 rpm) at room temperature overnight to separate the Fab-azide-PEG. 24 -CO[N(PN)2[Lys]4[(α-NH-DGA-3'NH-Dox)(ε-NH-COPEG 1100 )]4 was generated. SDS-PAGE analysis The SDS-PAGE was performed and scanned at 700 and 800 nm using an Odyssey scanner. The SDS-PAGE scanned at 700 nm showed a band for DBCO-Fab corresponding to 50 kDa and the expected band for Fab-[dendrimer-Dox / PEG] at approximately 65 kDa. 1100 A band for ]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 Azide-PEG 24 CO-[N(PN)2[Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 A solution of )]4 (10 μL of a 500 μM solution in PBS) was added to a solution of Fab-DBCO* (40 μL of a 13.4 μM solution in HEPES buffer). The resulting reaction mixture was then shaken (650 rpm) at room temperature overnight to obtain Fab-DBCO / N3-PEG. 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 and scanned at 700 nm and 800 nm using an Odyssey scanner. SDS-PAGE scanned at 700 nm showed a band for DBCO-Fab corresponding to 50 kDa and the expected Fab-[dendrimer-Nemo / PEG] at approximately 65 kDa. 1100 A band for ]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 Affibody 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 )]8 (600 μL of a 365 μM solution) according to general procedure G. The purified material was lyophilized to yield a fluffy white powder (2.06 mg, 33%). SDS-PAGE analysis showed a band corresponding to the affibody-dendrimer complex at approximately 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 according to Example 4 below using -TCO and nanobody 2D3 N-terminal tag, TEV, C-terminal azide.

[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 according to Example 4 below using -TCO and nanobody 2D3 N-terminal tag, TEV, C-terminal azide.

[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 Nanobody constructs were prepared according to Example 4 below using -TCO and nanobody 2D3 N-terminal tag, TEV, C-terminal azide

[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 Nanobody constructs were prepared according to Example 4 below using -TCO and nanobody 2D3 N-terminal tag, TEV, C-terminal azide

[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 12Nanobody constructs were prepared according to Example 4 below using -TCO and nanobody 2D3 N-terminal tag, TEV, C-terminal azide

[0374] 3.16 Nanobody-N3 / DBCO-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 74 DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazine 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) according to general procedure H. SDS-PAGE analysis showed a band corresponding to the nanobody-dendrimer complex of approximately 37 kDa (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), tetrazine dendrimer (MeTzPh)PEG4CO-NHPEG 24Prepared according to general procedure H using 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 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), tetrazine 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 ) 8], compound 67 (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 the nanobody-dendrimer complex of approximately 37 kDa (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 24CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazine dendrimer (MeTzPh) PEG4CO-NHPEG 24 Prepared according to general procedure H 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). SDS-PAGE analysis showed a band corresponding to the nanobody-dendrimer complex at approximately 37 kDa (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 ], compound 70 (1 equivalent), and nanobody-N3 ("nanobody-N3-C-terminal tag") (1 equivalent) using general procedure H. SDS-PAGE analysis showed a band corresponding to the nanobody-dendrimer complex at approximately 95 kDa (Figure 2).

[0379] 3.21 BHA[Lys]4[((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)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 )4], G2, was prepared according to general procedure H using compound 99 with the following exceptions.

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

[0381] The purification method was the same as that used for purifying compounds 74 and 76, yielding nanobody-dendrimer conjugate compound 86 (232 μg; 0.45 μg / μL / HEPES buffer, pH 8) and nanobody-dendrimer conjugate compound 87 (60 μg; 0.30 μg / μL / HEPES buffer, pH 8).

[0382] 3.22 BHA[Lys]8[((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)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 )8], G3, Compound 100, except for the following: , was prepared according to general procedure H:

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

[0384] The purification method was similar to that used for purifying compounds 74 and 76 to give the nanobody-dendrimer conjugate 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-COPEG 24 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, Compound 101, except for the following: , was prepared according to general procedure H:

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

[0387] The purification method was similar to that used for purifying compounds 74 and 76 to give the nanobody-dendrimer conjugate 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 ], G4, compound 102 according to general procedure H with the following exceptions:

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

[0390] The purification method was similar to that used for purifying compounds 74 and 76, yielding nanobody-dendrimer conjugates compound 90 (203 μg; 0.975 μg / μL and 0.556 μg / μL in HEPES buffer, pH 8) and compound 91 (101 μg; 0.34 μg / μL in 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-NH 2) 30 (ε-NH-COPEG 1000 ) 32 ], G5, compound 92 and BHA[L ys] 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-COPEG1000 ) 32 ]G5, Compound 103 were used to Prepared according to general procedure H with the following exceptions:

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

[0393] The purification method was similar to that used for purifying compounds 74 and 76, yielding nanobody-dendrimer conjugate 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-nanobody 2], G2, compound 94a, BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO ))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhMeT z))1(ε-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24NH-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 24 NH-COPEG(PhMeTz)), G2 dendrimer, compound 24, was prepared according to general procedure H with the following exceptions:

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

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

[0397] In addition, DBCO-Glu-NHPEG 24A solution of CO-NHPEG3-TCO compound 80 (10 mg / mL solution in 57 μL of MQ water, 0.322 μmol) was added to a solution of nanobody-N3 ("nanobody-N3-C-terminal tag") (2.5 mg, 0.161 μmol) in 900 μL of Tris buffer at pH 8. After allowing the reaction mixture to stand at RT for 2 days, the reaction mixture was purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO; 10 × 450 μL) using Tris buffer, pH 8. A solution of compound 105 (230.0 μg / 500 μL of MQ water; 0.026 μmol) was added to the solution, and the reaction mixture was allowed to stand at RT overnight. The purification method was similar to that used for purifying compounds 74 and 76, yielding nanobody-dendrimer compound 95, compound 94a, and compound 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 of compound 106 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 of Tris buffer, pH 8) was added a solution of TCEP (0.5 M / PBS, pH 7) (2.3 μL, 1.174 μmol). The reaction mixture was heated at 37 °C for 1 h, followed by the addition of DMSO (673 μL), followed by the addition of a solution of linker compound 57 (93.0 μg, 0.117 μmol) in DMSO (47.0 μL). After 1.5 h, the reaction mixture was cooled to RT and centrifuged. A solution of compound 99 (100 μg, 17 μL, 1.78 mg / 300 μL of MQ water) was added to the precipitate, and the solution was left at 4 °C overnight. Using a method similar to that used to purify compounds 74 and 76, compound 97 (29 μg) was obtained as a solution in Tris buffer, pH 8 (final concentration = 1.78 mg / mL).

[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 100 0)4], G2, compound 98 A solution of TCO-PEG3-aldehyde (Conjugate Probe) linker (0.7 mg, 1.46 μmol) in DMSO (50 μL) was diluted with a solution of nanobody ("Nanobody-N3-C-") (compound 79) (1.07 mg / mL stock solution in PBS buffer, pH 6 The mixture was added to a 500 ml solution (0.5 ml, 931 μL) followed by the addition of a solution of NaBHCN (0.09 mg, 1.5 μmol) in water (19 μL). The reaction mixture was cooled to 4 °C and monitored by UPLC analysis. After 16 h, the reaction mixture was diluted with PBS buffer (pH 6.5) to a total volume of 2.0 mL and purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO; 14 × 450 μL) using PBS buffer pH 6.5. UPLC: 5-20-30% ACN%, 15 min, 0.01% TFA buffer; nanobody (compound 79) Rt = 8.46 min, m / z 13802; product: 9.99 min, m / z 14263. To the nanobody-PEG3-TCO solution (500 μL PBS pH 6.5) was added a solution of G2 dendrimer compound 107 (179 μg, 0.020 μmol) in MQ water (15 μL). After standing overnight at 4 °C, the reaction mixture was purified using the same method used to purify compounds 74 and 764 to give compound 99 (12 μg; Figure 1i, lane 1) as a solution in 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 conjugates a) Nanobody sequence information Nanobody 2D3 array compound 79 EVQLESGSLVQPAGSLLSCAASGFTFDDYAMSWVLVPGKLEWVSSINWSGTHTDYADSVKGRFTISRNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSAGGTQTVSS Nanobody 2D3 N-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 #=unnatural amino acid.

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

[0403] To incorporate unnatural amino acids into recombinant proteins, an amber stop codon (TAG) was inserted in-frame at the end of the nanobody coding sequence, followed by either an ochre (TAA) or opal (TGA) stop codon to terminate translation. To incorporate a C-terminal his6 tag, an amber stop codon (TAG) was inserted in-frame at the end of the nanobody coding sequence, followed by a sequence encoding a TEV protease cleavage site and a his6 tag, followed by either an ochre (TAA) or opal (TGA) stop codon to terminate translation.

[0404] c) Expression and purification of nanobody-N3 Expression of the anti-HER2 nanobody 2D3 was performed in E. coli strain B95(DE3) (Mukai et al., Scientific Reports (2015), 5,9699) transformed with the nanobody expression plasmid and the orthogonal pair expression plasmid pEVOL-pAcFRS.2.t1 (Amiram et al., Nat. Biotechnol (2015), 33(12)). Cells were grown in Terrific broth (25 g / L tryptone, 30 g / L yeast, and 5 g / L glycerol, 0.017 M NaCl) in baffled shake flasks. 0.072M KH2PO4) at 37°C. 600The cells were grown to a cell density of 0.7–1.0. 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 allowed to proceed for 20 hours at 25°C. Cells were harvested by centrifugation, homogenized at high pressure, and then lysed with a protease inhibitor cocktail, lysozyme, and DNAse. The insoluble material was 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, the his6-tagged nanobody was purified by immobilized metal affinity chromatography (IMAC) using nickel-charged nitrilotriacetic acid-agarose. On-column refolding was achieved by first washing the bound protein with a column volume of 3 M guanidine HCl, 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8, followed by two column volumes of 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8. The bound protein was eluted with two column volumes of 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8. After IMAC, -nanobody-N3 was further purified by anion exchange chromatography using a HiTrap Q HP column (GE Healthcare, catalog 17115301). Binding and washing steps were performed with Tris buffer (20 mM, pH 8), and elution was performed with a 0% to 50% gradient of 1 M NaCl buffer (1 M NaCl, 20 mM Tris, pH 8). The resulting relevant nanobody fraction was buffer exchanged into 20 mM Tris, pH 8 using Amicon 10k MWCO filter units (Merck, catalogue UFC901008).

[0405] d) Conjugation of Nanobody-N3-C-Terminal Tag to Dendrimer Nanobody-N3-C-terminal tag was conjugated to dendrimer compounds 81–85 using bioorthogonal click chemistry following the procedure described for compounds 74 and 76, except that the linker DBCO-Glu-NHPEG24CO-NHPEG3-TCO (compound 51, Click Chemistry Tools) was used, generating compounds 86–95.

[0406] Gel electrophoresis of compounds 41–45 revealed bands at the appropriate molecular weight for the nanobody–dendrimer conjugates. The purity of each nanobody–dendrimer conjugate was confirmed using nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 3). After electrophoretic separation on a 4–15% polyacrylamide gel (Bio-Rad, catalog 4561086), the nanobody–dendrimer conjugates appeared as fluorescent bands roughly corresponding in size to the dendrimer plus the additional mass of the nanobody when imaged using a Typhoon Biomolecular Imager (GE Healthcare). Subsequent staining with Coomassie Brilliant Blue (CBB) confirmed the presence of the nanobody in the same location. No other bands were detected by CBB, indicating the absence of unreacted nanobody and the high purity of the preparation.

[0407] Example 5 SPR Binding Studies of Affibody-MMAE Dendrimers and Erb2 ECD Direct immobilization of ErbB2-ECD Using a ProteOn XPR36 instrument, ErbB2-ECD was immobilized on a GLC sensor chip surface (Bio-Rad) at 25 °C using HBS-P+ running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween 20). 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 in 10 mM sodium acetate, pH 5.0, and injected over the activated surface channel. Remaining activated sites were blocked with 1 M ethanolamine-HCl (pH 8.5). A mean response level of approximately 590 RU (1 RU = 1 pg protein / mm²) of protein binding was observed.

[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 device running buffer (RB). After the immobilization procedure, various concentrations of anti-ErbB2 affibody and its MMAE-dendrimer conjugate were injected over the immobilized ErbB2 protein at 60 μl / min, and the association was monitored for 90 s. Subsequently, running buffer was injected over the bound Ab-ErbB2 complex, and dissociation was observed for up to 60 min. Herceptin and affibody controls did not dissociate from the chip surface in the running buffer within a reasonable time frame (within 60 min). All binding measurements were performed in triplicate.

[0409] SPR data processing and analysis 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 binding model. Kinetic parameters (ka = association and kd = dissociation rate constants) and equilibrium dissociation constants (KD = kd / ka) were derived from the fits. All binding parameters are reported as mean values ​​+ / - standard deviation.

[0410] SPR results The affibody-MMAE-dendrimer conjugate specifically bound to ErbB2 protein in a process that yielded a classical kinetic binding profile. Kinetic binding analysis of the affibody-MMAE-dendrimer conjugate was then performed using a dose-response method. The kinetic and affinity parameters obtained from the SPR sensorgrams are summarized below. Note that because this affibody is a dimer, its binding constant is lower than that observed with the monomeric affibody used in the dendrimer conjugate. No binding was observed with compounds 20 and 21. This result clearly demonstrates nanomolar specific binding to the affibody-dendrimer target, despite the lower amounts compared to the native affibody. [Table 4]

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

[0412] All compounds were tested based on equivalent drug loading. The results show that PEG1100-targeted dendrimer conjugates are more effective at 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 Dendrimer-MMAE Dendrimer In Vivo Tolerance The dendrimer (0.1–0.3 ml of PBS solution) was intravenously injected into mice once a week for 3 weeks (days 1, 8, and 15). The mice were weighed daily and observed for signs of toxicity. Animals were monitored for up to 10 days after the final drug administration. Any mice that exceeded ethical endpoints (≥20% weight loss, poor general condition) were immediately sacrificed and observations recorded.

[0414] Compound 26 administered at 1 mg / kg was well tolerated in both nude SCID and balb / c mice, and no animals had to be sacrificed due to poor health.

[0415] Example 8: In vitro efficacy of the conjugate [Table 6]

[0416] GI 50 result The cytotoxicity of Herceptin®, Kadcyla®, lapatinib, compound 36 (target), and 2D3 nanobody-targeted dendrimer (compound 41) against MDA-MB-231, SKOV-3, SK-Br-3, NCI-N87, and OE-19 cells was evaluated using the MTT assay. Cells were seeded at a density of 5x103 in 96-well plates and incubated overnight. Cells were then treated with 1-log serial dilutions of the test composition for 72 hours (6 days for Herceptin). During the final 2 hours of incubation, 10% of the medium volume of MTT (thiazolyl blue tetrazolium bromide (Merck, Cat# M5655, 5 mg / ml sterile solution)) was added. Reduction of MTT in live cells produces an insoluble purple formazan metabolite. After 2 hours of incubation, all media was removed from the assay plates, 100 μl of DMSO was added, and the absorbance was immediately read at 570 nm. 50 is defined as the concentration that inhibits cell growth / proliferation by 50%. 50 Values ​​were determined from blank-corrected dose-response curves using a four-parameter nonlinear curve fit in GraphPad Prism 7.02. The results revealed that exemplary conjugates of the present disclosure have potent cytotoxic effects, especially 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 the HER2 WT plasmid (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 a MoFlo Astrios (Beckman Coulter). Clonal cell populations isolated during this process were further passaged in Geneticin and then subjected to a second round of cell sorting and transgene expression screening. Selected clones were then further expanded, and a stock of this cell line, designated MDA-MB-231 / HER2, was cryopreserved to create a master stock of stably transfected cells.

[0418] IC50 results in the lo / hi expressing 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. 5x10 cells were placed in a 96-well plate. 3 Cells were seeded at a density of 0.01 μg / ml and incubated overnight. Cells were then treated with the indicated concentrations of test compositions for 48 hours. AlamarBlue (Thermo Fisher, DAL1025) was added during the final 4 hours of incubation. Reduction of alamarBlue in live cells produces a red fluorescent metabolite that can be read on a plate reader (excitation 560 nm / emission 610 nm). Cell viability and subsequent IC 50 The IC values ​​were calculated from blank-corrected dose-response curves using a four-parameter nonlinear curve fit with variable slope (four parameters) in GraphPad Prism 7.02. The table below shows the IC values ​​of compound 36 (control) and compound 41 (target) with MDA-MB-231 cells and MDA-MB-231 / HER2 cells. 50The values ​​are shown. IC of 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) had an approximately 140-fold increased growth inhibitory effect on MDA-MB-231 / HER2 cells compared to compound 36 (control).

[0419] Dose-response curves and IC of Compound 36 (control) against MDA-MB-231 and MDA-MB-231 / HER2 at different MMAE concentrations 50 The values ​​are shown below, and are means ± standard deviations (sd; n = 4). [Table 8]

[0420] Dose-response curves and IC of compound 41 (target) against MDA-MB-231 and MDA-MB-231 / HER2 at different MMAE concentrations 50 Values ​​are means ± standard deviations (sd; n = 4). [Table 9]

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

[0422] For cell association measurements by fluorescence microscopy, 1 x 10 cells were placed in an 8-well chamber slide. 5 Cells were seeded at 1 / well and allowed to attach overnight. The following day, unconjugated dendrimer compounds 37, 38, and 39 (control) or nanobody dendrimer compounds 42, 43, and 44 were added to the medium to a final concentration of 0.5 μg / ml and incubated on ice for 1 hour. Cells were stained to visualize the cell membrane (wheat germ agglutinin-Alexa Fluor 488) and nuclei (Hoechst 33342). Cells were washed three times with 400 μl of cold Fluorobrite medium supplemented with 10% FBS. Cells were imaged using an Olympus IX83 microscope equipped with a 40x 0.9 NA air objective and a standard "Pinkel" DAPI / FITC / CY5 filter set.

[0423] For measurement of cell association by flow cytometry, cell suspensions were added to 96-well assay plates at 1 × 105 cells / well. After incubation with the nanobody dendrimers on ice for 1 hour, cells were washed three times with 200 μl of DMEM containing 10% FBS to remove unbound dendrimers. Cells were then resuspended in cold Fluorobrite medium supplemented with 10% FBS, and Cy5 fluorescence was measured using excitation at 642 nm and emission collected between 661 and 691 nm. To measure internalization rates, cells were incubated with the nanobody dendrimers for 24, 4, 1, 0.5, and 0.1 hours at 37°C. Unbound nanobody dendrimers were then removed by washing, and the cells were analyzed by flow cytometry.

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

[0425] Flow cytometry: These results demonstrate that nanobody-dendrimer compounds 42, 43, and 44 bind to HER2-positive MDA-MB-453 cells. Unconjugated dendrimer compounds 37, 38, and 39 did not bind to MDA-MB-453 cells. Data shown are the mean MFI of triplicate wells treated 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 a 24-hour period. Compound 41 (target) showed increased binding depending on the expression level of the HER2 receptor in the cell line. Flow cytometry revealed that by 24 hours, MDA-MB-231 / HER2 cells treated with compound 41 (146.1 ± 9.6) exhibited approximately 9-fold greater fluorescence compared to cells treated with compound 36 (16.05 ± 1.89). SKOV-3 cells treated with compound 41 (277.5 ± 5.9) also exhibited approximately 16-fold greater fluorescence compared to cells treated with compound 36 (16.4 ± 6.86). The fluorescence intensity was 2 times stronger than that of the control. The results are shown in Figure 4.

[0427] Dendrimer sizes G3~G5: Additional dendrimer generations were tested: compounds 37 and 42 (G3), compounds 38 and 43 (G4), and compounds 39 and 44 (G5), unconjugated and conjugated to 2D3, respectively. The unconjugated control dendrimers, compounds 37, 38, and 39, showed significantly less association with MDA-MB-231 (HER2-negative) and MDA-MB-231 / HER2 (HER2-positive). 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 compounds 42 (G3, 60.73 ± 0.21) and 43 (G4, 56.27 ± 1.02). Regardless of the dendrimer formation, dendrimer-conjugated 2D3 HER2-nanobodies exhibit significantly improved binding to cells overexpressing the HER2 receptor.

[0428] Percent cell association values ​​of compound 42 (G3), compound 43 (G4), and compound 44 (G5) with MDA-MB-231 and MDA-MB-231 / HER2 cells over 24 hours. Values ​​are means ± standard deviation (SD; n = 3). [Table 11] [Table 12] [Table 13] [Table 14]

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

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

[0431] The results of flow cytometry of these complexes are shown in FIG.

[0432] Example 10: Studies demonstrating internalization of the complex into HER2-overexpressing cells Confocal cellular uptake MDA-MB-231, MDA-MB-231 / HER2, and SKOV-3 cells were plated at 1.0 x 104 cells / well on a μ-slide 8-well chambered coverslip (ibidi) and allowed to attach overnight at 37°C and 5% CO2. Compounds 36 and 41 (3.33 nM) were then added and incubated for 1, 3, 6, and 24 hours. After washing, the cells were fixed with 1% paraformaldehyde for 20 minutes at room temperature. Cell membranes were stained with Alexa Fluor 488-wheat germ agglutinin (AF488-WGA, 5 μg mL). -1The cells were stained with 4',6-diamidino-2-phenylindole (DAPI, 2 μg mL ) / DPBS for 10 minutes at room temperature. -1 ) / DPBS for 10 minutes at room temperature. Fluorescent 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. The confocal images show that even after 24 hours of incubation, compound 36 (Figure 6a) and compound 41 (Figure 6b) barely associate with MDA-MB-231 cells. Meanwhile, target compound 41 was internalized by MDA-MB-231 / HER2 and SKOV-3 cells after 24 hours of incubation (Figures 7a and 8a), whereas compound 36 was not internalized (Figures 7b and 8b).

[0434] Example 11 Tumor Distribution of Tritium-Labeled MMAE-Conjugated Dendrimers (Compounds 40 and 45) MDA-MB-231 / HER2 cells (5 x 10 cells in 50 µL of PBS:Matrigel) were implanted subcutaneously into the fourth mammary fat pad of female NOD / SCID mice (5-7 weeks old). Solid tumors were grown to a volume of 100 mm. 3 The mice were allowed to grow until maturity (approximately 3-4 weeks). The mice were divided into two groups of six mice each, and Compound 40 (control group) or Compound 45 (targeting group) (0.5 μCi in 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 dislocation. Selected organs (tumor, liver, spleen, kidney, pancreas, lung, heart, and brain, etc.) were then removed, weighed, and analyzed for tritium ( 3 H) were processed for biodistribution.

[0435] The results are shown in Figure 9. Targeted dendrimer compound 45 accumulated in HER2-positive tumors (3.53% dose / g ± 0.43) to a greater extent than the unconjugated control compound 40 (1.88% dose / g ± 0.28), representing an approximately 80% increase in tumor uptake (p < 0.05). The difference in blood concentrations between the targeted and control dendrimers was not significant: 4.52% dose / g ± 0.24 and 3.69% dose / g ± 0.31, respectively. Because pharmacokinetic differences are unlikely to affect tumor retention, 2D3HER2-nanobody targeting improved dendrimer retention.

[0436] Example 12 Efficacy and confocal imaging of tumor uptake of Cy5-labeled MMAE-conjugated dendrimers (compounds 36 and 41) in a SKOV3 tumor model 3x10 6 SKOV3 cells / PBS:Matrigel (1:1) were inoculated subcutaneously. Solid tumors were 200 mm 3 The mice were allowed to grow until maturity (approximately 3 weeks). Mice were divided into five different groups and administered Compound 36 (control group) or Compound 41 (target group) (0.5 mg / kg MMAE in 250 μL of PBS, pH 7), Kadcyla® 40 mg / kg, and Herceptin® (40 mg / kg) via tail vein injection under isoflurane sedation.

[0437] (a) Confocal imaging Mice were anesthetized with isoflurane for 48 hours, and blood was collected by cardiac puncture immediately prior to cervical dislocation (n = 2 per group). Tumors were then excised and fixed overnight in 4% paraformaldehyde, then washed with PBS and embedded in agarose. Tumors were then sectioned to 100 μm using a vibratome, and 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. The targeted dendrimer (compound 41) showed uptake in the central and peripheral areas of the tumor, whereas compound 36 did not.

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

[0439] Example 13. Kinetics of 4th generation multi-nanobody dendrimer internalization in MDA-MB-231 / HER2+ cell line The fourth generation consists of conjugated anti-HER2 2D3 nanobodies, either one (single nanobody) or two to four (multiple nanobodies), compounds 90 and 91, isolated as described above. Cy5-labeled nanobody-dendrimer conjugates bearing the conjugated siderophore-derived chelator desferrioxamine (DFO) were used to compare binding to HER2+ human cell lines with different numbers of nanobodies attached per dendrimer.

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

[0441] For cell binding assays by flow cytometry, 20,000 cells / well were plated overnight in 96-well culture plates in DMEM medium supplemented with 10% FBS. Single or multiple nanobody dendrimers were added to the cells at 3 nM, 6 nM, 12 nM, and 30 nM and incubated at 37°C for 0.5, 1, 2, 4, and 6 hours. Control cells prepared in the same manner were pre-chilled and incubated with 30 nM single or 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 plates using TrypLE™ Express Enzyme (1X) with 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 Cy5 signal. To estimate the amount of dendrimer internalized at each concentration and time point, the Cy5 signal of cells incubated with dendrimers on ice was taken as the maximum surface-bound dendrimer, assuming no internalization. This signal was subtracted from the signal measured in cells incubated at 37°C (representing surface-bound and internalized dendrimers) to isolate only the signal from internalized dendrimers (see Figure 15).

[0443] The multi-conjugated (compound 91) anti-HER2 nanobody dendrimer is internalized more rapidly in the body at low concentrations than the single (compound 90) dendrimer. At high concentrations, compound 90 exhibits 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 cultured on µ-slide 8-well chamber coverslips. (ibidi) 1.0x10 4 Cells were plated at 1000 x g / well and allowed to attach overnight at 37°C and 5% CO2. Compound 91 (multiple 2D3-dendrimer conjugates) and compound 90 (single 2D3-dendrimer conjugates) (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. Cell membranes were stained with Alexa Fluor 488® conjugates of wheat germ agglutinin (AF488-WGA, 5 μg mL). -1 ) / DPBS at room temperature for 10 minutes. Cell nuclei were stained with Hoechst 33342 (2 μg mL -1 ) / PBS for 10 minutes at room temperature. Fluorescent 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 are internalized by SKOV-3 cells after 24 hours of incubation. Notably, compound 91 exhibits a greater degree of binding and internalization than compound 90 at 3 and 6 hours.

[0446] The multi-conjugated (compound 91) anti-HER2 nanobody G4 dendrimer is internalized more rapidly than the single nanobody G4 dendrimer (compound 90) at this concentration, with differences visible up to 6 hours, but no longer apparent at 24 hours.

[0447] Example 15 Targeted Zr Radionuclide-Containing Dendrimers - Imaging Studies with SKOV3 Breast Cancer Xenografts The accumulation of 89Zr-labeled HER2-targeted and non-targeted dendrimer constructs was investigated in the SKOV3 mouse xenograft model of ovarian cancer. Biodistribution was measured by PET-CT up to 9 days after injection and verified (when 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 µL of 50:50 Matrigel:PBS) were injected SC into the right flank of healthy female NOD-SCID (approximately 20 g) 8-week-old mice. Tumors were allowed to grow for 4 weeks before injection with imaging compounds. All tumors were palpable at the time of imaging and measured approximately 3–5 mm in size at the time of imaging experiments.

[0449] Test Compound The compounds in the table below were labeled as follows: Table Test Compounds [Table 21]

[0450] Radiolabeling of test compounds with Zr-89 and RadioTLC analysis All constructs (pretreated for iron removal as needed: http: / / jnm.snmjournals.org / content / 44 / 8 / 1271.long) were incubated in 0.1 M pH 7.4 HEPES buffer for 1 min at 25°C. Excess amount of drimer (see Table 1) 89The samples were incubated with Zr at 37°C for 45 minutes. A sample of each solution was taken and mixed 1:1 with 50 mM DTPA. Five microliters of each solution was spotted onto TLC paper (silica gel-impregnated Agilent iTLC-SG glass microfiber chromatography paper) and run in 50:50 HO:ethanol. The plates were then imaged on an Eckert & Ziegler Mini-Scan and Flow-Count iTLC Reader. Where necessary, unbound zirconium was removed by purification using 7K MWCO Zeba Spin Columns (Thermo Scientific) according to the manufacturer's protocol. All samples showed greater than 95% labeling. For quality control, free zirconium was removed. 89 Bound to Zr and DTPA 89 Control experiments were carried out to monitor the elution behavior of Zr, and 89 To check for any unbound Zr-labeled chelator, each sample was run with and without DTPA. The TLC images are shown in Figure 18, which show: 89 All Zr is bound to the dendrimer and is free 89 RadioTLC shows that Zr was not present: [Table 22]

[0451] Test injection details For in vivo imaging experiments, two mice were injected with 100 µL of test compound via the tail vein (29G needle, approximately 1.5–3.5 MBq), and tumor accumulation and biodistribution were observed at various time points. Gamma counting was used to determine organ distribution on day 9 after injection. In vivo quantification was performed. In biodistribution experiments, two mice were injected with the constructs and ex vivo tumor accumulation and biodistribution were monitored by gamma counting 48 hours post-injection.

[0452] result PET-CT imaging Thirty- to 90-minute still images were acquired at 4, 24, 48 hours, 5, 7, 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 fusion of CT and PET images and definition of regions of interest (ROIs). The CT and PET datasets for each animal were aligned using IRW software (Siemens) to ensure overlap of organs of interest. 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 cm of tissue. 3 The activity is expressed as a percentage of decay-corrected injected activity per gram and can be approximated as percent injected dose / g (ID / g%). Results are shown in Figure 19, which is a graph depicting the zirconium injected dose rate (percentage) per gram for compounds 89, 90, and 92 over a 9-day period in (a) kidney, (b) liver, and (c) tumor. Representative PET images are shown in Figure 20. Representative maximum intensity projections of radiolabeled conjugates. All PET data are expressed in becquerels / voxel (cm3) and thresholded to highlight tumor uptake.

[0453] Organs were removed on days 2 and 9 post-injection, and signal intensity was quantified and imaged by ex vivo gamma analysis. Additionally, n=2 per cohort was collected 48 hours later to assess biodistribution by gamma analysis. Ex vivo biodistribution results are shown in the table in Figure 21, which shows the tumor:organ ratios of ex vivo signal at days 2 and 9 for injected dose / g.

[0454] Results show a higher tumor-to-blood ratio of targets compared to non-targets, especially for compound 88 (small G3 dendrimer), at day 2. At day 9, compounds 90 and 92 (larger G4 and G5 dendrimers) show better tumor-to-blood ratios of targets compared to non-targets.

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

[0456] The results show higher signals in targeted tumors compared to non-targeted tumors, particularly at day 2 for compounds 90 and 92 (larger G4 and G5 dendrimers) and at day 9 for compound 89 (G3 dendrimer).

[0457] conclusion 1. The larger the dendrimer, the better the accumulation in tumors. 2D3 showed rapid clearance and low accumulation. 2. In targeted therapy, they accumulate in tumors more easily than non-targeted dendrimers. 3. Compound 88 (G3 1K-nanobody) shows significantly enhanced signal in tumors compared to other small molecule dendrimers. 4. Compounds 90 and 92 (G4 and G5 nanobodies) showed >12% ID / g in tumors at 48 hours and >4% and >8% ID / g at 9 days, respectively. 5. Small molecule nanobodies containing dendrimers and nanobodies alone exhibited high renal retention. No abnormal accumulation in clearance organs was observed, and liver and spleen signals were within the expected concentration range typically observed in similar systems.

[0458] Example 16: G5, PEG 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 in 24-well plates (1 x 10 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. The cells were incubated with compound 70 (G5, PEG1000, CTX) or 78 (2D3, G5, PEG1000, CTX) at 3.33 nM at 37 °C in a 5% CO2 humidified atmosphere for incubation times ranging from 1 to 24 hours. After incubation, unbound / unassociated particles were removed from the adherent cells by gently washing three times with DPBS (300 µL / well). Cells were removed from the plate by treatment with TrypLE™ Express Enzyme (1X), no phenol red (150 μL / well) at room temperature for 5-10 minutes. The plate was then placed on ice. Cell binding and association of the samples were then determined by acquiring Cy5 signal by flow cytometry.

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

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

[0461] It will be apparent to those skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore considered in all respects to be illustrative and not restrictive.

[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 Health, 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.

Claims

1. 1. A dendrimer-targeting agent conjugate comprising: a) i) a core unit (C), and ii) a dendrimer comprising building blocks (BU), each building block being a lysine residue or analog thereof; a dendrimer in which the core unit is covalently attached to at least two building blocks via amide linkages, each amide linkage being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the building block; b) a HER2 targeting agent, which is a peptide moiety having a molecular weight of up to about 80 kDa and comprising an antigen-binding site, said peptide moiety being covalently linked to said dendrimer by a spacer group; c) a therapeutic agent covalently linked to a surface building unit of the dendrimer; d) hydrophilic polymer groups covalently linked to the surface building units of the dendrimer; The complex comprising:

2. The conjugate of claim 1 , wherein the peptide moiety is selected from a heavy chain antibody, a Fab, an Fv, an scFv, or a single domain antibody.

3. The peptide portion is a heavy chain variable (V Ｈ ) domain or heavy chain variable (V Ｈ 3. The complex according to claim 1 or 2, comprising a .gamma.-.gamma.-.alpha ...

4. The peptide portion is a light chain variable (V Ｌ ) domain or a light chain variable (V Ｌ 4. The complex according to claim 1, comprising a .gamma.-domain.

5. The conjugate of any one of claims 1 to 4, wherein the targeting agent has a molecular weight of about 5 kDa to about 30 kDa.

6. 6. The conjugate of claim 5, wherein the targeting agent has a molecular weight of about 5 kDa to about 15 kDa.

7. 7. The conjugate of claim 6, wherein the targeting agent has a molecular weight of about 10 kDa to about 16 kDa.

8. The conjugate of any one of claims 1 to 7, wherein the targeting agent comprises fewer than 120 amino acid residues.

9. The conjugate of any one of claims 1 to 8, wherein the targeting agent comprises or consists of any of the amino acid sequences described herein.

10. The conjugate of any one of claims 1 to 9, wherein one targeting agent is covalently linked to the dendrimer.

11. 10. The conjugate of any one of claims 1 to 9, wherein two or more targeting agents are covalently linked to the dendrimer.

12. The covalent linkage between the targeting agent and the spacer group is formed by reaction between complementary reactive functional groups present on a targeting agent precursor and a spacer group precursor. The complex according to any one of claims 1 to 11.

13. 13. The conjugate of claim 12, wherein the targeting agent precursor comprises an unnatural amino acid residue, the unnatural amino acid residue having a side chain comprising a reactive functional group.

14. the unnatural amino acid residue is 【Chemistry 1】 14. The conjugate of claim 13, wherein the residue is:

15. 15. The conjugate of any one of claims 1 to 14, wherein the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting agent.

16. The conjugate of any one of claims 12 to 15, wherein the spacer group precursor comprises a reactive functional group that is an alkyne group.

17. 17. The conjugate of claim 16, wherein the alkyne group is a dibenzocyclooctyne group.

18. The conjugate of any one of claims 1 to 15, wherein the therapeutic agent is a chemotherapeutic agent.

19. The conjugate of any one of claims 1 to 18, wherein the therapeutic agent is a cytotoxic agent.

20. The conjugate of any one of claims 1 to 19, wherein the therapeutic agent is a super cytotoxic agent.

21. 21. The conjugate of claim 20, wherein the therapeutic agent is an auristatin or a maytansinoid.

22. 22. The conjugate of claim 21, wherein the therapeutic agent is monomethyl auristatin E.

23. 22. The conjugate of claim 21, wherein the therapeutic agent is monomethyl auristatin F.

24. The conjugate of any one of claims 1 to 23, wherein the therapeutic agent is covalently linked to a surface building unit of the dendrimer via a linker.

25. The conjugate of any one of claims 1 to 24, wherein the therapeutic agent is covalently linked to a surface building unit of the dendrimer via a cleavable linker.

26. 26. The conjugate of claim 25, wherein the cleavable linker comprises either a Val-Cit-PAB group or a Val-Arg-PAB-P-Trigger group.

27. 27. The method according to claim 1, wherein the hydrophilic polymer group is selected from the group consisting of polyethylene glycol (PEG), polyethyloxazoline (PEOX), and polysarcosine. The complex according to any one of claims 1 to 4.

28. 28. The conjugate of any one of claims 1 to 27, comprising PEG groups covalently linked to surface building units of the dendrimer.

29. 29. The conjugate of claim 28, wherein the PEG group has an average molecular weight in the range of about 500 to about 2500 g / mol.

30. 30. The conjugate of any one of claims 1 to 29, wherein the spacer group comprises a PEG group.

31. The core unit has the structure: 【Chemistry 2】 The complex of any one of claims 1 to 30, comprising:

32. 32. The conjugate of claim 1, wherein the dendrimer has constitutional units of generations 1 to 5.

33. 33. The conjugate of claim 32, wherein the dendrimer has three generations of building blocks.

34. The constituent units are each 【Transformation 3】 The complex according to any one of claims 1 to 33,

35. A conjugate according to any one of claims 1 to 34 for administration in combination with a further active agent.

36. The conjugate of any one of claims 1 to 35, wherein the conjugate is internalized in HER2-expressing cells.

37. 37. The conjugate of any one of claims 1 to 36, wherein administration of the conjugate results in reduced side effects compared to administration of an equivalent dose of the free therapeutic agent.

38. 38. The conjugate of any one of claims 1 to 37, wherein administration of the conjugate results in at least a 50% decrease in maximum plasma concentration of the released therapeutic agent compared to administration of an equivalent dose of free therapeutic agent.

39. A composition comprising a plurality of the conjugates of any one of claims 1 to 38.

40. i) a complex according to any one of claims 1 to 39; ii) a pharmaceutically acceptable excipient; and A pharmaceutical composition comprising:

41. 41. The composition of claim 39 or 40, wherein the composition is formulated for parenteral delivery.

42. A conjugate according to any one of claims 1 to 38 or a composition according to any one of claims 39 to 41 for use in the treatment of cancer.

43. 43. A method of treating cancer, comprising administering a therapeutically effective amount of the conjugate of any one of claims 1 to 38 or the composition of any one of claims 39 to 42 to a subject in need thereof.

44. Use of a conjugate according to any one of claims 1 to 38 or a composition according to any one of claims 39 to 43 in the manufacture of a medicament for the treatment of cancer.

45. 45. The method, use, or complex or composition for use according to any one of claims 42 to 44, wherein the cancer is ovarian cancer, breast cancer, gastric cancer, uterine cancer, or another cancer characterized by abnormal expression of the ERBB2 gene.

46. 1. A method of killing HER2-expressing cells, comprising: contacting the complex of any one of claims 1 to 38 with a HER2-expressing cell, thereby allowing the complex to be internalized into the cell, and thereby allowing the therapeutic agent to kill the HER2-expressing cell. The method.

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