Targeted dendrimer conjugates

Abstract

Provided herein are dendrimer-targeting agent conjugates comprising a dendrimer comprising a core unit and a lysine or lysine analog structural unit, a HER2 targeting agent and a therapeutic agent, wherein the HER2 targeting agent is a peptide portion having a molecular weight of up to about 80 kDa and comprising an antigen binding site, the peptide portion being covalently linked via a spacer group, and the therapeutic agent being covalently linked to the surface structural unit of the dendrimer. Also provided herein are compositions comprising the conjugates, and methods of treatment using the conjugates, particularly for treating cancer.

Description

Technical Field

[0001] The present disclosure generally relates to therapies that can be used to target cancers that overexpress HER2. More specifically, the present disclosure relates to targeted delivery of a therapeutic moiety via a dendrimer-targeting moiety conjugate comprising a HER2 antibody fragment or mimetic and a therapeutic moiety such as a residue of an ultracytotoxic agent. Background Art

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

[0003] There have been many efforts to develop effective tumor therapies. However, the challenge of finding new therapies with sufficient efficacy at the target, appropriate pharmacokinetic properties (so that it can be delivered to the site of action) and sufficient duration of action as well as a good safety profile is extremely difficult, time consuming and expensive.

[0004] Monoclonal antibody therapies such as trastuzumab and rituximab The development of constitutes an approach with remarkable success. For example, It was approved in 1998 and is used to treat HER2-overexpressing breast cancer and gastric cancer. The antibody works by binding to the HER2 / ERBB2 receptor, which is overexpressed in some cancer forms, and blocking the signals that stimulate cancer cell growth. However, There are drawbacks. For example, known side effects include myocardial damage and heart failure. Even in the subset of patients whose cancers are HER2 positive it is not completely effective and several mechanisms of resistance have been proposed (Pohlmann, Mayer and Mernaugh, 2009).

[0005] Another contrasting approach to treatment involves the use of cytotoxic agents that are active against rapidly dividing cells. A long-established example is cisplatin, which is used to treat a variety of cancers, including ovarian cancer, bladder cancer, testicular cancer, and squamous cell carcinoma. Another group of cytotoxic agents are super-cytotoxic agents, including auristatins such as monomethyl auristatin E (MMAE), which are extremely effective agents but may also cause severe toxic and damaging side effects in some patients. Therefore, such super-cytotoxic agents cannot be safely administered to patients as chemotherapeutic agents in their own right. Research has been conducted to identify means of utilizing such super-cytotoxic agents, leading to the development of antibody drug conjugates such as brentuximab vedotin. It contains MMAE conjugated to brentuximab (a CD30 antibody) via a linear linker and is approved for the treatment of Hodgkin lymphoma and anaplastic large cell lymphoma.

[0006] Many drug candidates fail in clinical trials due to poor therapeutic index (i.e., the ratio of therapeutic effect to toxicity). Some potential reasons for these failures include lack of efficacy, lack of specificity, poor drug absorption / bioavailability, inability to control biodistribution, rapid metabolism and clearance, and instability during storage. In practice, a balance is struck between achieving the necessary therapeutic cytotoxicity to kill cancer cells and mitigating deleterious toxicity and side effects to a level that can be tolerated by patients.

[0007] Various approaches have been explored in an attempt to improve the therapeutic effects of compounds with suboptimal properties. For example, for rapidly cleared compounds, sustained release formulations can be developed. One such example in the field of chemotherapeutic agents is the product (liposomal formulation of the active ingredient irinotecan), which has a greatly reduced rate of elimination from plasma compared to conventional formulations (Messerer et al., 2004). Other techniques include the use of depot injection formulations (e.g., Lupron Dendrimer technology has also been investigated in an attempt to improve the properties of pharmaceutically active agents and circumvent formulation difficulties, for example WO 2012 / 167309 describes the provision of drug-dendrimer conjugates containing, inter alia, the poorly soluble agent docetaxel.

[0008] Despite these and other advances in the art, there remains a need to develop additional therapies that are safe and effective in treating diseases such as cancer.

[0009] Any discussion of documents, acts, materials, devices, articles of manufacture or the like included in this specification should not be taken as an admission that any or all of these form part of the prior art base or are common general knowledge in the field relevant to the present disclosure. Summary of the invention

[0010] In a first aspect, there is provided a dendrimer-targeting agent conjugate comprising:

[0011] a) a dendrimer, said dendrimer comprising

[0012] i) Core Unit (C); and

[0013] ii) building blocks (BU), each building block being a lysine residue or an analog thereof;

[0014] wherein the core unit is covalently attached to at least two structural units via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a structural unit;

[0015] 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 via a spacer group;

[0016] c) a therapeutic agent covalently attached to a surface moiety of the dendrimer; and

[0017] d) a hydrophilic polymer group covalently attached to the surface structural unit of the dendrimer.

[0018] In some embodiments, the peptide portion is selected from: a heavy chain antibody, a Fab, a Fv, a scFv, or a single domain antibody. In some embodiments, the peptide portion comprises a heavy chain variable (V H ) domain. In some embodiments, the peptide portion comprises a light chain variable (V L ) domain or consist of it.

[0019] In some embodiments, the targeting agent has a molecular weight of about 5kDa to about 30kDa. In some embodiments, the targeting agent has a molecular weight of about 5kDa to about 18kDa. In some embodiments, the targeting agent has a molecular weight of about 5kDa to about 15kDa. In some embodiments, the targeting agent has a molecular weight of about 10kDa to about 16kDa. In some embodiments, the targeting agent has a molecular weight of about 14kDa to about 18kDa.

[0020] In some embodiments, the targeting agent comprises less than 120 amino acid residues.

[0021] In some embodiments, the targeting agent comprises or consists of any one of the amino acid sequences as defined herein.

[0022] In some embodiments, one targeting agent is covalently attached to the dendrimer. In some embodiments, more than one targeting agent is covalently attached to the dendrimer.

[0023] In some embodiments, the targeting agent precursor comprises a non-natural amino acid residue having a side chain comprising a reactive functional group. In some embodiments, the non-natural amino acid residue is a residue of:

[0024]

[0025] In some embodiments, the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting moiety.

[0026] In some embodiments, the spacer group precursor comprises a reactive functional group that is an alkyne group. In some embodiments, the alkyne group is a dibenzocyclooctyne group.

[0027] In some embodiments, the spacer group is covalently attached to the surface building block of the dendrimer. In some embodiments, the spacer group is covalently attached to the core unit.

[0028] In some embodiments, the covalent bond between the targeting agent and the spacer group has been formed by reaction between complementary reactive functional groups present on a targeting agent precursor and a spacer group precursor. In some embodiments, the spacer group comprises an olefin group.

[0029] 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 super cytotoxic agent. In some embodiments, the therapeutic agent is auristatin or 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.

[0030] In some embodiments, the therapeutic agent is covalently attached to the surface building block of the dendrimer via a linker. In some embodiments, the therapeutic agent is covalently attached to the surface building block of the dendrimer via a cleavable linker. In some embodiments, the cleavable linker comprises a Val-Cit-PAB group.

[0031] In some embodiments, the conjugate comprises a hydrophilic polymer group covalently attached to a surface structural unit of the dendron. In some embodiments, the hydrophilic polymer group is a PEG group covalently attached to a surface structural unit of the dendron. In some embodiments, the PEG group has an average molecular weight in the range of about 500 to about 2500 g / mol.

[0032] In some embodiments, the spacer group comprises a PEG group.

[0033] In some embodiments, the core unit comprises the following structure:

[0034]

[0035] In some embodiments, the core unit comprises the following structure:

[0036]

[0037] In some embodiments, the core unit is BHALys.

[0038] In some embodiments, the dendrimer has one to five generations of structural units. In some embodiments, the dendrimer has three to five generations of structural units. In some embodiments, the dendrimer has three generations of structural units. In some embodiments, the dendrimer has four generations of structural units. In some embodiments, the dendrimer has five generations of structural units.

[0039] In some embodiments, each of the structural units is:

[0040]

[0041] In some embodiments, the conjugate is for administration in combination with an additional active agent.

[0042] In some embodiments, the conjugate is internalized within a HER2-expressing cell.

[0043] In some embodiments, administration of the conjugate results in reduced side effects compared to administration of an equivalent dose of the free therapeutic agent.

[0044] In some embodiments, administration of the conjugate provides at least a 50% reduction in the maximum plasma concentration of the released therapeutic agent compared to administration of an equivalent dose of the free therapeutic agent.

[0045] In a second aspect, there is provided a composition comprising a plurality of conjugates as defined herein.

[0046] In a third aspect, a pharmaceutical composition is provided, comprising:

[0047] i) a conjugate as described herein; and

[0048] ii) a pharmaceutically acceptable excipient.

[0049] In some embodiments, the composition is formulated for parenteral delivery.

[0050] In some embodiments, the conjugate or composition is for use in treating cancer.

[0051] In a fourth aspect, provided is a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition as described herein.

[0052] In a fifth aspect, there is provided use of a conjugate or composition as described herein in the manufacture of a medicament for treating cancer.

[0053] 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.

[0054] In a sixth aspect, a method for killing HER2-expressing cells is provided, comprising:

[0055] Contacting a conjugate as defined herein with a HER2 expressing cell causes said conjugate to be internalized within said cell, whereby said therapeutic agent kills said HER2 expressing cell.

[0056] It should be understood that further aspects, embodiments, and examples are described herein that may include one or more embodiments or features as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 SDS Page analysis of compounds 74, 75 and 76 stained with Coomassie blue is shown.

[0058] Figure 2 SDS Page and fluorescence imaging analysis of compounds 77 and 78 are shown.

[0059] Figure 3 SDS Page and fluorescence imaging analysis of compounds 41 (lane A: crude mixture; lane B: purified conjugate), 42 (lane C: crude mixture; lane D: purified conjugate), 43 (lane E: crude mixture; lane F: purified conjugate), 44 (lane G: crude mixture; lane H: purified conjugate) and 45 (purified conjugate) are shown.

[0060] Figure 4 Mean fluorescence intensity values ​​for compound 36 (control) and compound 41 (targeted) with MDA-MB-231, MDA-MB-231 / HER2 and SKOV-3 cells over 24 h are shown. At least 10,000 cells were counted for each measurement. Values ​​are mean ± standard deviation (SD; n=3).

[0061] Figure 5 Shown is flow cytometry analysis of dendrimers incubated at 3.33 nM with HER2-positive cells (MDA-MB-231 / HER2) and HER2-negative cells (MDA-MB-231) at 37°C over a 24 h incubation. At least 10,000 cells were counted for each measurement. Values ​​are mean ± standard deviation (SD; n=3).

[0062] Figure 6 Confocal microscopy images of MDA-MB-231 cells treated with a) Compound 36 (control) or b) Compound 41 (targeted) at a concentration of 3.33 nM for 24 h are shown. Green fluorescence, blue fluorescence, and red fluorescence represent cell membranes stained with AF-488-WGA, nuclei stained with DAPI, and dendrites labeled with Cy5, respectively. Scale bar = 50 μm.

[0063] Figure 7 Confocal microscopy images of MDA-MB-231 / HER2 cells treated with a) Compound 36 (control) or b) Compound 41 (targeted) at a concentration of 3.33 nM for 24 h are shown. Green fluorescence, blue fluorescence, and red fluorescence represent cell membranes stained with AF-488-WGA, nuclei stained with DAPI, and dendrites labeled with Cy5, respectively. Scale bar = 50 μm.

[0064] Figure 8 Confocal microscopy images of SKOV-3 cells treated with a) compound 36 (control) or b) compound 41 (targeted) at a concentration of 3.33 nM for 24 h are shown. Green fluorescence, blue fluorescence, and red fluorescence represent cell membranes stained with AF-488-WGA, nuclei stained with DAPI, and dendrites labeled with Cy5, respectively. Scale bar = 50 μm.

[0065] Fig. 9 Tumor and blood distribution data of 3H-labeled compound 40 and compound 45 dendrimers after sacrifice at 48 h are shown. All data are normalized by tissue mass. All data represent mean ± SEM (n = 5); (NS = not significant; * = p value < 0.05).

[0066] Fig.10 Representative ex vivo tumor distributions of Compounds 36 and 41 after sacrifice at 48 h are shown. Data represent typical fields of view of (left) non-targeted dendrimers (Compound 36) and (right) targeted dendrimers (Compound 41).

[0067] Fig.11 Images showing that the targeted dendrimer (Compound 41) was taken up into the core and peripheral regions of the tumor, and that the control Compound 36 was not, are shown.

[0068] Fig.12 Shown is a graph of mean tumor volume over time for mice vaccinated with SKOV3 cells treated with: vehicle, control compound 36, targeted conjugate 41, or

[0069] Fig.13 Shown is the percentage survival over time for mice vaccinated with SKOV3 cells treated with: vehicle, control compound 36, targeted conjugate 41, or

[0070] Fig.14 Shown are the mean % weight change over time for mice vaccinated with SKOV3 cells treated with: vehicle, control compound 36, targeted conjugate 41, or

[0071] Fig.15 The internalization kinetics of 4th generation dendrimers with single (Compound 90; MFI single) or multiple (Compound 91; MFI multiple) conjugated anti-HER2 Nanobodies are shown.

[0072] Fig.16 Confocal microscopy images of SKOV-3 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 are shown. Compound 91 was labeled with Cy5 (magenta), cell membranes were stained with AF488-WGA (green), and nuclei were labeled with Hoechst 33342 (blue). Scale bar = 30 μM.

[0073] Fig.17 Confocal microscopy images of SKOV-3 after incubation with compound 90 (single 2D3-dendrimer conjugate) at 37°C for a) 1 h, b) 3 h, c) 6 h, or d) 24 h are shown. Compound 91 was labeled with Cy5 (magenta), cell membranes were stained with AF488-WGA (green), and nuclei were labeled with Hoechst 33342 (blue). Scale bar = 30 μM.

[0074] Fig.18 is a radioactive TLC image of the compound of the present disclosure, which shows 89 Zr is bound to the dendrons.

[0075] Fig.19 Graphs showing the percentage of zirconium dose / gram injected in (a) kidney, (b) liver, and (c) tumor over 9 days for compounds 89, 90, and 92.

[0076] Fig. 20 Representative maximum intensity projections of radiolabeled conjugate PET images of animals at 4 hours to 9 days are shown. Data are expressed as becquerel / voxel (cm3) and have been thresholded to highlight tumor uptake.

[0077] Fig.21 A table providing tumor:organ ratios for ex vivo signals of compounds of the present disclosure at day 2 and day 9 injected zirconium dose / gram is shown.

[0078] Fig. 22 A table providing the percentage of injected zirconium dose per gram in ex vivo tumors and organs at day 2 and day 9 is shown.

[0079] Fig.23 Shown are flow cytometry analysis and mean fluorescence intensity values ​​of compound 70 (control) and compound 78 (targeted) with MDA-MB-231 / HER2 cells over 24 h.

[0080] Fig.24 Shown are flow cytometry analysis and mean fluorescence intensity values ​​of compound 70 (control) and compound 78 (targeted) with MDA-MB-231 cells over 24 h.

[0081] Fig.25 Shown are flow cytometry analysis and mean fluorescence intensity values ​​for compound 70 (control) and compound 78 (targeted) over 24 h on SKOV-3 cells.

[0082] Description of the sequence listing

[0083] SEQ ID NO.1: 2D3 nanobody.

[0084] SEQ ID NO.2: 2D3 nanobody with N-terminal tag, TEV, and C-terminal azide.

[0085] SEQ ID NO.3: 2D3 nanobody with C-terminal tag, TEV, azide. DETAILED DESCRIPTION

[0086] General Definition

[0087] Unless specifically defined otherwise, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art (eg, chemistry, biochemistry, medicinal chemistry, polymer chemistry, etc.).

[0088] Throughout this specification, the word "comprise" or variations such as "comprise / comprising" should be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but 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") should be understood to mean "X and Y" or "X or Y", and should be considered to provide explicit support for both meanings or for either meaning.

[0089] As used herein, unless stated to the contrary, the term about means + / - 10%, more preferably + / - 5% of a specified value.

[0090] As used herein, the terms "a," "an," and "the" include both singular and plural aspects unless the context clearly dictates otherwise.

[0091] As used herein, the term "subject" refers to any organism susceptible to HER2 (human epidermal growth factor receptor 2) related diseases or conditions. In some embodiments, 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 suffers from cancer. In one embodiment, the subject suffers from HER2 positive cancer.

[0092] As used herein, the term "treating" includes relieving symptoms associated with a particular disorder or condition. For example, as used herein, the term "treating cancer" includes relieving symptoms associated with cancer. In one embodiment, the term "treating cancer" refers to a reduction in the size of a cancerous tumor. In one embodiment, the term "treating cancer" refers to a reduction in the aggressiveness of a cancer. In one embodiment, the term "treating cancer" refers to an increase in progression-free survival. As used herein, the term "progression-free survival" refers to the length of time during and after treatment of cancer that a patient is alive with the disease, i.e., cancer, but does not have a recurrence of the disease or an increase in symptoms.

[0093] As used herein, the term "prevent" includes preventing a particular disorder or condition. For example, as used herein, the term "preventing cancer" refers to preventing the onset or continuation 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.

[0094] As used herein, the term "therapeutically effective amount" refers to an amount of a conjugate containing a therapeutic agent that is sufficient to alleviate or prevent one or more symptoms of the disorder or condition being treated to a certain extent. The result can be a reduction and / or alleviation of the signs, symptoms or causes of a disease or condition or any other desired change in a biological system. In one embodiment, the term "therapeutically effective amount" refers to an amount of a conjugate that is sufficient to cause a reduction in the size of a cancerous tumor. In one embodiment, the term "therapeutically effective amount" refers to an amount of a conjugate that is sufficient to cause an increase in progression-free survival. As used herein, the term "effective amount" refers to an amount in which a conjugate effectively achieves a desired pharmacological effect or therapeutic improvement without excessive adverse side effects or achieves a desired pharmacological effect or therapeutic improvement and has a reduced side effect profile. The therapeutically effective amount can be determined, for example, by routine experiments including, but not limited to, dose escalation clinical trials. The term "therapeutically effective amount" includes, for example, a preventive effective amount. In one embodiment, a preventive 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 due to variations in the metabolism of the compound and any of the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. One of ordinary skill in the art can determine an appropriate "effective amount" in any individual case using routine experimentation.

[0095] As used herein, the term "attached" refers to the connection between chemical components through covalent bonding. The term "covalent bonding" is used interchangeably with the term "covalently attached".

[0096] Dendrimer

[0097] In a first aspect, there is provided a dendrimer-targeting agent conjugate comprising:

[0098] a) a dendrimer, said dendrimer comprising

[0099] i) Core Unit (C); and

[0100] ii) building blocks (BU), each building block being a lysine residue or an analog thereof;

[0101] wherein the core unit is covalently attached to at least two structural units via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a structural unit;

[0102] 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 via a spacer group;

[0103] c) a therapeutic agent covalently attached to a surface moiety of the dendrimer; and

[0104] d) a hydrophilic polymer group covalently attached to the surface structural unit of the dendrimer.

[0105] Dendrimer conjugates have been shown to be highly effective anticancer agents in in vitro and in vivo tests and have shown promising results relative to and (HER2-emtansine antibody-drug conjugate) showed improved effects. Compared with itself, the conjugate is rapidly internalized into HER2 overexpressing cancer cells. It is expected that rapid internalization characteristics contribute to the delivery of a high proportion of therapeutic agents administered via the conjugate to the desired site of action. The ability of the therapeutic moiety to be specifically delivered to the target cell (wherein the therapeutic moiety is rapidly internalized) contributes to reducing the undesirable side effects (i.e., toxicity) associated with other non-cancerous cells being exposed to cytotoxic agents. This method is particularly advantageous in the case of delivering cytotoxic therapeutic moieties (e.g., super cytotoxic therapeutic moieties) (in which the free circulation in the plasma may have toxic effects in the case).

[0106] As used herein, the term "dendron" refers to a molecule containing a core and a dendron attached to the core. Each dendron consists of several generations of branching structural units, resulting in a branched structure with an increasing number of branches with each generation of structural units. A "conjugate" may include a pharmaceutically acceptable salt or solvate as defined herein.

[0107] As used herein, the term "building block" refers to a branched molecule, which is a lysine residue or an analog thereof having three functional groups, one functional group (e.g., derived from a carboxylic acid group) for attachment to the core or the previous generation building block, and at least two functional groups (e.g., derived from amines) for attachment to the next generation building block or the surface of the dendrimer-forming molecule.

[0108] It will be appreciated by those skilled in the art that the conjugates may be produced in a variety of forms, including, for example, salt forms (e.g., in the case of the presence of ionizable groups in the conjugate), as well as different solvates. It will be appreciated that the present disclosure relates to all such forms of dendrimer-targeting moiety conjugates.

[0109] Suitable salts of the conjugate include salts formed with organic or inorganic acids or bases. As used herein, the phrase "pharmaceutically acceptable salts" refers to pharmaceutically acceptable organic or inorganic salts. Exemplary acid addition salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugarates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoic acid (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoic acid)) salts. Exemplary base addition salts include, but are not limited to, ammonium salts; alkali metal salts, such as those of potassium and sodium; alkaline earth metal salts, such as those of calcium and magnesium; and salts with organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di- or tri-lower alkylamines (e.g., ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl-propylamine), or mono-, di- or tri-hydroxy lower alkylamines (e.g., mono-, di- or triethanolamine). Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate ions, succinate ions or other counterions. Counterions may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, pharmaceutically acceptable salts may have more than one charged atom in their structure. Cases where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions. It is also understood that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure, as these may be used as intermediates in the preparation of pharmaceutically acceptable salts, or may be useful during storage or transportation.

[0110] Those skilled in the art of organic and / or medicinal chemistry will appreciate that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are referred to as "solvates". For example, a complex with water is referred to as a "hydrate". As used herein, the phrase "pharmaceutically acceptable solvate" or "solvate" refers to the association of one or more solvent molecules and 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.

[0111] Core Unit

[0112] The core unit (C) of the dendrimer is covalently attached to the structural unit via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the structural unit. Thus, the core unit can be formed, for example, from a core unit precursor comprising an amino group. Any suitable amino-containing molecule can be used as a core unit precursor.

[0113] Typically, the core unit provides additional functionality for the nitrogen atom for covalent attachment of the building block, for example, it may include a functional group for attaching a targeting moiety via a spacer group. In other words, the HER2 targeting agent is typically covalently linked to the core unit of the dendrimer via a spacer group. In some embodiments, the core unit may be derived from a precursor having three reactive nitrogen atoms, two of which may be used to attach a building block, and one of which may be used to attach a spacer group.

[0114] In some embodiments, the core unit has the following structure:

[0115]

[0116] In some embodiments, the core unit is a lysine molecule with three attachment points, having the following structure:

[0117]

[0118] In some embodiments, the core unit can be derived from a precursor having two reactive nitrogen atoms that can be used to attach structural units. 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:

[0119] That is, the core unit thus comprises a lysine residue, wherein the acid moiety has been terminated with benzhydrylamine (BHA-Lys) to form the corresponding amide, and can be formed, for example, from the following core unit precursor having two reactive (amino) nitrogens:

[0120]

[0121] In the case of using a core unit precursor having only two reactive nitrogen atoms (such as BHA-Lys), the two amino groups are typically functionalized with the building block, and the spacer group (and therefore the targeting agent) is typically attached via the surface building block. The dendrimer-targeting agent conjugate of the present invention allows multiple terminal groups to be presented on the surface of the dendrimer-targeting agent conjugate in a controlled manner. In particular, in the case of using a lysine building block, the placement of the targeting agent and / or hydrophilic polymer group and / or therapeutic agent on the α or ε 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 through the building block. In other words, in those embodiments, the core unit does not provide an attachment point for the targeting agent other than via the building block. It should be understood that in such embodiments, any functional group present in the core unit that is not used for covalent attachment to the building block will be unreactive (i.e., unreactive under the conditions to which the conjugate has been exposed), or will have been capped with a suitable capping group to prevent further reaction. An example of such a core unit is the BHA-Lys group discussed above.

[0122] Structural unit

[0123] The building block (BU) is a lysine residue or an analog thereof and can be formed from a suitable building block precursor (e.g., lysine or a lysine analog containing an appropriate protecting group). Lysine analogs have two amino nitrogen atoms for bonding to subsequent building blocks and an acyl group for bonding to the previous building block or core unit. Examples of suitable building blocks include

[0124] wherein the acyl group of each building block provides a covalent attachment point for attachment to the core or previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for attachment to a subsequent generation building block or a terminal group (such as a linked therapeutic agent).

[0125] In some preferred embodiments, the structural units are each:

[0126]

[0127] wherein 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 wherein each nitrogen atom provides a covalent attachment point for attachment to a subsequent generation building block or a terminal group (such as a linked therapeutic agent).

[0128] In some preferred embodiments, the structural units are each:

[0129]

[0130] wherein the acyl group of each building block provides a covalent attachment point for attachment to the core or previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for attachment to a subsequent generation building block or a terminal group (such as a linked therapeutic agent).

[0131] The outermost structural unit (BU 外 ) can be formed from lysine or lysine analog building blocks as used in the other generation building blocks (BU) as described above. The outermost generation building block (BU 外 ) is the outermost generation structural unit of the core unit of the dendron, i.e., no further generation structural units are attached to the outermost generation structural unit (BU 外 ).

[0132] It will be appreciated that the dendrons of the dendron can be synthesized to the desired number of generations, for example by attaching the building blocks (BU) accordingly. In some embodiments, each generation of building blocks (BU) can be formed from the same building blocks, for example, the building blocks of all generations can be lysine building blocks. In some other embodiments, one or more generations of building blocks can be formed from building blocks that are different from the building blocks of other generations.

[0133] Dendrimer moieties typically have one to five generations of structural units. In some embodiments, the dendrimer is a one generation structural unit dendrimer. In some embodiments, the dendrimer is a two generation structural unit dendrimer. In some embodiments, the dendrimer is a three generation structural unit dendrimer. In some embodiments, the dendrimer is a four generation structural unit dendrimer. In some embodiments, the dendrimer is a five generation structural unit dendrimer. For example, a three generation structural unit dendrimer is a dendrimer having a structure that includes three structural units covalently linked to each other, such as in the case where the structural unit is lysine, and it may include the following substructures:

[0134]

[0135] In some embodiments, the dendron has a complete generation of structural units. For example, a three-generation dendron has three complete generations of structural units. In the case where the core has two reactive amine groups, such a three-generation dendron will contain 14 structural units (i.e., core unit + 2BU + 4BU + 8BU). However, it should be understood that due to the nature of the synthetic method used to produce the dendron, one or more reactions performed to produce the dendron may not be fully carried out to completion. Therefore, in some embodiments, the dendron may contain incomplete generations of structural units. For example, a population of dendrons can be obtained, wherein the dendrons have a distribution of the number of structural units / dendron. In some embodiments, a population of dendrons is obtained, which has an average number of structural units / dendron 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 dendrons is obtained, wherein at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrons have 10 or more structural units. In some embodiments, a population of dendrimers is obtained wherein at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 12 or more building blocks.

[0136] In some embodiments, each generation of structural units in each dendron (X) can be represented by the formula [BU]2 (b-1) , where b is the number of generations. A dendron (X) having three complete generations of building blocks is represented by [BU]1-[BU]2-[BU]4.

[0137] Targeted Agents

[0138] The dendrimer-targeting agent conjugates as described herein comprise a HER2 targeting agent, i.e., a targeting agent capable of binding to human epidermal growth factor receptor 2 (HER2, also known as ERBB2; Gene ID No. 2064, NCBI). The HER2 targeting agents as described herein can be used to target the disclosed dendrimer conjugates to tumors and cancer cells.

[0139] The targeting agent comprises a peptide portion with a molecular weight of up to about 80 kDa and comprises an antigen binding site that specifically binds to a molecule (i.e., HER2) and / or has affinity for the molecule. The interaction may occur by any type of bonding or association (including, for example, covalent, ionic and hydrogen bonding, van der Waals forces).

[0140] In one embodiment, a HER2 targeting agent as described herein has a molecular weight of about 3 kDa to about 80 kDa, or about 3 kDa to about 60 kDa, or about 3 kDa to about 50 kDa, or about 3 kDa to about 40 kDa, or about 3 kDa to about 30 kDa, or about 3 kDa to about 20 kDa, or about 3 kDa to about 15 kDa, or about 3 kDa to about 13 kDa, or about 5 kDa to about 15 kDa, or about 5 kDa to about 12 kDa, or about 5 kDa to about 10 kDA.

[0141] In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 80kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 60kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 50kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 40kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 30kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 20kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 15kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 3kDa to about 13kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 5kDa to about 15kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 5kDa to about 12kDa. In some embodiments, the HER2 targeting agent has a molecular weight of about 5kDa to about 10kDa.

[0142] As used herein, "kDA" or "kilodalton" refers to the unit of molecular mass consisting of 1000 Daltons.

[0143] In some embodiments, the targeting agent is an antibody fragment. As used herein, the term "antibody fragment" should be taken to mean a portion or fragment of an antibody that is capable of specifically binding to an antigen, including, for example, an F-terminally defined herein. V 、V H 、V L Or variable region. This term should be understood to cover fragments directly derived from antibodies as well as proteins produced using recombinant means. In one embodiment, the antibody fragment is selected from Fab, Fv, scFv, heavy chain antibody, domain antibody, heavy chain antibody, diabody or triabody.

[0144] As used herein, the term "Fv" should be taken to mean any protein, whether it consists of multiple polypeptides or a single polypeptide (scFv), in which V L and V HThe V that forms the antigen binding domain H and V L The Fv of the present disclosure (and any protein of the present disclosure) may have multiple antigen binding sites, which may or may not bind to the same antigen. This term should be understood to cover fragments directly derived from antibodies as well as proteins produced using recombinant means. In some examples, V H Not linked to the heavy chain constant domain CH1, and / or V L Not linked to a light chain constant domain (CL), such as a domain antibody. Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, "Fab fragments" consist of monovalent antigen-binding fragments of immunoglobulins and can be produced by digesting whole antibodies with papain to produce fragments consisting of a complete light chain and a portion of a heavy chain, or can be produced using recombinant means. Fab fragments generally contain V H and C H 1 and V L and C L The "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin followed by reduction to produce a fragment consisting of a complete light chain and a portion of a heavy chain (including V H and a single constant domain). Two Fab' fragments are obtained for each antibody treated in this way. Fab' fragments can also be produced by recombinant means. "Single-chain Fv" or "scFv" is a recombinant molecule containing the variable region fragment (Fv) of an antibody, in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0145] In some embodiments, the antibody fragment is selected from: a heavy chain antibody, a Fab, a Fv, a scFv, or a single domain antibody.

[0146] As used herein, a "single domain antibody (sdAb)" (also referred to as a "domain antibody (dAb)" or "nanobody") comprises a heavy chain V H or light chain V L In one embodiment, the variable region is derived from a camelid. In one embodiment, the variable region is derived from a shark. In one embodiment, V H It is a camelid-derived V H .

[0147] 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.

[0148] In one embodiment, the single domain antibody comprises a single domain amino acid sequence as described in, for example, EP2215125A1, US20110028695, Hussack et al. (2018), or Arezumand et al. (2017). In one embodiment, the single domain antibody comprises a single domain amino acid sequence as described in US20110028695. 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, which comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% homology to the amino acid sequence shown in US20110028695.

[0149] In one embodiment, the single domain antibody comprises a single domain amino acid sequence as disclosed in Example 7. In one embodiment, the single domain antibody is 2D3, comprising the amino acid sequence as shown in SEQ ID NO: 1986 of US20110028695.

[0150] In one embodiment, a single domain antibody comprises the amino acid sequence

[0151] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNANNNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS.

[0152] 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 HER2 extracellular domain I, II, III and / or IV. In one embodiment, the targeting agent binds to HER2 extracellular domain III. In some embodiments, the targeting agent binds to the dimerization arm of ErbB2. In the case where the targeting agent binds to the dimerization arm of ErbB2, the targeting agent can compete with pertuzumab for HER2 binding. In some embodiments, the targeting agent binds to HER2 with the following binding affinity: at least 1nM, at least 2nM, at least 5nM, at least 10nM, as determined by SPR studies.

[0153] 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.

[0154] In some embodiments, the HER2 targeting agent comprises less than about 500, less than about 400, less than about 300, less than about 200, less than about 150, less than about 140, less than about 130, less than about 120, less than about 110, less than about 100 amino acid residues. In some embodiments, the HER2 targeting agent comprises more than about 50, more than about 75, more than about 100 or more than about 120 amino acid residues. In some embodiments, the HER2 targeting agent comprises less than 120 amino acid residues. In some embodiments, the HER2 targeting agent comprises about 100 to about 120 amino acid residues.

[0155] In some embodiments, the HER2 targeting agent is a mimetic of an antibody or antibody fragment. As used herein, the term "mimetic / mimetics" refers to a compound that can bind to an antigen like an antibody or antibody fragment, but is not structurally related to an antibody. The term should be understood to not cover antibodies or antibody fragments as described herein. The term should be understood to cover synthetic mimics (generated in vitro) and mimics produced using recombinant means. The term should be understood to cover protein mimics.

[0156] In one embodiment, the mimetic is selected from the group consisting of: affibody, aptamer, affilin, affimer, affitin, anticalin, avimer, alpha body, monomer, DARPin, Fyomer, type III fibronectin derived protein scaffold, plant cystatin derived protein scaffold and paratope mimetic peptide. Like antibodies, mimetic can be used as targeting moiety.

[0157] In one embodiment, the mimetic is derived from one of the following protein scaffolds: the z domain of protein A, gamma-B crystallin, ubiquitin, cystatin, sac7d, a triple helix, a coiled coil, a lipocalin, a cyclic peptide, the A domain of a membrane receptor, an ankyrin repeat motif, the sh3 domain of Fym, the Kunits domain of a protease inhibitor, the type III domain of fibronectin, and the IgG-like thermostable carbohydrate binding module family 32 (CBM32) from Clostridium perfringens.

[0158] In one embodiment, the analog is about 3kDa to about 20kDa, or about 4kDa to about 18kDa, or about 6kDa to about 16kDa, or about 6kDa to about 14kDa, or about 6kDa to about 12kDa, or about 6kDa to about 10kDa, or about 6kDa to about 8kDa. In one embodiment, the analog is about 3kDa to about 20kDa, and in one embodiment, the analog is about 3kDa to about 20kDa. In one embodiment, the analog is about 4kDa to about 18kDa. In one embodiment, the analog is about 6kDa to about 16kDa. In one embodiment, the analog is about 6kDa to about 14kDa. In one embodiment, the analog is about 6kDa to about 12kDa. In one embodiment, the analog is about 6kDa to about 10kDa. In one embodiment, the analog is about 6kDa to about 8kDa.

[0159] In some embodiments, the targeting agent is an affibody. As used herein, the term "affimer" refers to any class of very small (about 6 kDa) polypeptide antibody mimics based on a three alpha helical bundle domain of about 58 amino acids in length called a "Z domain". Typically, the scaffold for an affibody is based on a modified form of the B-domain of protein A. Affibodies are characterized by very high stability (tolerance to temperatures up to 90 ° C and target affinity in the range of nanomolar to picomolar). See, for example, Nord et al. (1995), Protein Eng., 8: 601-608. Examples of known affibodies include, for example, affibodies for HER2 (e.g., anti-HER2 AFFIBODY AB, Bromma, Sweden; U.S. Patent No. 7,993,650).

[0160] In some embodiments, the targeting agent is an affibody and has a molecular weight ranging from about 3 kDa to about 10 kDa, or about 3 kDa to about 8 kDa, or about 4 kDa to about 8 kDa, or about 4 kDa to about 7 kDa, or about 5 kDa to about 7 kDa, or about 6 kDa.

[0161] In some embodiments, the targeting agent is an affibody and has less than 80 amino acid residues, or less than 70 amino acid residues, or less than 65 amino acid residues, or less than 60 amino acid residues. In some embodiments, the targeting agent consists of 40 to 80 amino acid residues, or 50 to 70 amino acid residues, or 55 to 65 amino acid residues, or 56 to 60 amino acid residues, or about 58 amino acid residues.

[0162] For the purposes of this disclosure, the term "antibody" includes four-chain proteins, which include, for example, two light chains and two heavy chains, including recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, deimmunized antibodies and half antibodies, bispecific antibodies) that can specifically bind to one or several closely related antigens through Fv. Antibodies generally include constant domains, which can be arranged into constant regions or constant fragments or crystallizable fragments (Fc). An exemplary form of an antibody includes a four-chain structure as its basic unit. A full-length antibody includes two heavy chains (about 50-70 kDa) and two light chains (about 23 kDa each) covalently linked. The light chain generally includes a variable region and a constant domain, and is a kappa light chain or a lambda light chain in mammals. The heavy chain generally includes a variable region and one or two constant domains connected to another constant domain by a hinge region. The heavy chain of a mammal has one of the following types: α, δ, ε, γ or μ. Each light chain is also covalently linked to one of the heavy chains. The antibody of the present invention can be used for the preparation of the antibody of the present invention.For example, two heavy chains and heavy chain and light chain are kept together by interchain disulfide bond and by non-covalent interaction.The number of interchain disulfide bonds can change in different types of antibodies.Each chain has an N-terminal variable region (VH or VL, wherein each length is about 110 amino acids) and one or more constant domains at the C-terminal.The constant domain of light chain (CL, its length is about 110 amino acids) is aligned with the first constant domain of heavy chain (CH, its length is -330-440 amino acids) and disulfide bonded to the first constant domain of heavy chain.The variable region of light chain is aligned with the variable region of heavy chain.Antibody heavy chain can include 2 or more other CH domains (such as CH2, CH3 etc.) and can be included in the hinge region between CH1 constant domain and CH2 constant domain. 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 or a deimmunized or germlined form thereof, or an affinity matured form thereof.

[0163] The terms "full-length antibody" or "whole antibody" are used interchangeably to refer to an antibody in substantially complete form, as opposed to an antigen-binding fragment of an antibody. Specifically, a whole antibody includes an antibody having a heavy chain and a light chain (including a constant region). The constant region can be a wild-type sequence constant region (e.g., a human wild-type sequence constant region) or an amino acid sequence variant thereof.

[0164] As used herein, the term "variable region" refers to the portion of the light chain and / or heavy chain of an antibody as defined herein, or only a heavy chain antibody (e.g., a camelid antibody or a cartilaginous fish immunoglobulin new antigen receptor (IgNAR)), which is capable of specific binding to an antigen and includes the amino acid sequences of the complementary determining regions (CDRs) (i.e., CDR1, CDR2, and CDR3) and framework regions "FR". FRs are those variable region residues that are different from the CDR residues. For example, a variable region comprises three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. V H V refers to the variable region of the heavy chain. L Refers to the variable region of the light chain.

[0165] As used herein, the term "complementarity determining region" (synonymous CDR, i.e., CDR1, CDR2, and CDR3) refers to the following amino acid residues of the variable region of an antibody, the presence of which is a major contributing factor to specific antigen binding. Each variable region generally has three CDR regions, identified as CDR1, CDR2, and CDR3. Each complementary determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat et al., (1987 and / or 1991). For example, in the heavy chain variable region, CDRH1 is between residues 31-35, CDRH2 is between residues 50-65, and CDRH3 is between residues 95-102. In the light chain, CDRL1 is between residues 24-34, CDRL2 is between residues 50-56, and CDRL3 is between residues 89-97. These CDRs may also comprise multiple inserts, such as described in Kabat (1987 and / or 1991). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including the classical numbering system of Chothia and Lesk (1987), Chothia et al. (1989) and / or Al-Lazikani et al., (1997); the numbering system of Honnegher and Plükthun (2001); the IMGT system discussed in Giudicelli et al., (1997); or the enhanced Chothia numbering scheme (http: / / www.bioinfo.org.uk / mdex.html). In one example, CDRs and / or FRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the five C-terminal amino acids listed herein, or any one or more of those amino acids are substituted by another naturally occurring amino acid. In an additional or alternative selection, the light chain CDR1 does not include the four N-terminal amino acids listed herein, or any one or more of those amino acids are substituted by another naturally occurring amino acid. In this regard, Padlan et al., 1995 established that the five C-terminal amino acids of the heavy chain CDR2 and / or the four N-terminal amino acids of the light chain CDR1 are generally not involved in antigen binding. In one example, CDRs and / or FRs are defined according to the Chothia numbering system.

[0166] As used herein, the term "Kabat numbering system" refers to the scheme for numbering antibody variable regions and identifying CDRs (hypervariable regions) as set out in Kabat (Kabat et al., 1987 and / or 1991).

[0167] As used herein, the term "Chothia numbering system" refers to the scheme for encoding antibody variable regions and identifying CDRs (structural loops) as set out in Chothia and Lesk (Chothia and Lesk, 1987) or Al-Lazikani (Al-Lazikani et al., 1997).

[0168] As used herein, the term "antigen binding domain" should be taken 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 comprises an F-terminal domain as described herein. V 、V H or V L One or more of .

[0169] As used herein, the term "binds" or "binding" with respect 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., antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure rather than binding to a protein extensively. If an antibody binds to epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope "A" (or free unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0170] As used herein, the term "specifically binds (specifically binds / binds specifically)" or similar phrases should be considered to mean that the protein of the present disclosure reacts or associates more frequently and more rapidly with a specific antigen (such as HER2) or a cell expressing the specific antigen with a longer duration and / or with greater affinity than with an alternative antigen or cell. For example, compared with the situation of binding to other antigens, the protein that specifically binds to an antigen binds to the antigen more easily and / or with a longer duration with greater affinity (e.g., 20 times or 40 times or 60 times or 80 times to 100 times or 150 times or 200 times greater affinity), avidity. It should also be 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. Therefore, "specific binding" does not necessarily require exclusive binding or undetectable binding to another antigen, which is explained by the term "selective binding".

[0171] In some embodiments, one targeting agent is covalently attached to the dendrimer. In some embodiments, more than one targeting agent is covalently attached to the dendrimer. In some embodiments, two targeting agents are covalently attached to the dendrimer. In some embodiments, three targeting agents are covalently attached to the dendrimer. In some embodiments, four targeting agents are covalently attached to the dendrimer.

[0172] The targeting agent is attached to the rest of the conjugate via the spacer. In some embodiments, the covalent attachment or connection between the targeting agent and the spacer group has been formed by reaction between complementary reactive functional groups present on a targeting agent precursor and a spacer group precursor, which may or may not be attached to the rest of the conjugate when it reacts with the HER2 targeting moiety precursor, depending on the process used to prepare the conjugate.

[0173] In some embodiments, the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting moiety.

[0174] The covalent attachment site of the targeting agent precursor can be, for example, cysteine, lysine, N-terminal amine, tyrosine, carbohydrate, non-natural amino acid or transaminase (e.g., transglutaminase or sortase A) or recognition sequence. The binding site for covalent attachment to protein is known in the art (e.g., Milla P. et al., 2012). It can be used to identify the position in the targeting agent polypeptide sequence via computer simulation crystal structure prediction to find out the covalent attachment site (e.g., away from the paratope, CDR, for correct folding and conformation key features and / or with highly charged, polar or bulky side groups) that is suitable for conjugation in structure. According to these candidate positions, empirical screening can be performed to determine the best preservation function while allowing robust conjugation of alternative sites. In one embodiment, the 2D3 position is selected from the group consisting of positions 13, 16, 42 and 126 (c-terminal). In some embodiments, the targeting agent precursor includes non-natural amino acid residues for attachment to the spacer. The non-natural amino acid residue may have a side chain with a reactive functional group that is complementary to the reactive functional group of the spacer group precursor. In some embodiments, the non-natural amino acid residue is a residue containing an azide group, for example, it may be a 4-azidophenylalanine residue, for example The azide group is capable of undergoing a cycloaddition reaction with an alkyne group that may be present on the spacer precursor group. In some embodiments, the non-natural amino acid is a diene-containing amino acid, for example a spirocyclopentadiene-containing amino acid, such as: Diene groups are capable of cycloaddition (e.g., Diels-Alder) reactions with olefin groups (such as those present on maleimide). Additional examples of non-natural 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 methylcyclopropene group. Additional examples of non-natural amino acids include:

[0175]

[0176]

[0177] In some embodiments, the targeting agent comprises or consists of any one of the amino acid sequences as defined herein.

[0178] Spacer Group

[0179] As used herein, the term "spacer group" refers to a chemical entity used to attach a targeting agent to a dendrimer. That is, the spacer group joins the targeting agent to the dendrimer (e.g., by covalent attachment). The spacer group is intended to position the targeting agent so that it can bind to the HER2 receptor without excessive detrimental interference from other components of the dendrimer.

[0180] As discussed above, the targeting agent can be covalently attached to the dendron, for example, through the core of the dendron. In some embodiments, the spacer group is covalently attached to the core unit. In some embodiments, the targeting agent is covalently attached to the dendron through the core of the dendron. That is, the targeting agent (such as an antibody fragment) is covalently attached to the core of the dendron through the spacer group. If the dendron is sterically crowded, it may be beneficial to covalently attach the targeting agent to the dendron via a spacer attached to the core of the dendron, wherein the spacer group may be of sufficient length to protrude beyond the surface of the dendron, thereby allowing the targeting agent to bind to its receptor or analog in vivo.

[0181] In some other embodiments, the targeting agent is covalently attached to the dendron via the surface structural unit of the dendron. In some embodiments, the spacer group is covalently attached to the surface structural unit. For example, the targeting agent can be connected to the surface nitrogen of the lysine residue via the spacer group, such as through an amide bond formed between the amine group of the lysine residue and the carboxylic acid group present on the 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 the spacer group, thereby forming an amide bond between the amine group of the lysine residue and the carboxylic acid group present on the intermediate comprising the targeting agent and the spacer group.

[0182] Exemplary spacer groups include polyethylene glycol (PEG), polypropylene glycol, polyaryl, peptide, amino acid, alkyl and alkenyl chains and carbohydrates (monosaccharides, oligosaccharides and polysaccharides) or their residues. In some embodiments, the spacer group includes a PEG chain, such as 2 to 60 ethyleneoxy repeating units, for example 2 to 20 or 20 to 48 repeating units. In one embodiment, PEG is 8 to 36 repeating units. In other embodiments, PEG is 12, 16, 20, 24 or 36 repeating units.

[0183] In some embodiments, the spacer group comprises multiple PEG groups interspersed with other functional groups. For example, the spacer group can comprise a PEG group attached via, for example, an amide group or other functional group that can be used to attach a moiety of the spacer group.

[0184] In order to attach the spacer group to the targeting agent and / or dendrimer, the spacer group precursor may comprise one or more reactive functional groups. In certain embodiments, the reactive functional groups include groups such as hydroxyl, carboxyl, active esters (such as NHS or pentafluorophenol esters), amino, azide, maleimide (including sulfo-maleimide), diene (such as cyclopentadiene, e.g., spiro[2.4]hept-4,6-diene group), tetrazine, citraconimide, alkyne-containing groups (including BCN (bicyclo[6.1.0]non-4-ene-9-yl), DBCO (dibenzocyclooctyne-amine)), activated olefins (such as methyl-cyclopropene-containing moieties), thiols, carbonyl groups (such as aldehydes and ketones), alkoxyamines, haloacetates, biotin, tetrazine, olefin-containing groups (including TCO (trans-cyclooctene), methyl-cyclopropene groups), and PTAD or other tyrosine reactive groups.

[0185] For example, in some embodiments, the spacer group intermediate may comprise two reactive groups, e.g., one at each end, the two reactive groups being orthogonal, i.e., at least one of the reactive groups is capable of reacting with a complementary group present on an intermediate comprising a targeting agent or an intermediate comprising a dendrimer under conditions where the other reactive group is stable and substantially unreactive, to attach the spacer group to that component of the conjugate. This allows the spacer to be attached (e.g., covalently attached) to the dendrimer or targeting agent, and then subsequently by reaction of the other reactive group with a complementary group present on the remaining component, to link the targeting agent to the dendrimer.

[0186] In some embodiments, the spacer group is attached (e.g., covalently attached) to the targeting agent by reaction of precursors containing alkyne and azide groups, respectively (e.g., the intermediate containing the targeting group can contain the azide group, and the intermediate containing the spacer group can contain the alkyne group). This reaction results in the formation of a triazole-containing group, for example, as follows:

[0187] And the triazole-containing group can be formed by reacting a precursor having the following structure:

[0188]

[0189] As another example, the spacer group can be attached via the formation of a triazole-containing group such as the following:

[0190] And the triazole-containing group can be formed by reacting a precursor having the following structure:

[0191]

[0192] In some embodiments, the spacer group is attached (e.g., covalently attached) to the targeting agent by reaction of precursors containing an olefin (e.g., a strained olefin such as trans-cyclooctene) and a tetrazine group, respectively. This reaction results in the formation of a pyridazine-containing group, such as the following, with the expulsion of nitrogen:

[0193]

[0194] And the pyridazine-containing group can be formed by reacting a precursor having the following structure:

[0195]

[0196] In some embodiments, the spacer group is attached (e.g., covalently attached) to the targeting agent by reaction of precursors containing an olefin (e.g., a strained olefin such as methylcyclopropene) and a tetrazine group, respectively. This reaction results in the formation of a pyridazine-containing group with the expulsion of nitrogen.

[0197] In order to attach (e.g., covalently attach) the spacer group to the dendrimer, the spacer group precursor may comprise additional functional groups. For example, the spacer group precursor may comprise a carboxylic acid group that is capable of reacting (e.g., forming an amide bond) with an amino group forming part of the core unit precursor.

[0198] In some embodiments, the spacer group is attached (e.g., covalently attached) to the dendrimer by reaction of precursors comprising carboxylic acid and amine groups, e.g., the spacer group intermediate may contain a carboxylic acid group that can react (e.g., form an amide bond) with an amine group present as part of or extending from a core unit, and the spacer group is attached to the targeting agent by reaction of precursors containing alkyne and azide groups, 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).

[0199] As discussed above, the targeting agent precursor can include non-natural amino acid residues. In some embodiments, the targeting agent includes non-natural amino acid residues. The non-natural amino acid residue can be, for example, a non-natural amino acid that can present a reactive side chain, wherein the reactive side chain carries a functional group complementary to those functional groups present on the spacer group precursor. In this way, the complementary functional groups can react, and therefore the attachment of the targeting moiety precursor to the spacer group precursor can occur. In some embodiments, the non-natural amino acid residue is a 4-azidophenylalanine residue. In some embodiments, the spacer group precursor contains an alkyne group for conjugation to a targeting moiety precursor containing a non-natural amino acid residue. In some embodiments, the spacer group precursor contains an alkyne group for conjugation to a targeting agent precursor containing a 4-azidophenylalanine residue. In some embodiments, the spacer group precursor contains an alkyne group as a dibenzylcyclooctane group for conjugation to a targeting moiety precursor containing a 4-azidophenylalanine residue. In some embodiments, one end of the spacer group is attached to the targeting moiety by cycloaddition reaction of the DBCO group with the azide moiety on a 4-phenylalanine residue that forms part of the targeting moiety, e.g., to form a triazole-containing group such as:

[0200]

[0201] or reaction of a BCN((bicyclo[6.1.0]non-4-en-9-yl)) group with an azide moiety on a 4-phenylalanine residue forming part of the targeting moiety, e.g. to form a triazole containing group such as:

[0202]

[0203] In some embodiments, one end of the spacer group is attached (e.g., covalently attached) to the dendrimer by an amidation reaction between an amino group present on the core or on a surface building block and a carboxyl group present on the spacer group (e.g., by reaction of an activated ester). In some embodiments, the spacer group precursor contains a tetrazine group. In some embodiments, the spacer group precursor comprises a maleimide group, e.g., for conjugation to a diene (such as a cyclopentadiene, e.g., a spiro[2.4]hepta-4,6-diene group).

[0204] In some embodiments, the spacer group precursor contains a PEG chain having a reactive carboxyl group for bonding to the amine at the core of the dendron and an azide group for conjugating to a targeting agent precursor containing a reactive alkyne moiety. In some embodiments, the spacer group precursor consists of a PEG chain having a reactive amine group for bonding to the carboxyl group at the core of the dendron and an azide group for conjugating to a targeting agent precursor containing a reactive alkyne moiety. In some embodiments, the spacer group precursor consists of a PEG chain having a reactive carboxyl group for bonding to the amine at the core of the dendron and a maleimide group for conjugating to a targeting agent precursor containing a reactive thiol moiety. In some embodiments, the spacer group precursor consists of a PEG chain having a reactive amine group for bonding to the carboxyl group at the core of the dendron and a thiol or masked thiol group for conjugating to a targeting agent precursor containing a reactive maleimide moiety. In some embodiments, the spacer group precursor contains a PEG chain having a reactive carboxyl group for conjugation to an amine at the core of the dendron and a tetrazine group for conjugation to a targeting agent precursor containing a reactive olefin moiety. In some embodiments, the spacer group precursor contains a PEG chain having a reactive carboxyl group for conjugation to an amine at the core of the dendron and a maleimide group for conjugation to a targeting agent precursor containing a reactive diene (such as a cyclopentadiene, e.g., a spiro[2.4]hepta-4,6-diene group).

[0205] In some embodiments, the attachment of the targeting agent to the dendrimer can be achieved by attaching (e.g., covalently attaching) a first spacer group to the targeting agent intermediate, attaching (e.g., covalently attaching) a second spacer group to the dendrimer (e.g., the core of the dendrimer), and then reacting the complementary functional groups present on the first spacer group and the second spacer group together to attach the targeting agent to the dendrimer. This approach can provide for easy attachment of the dendrimer to the targeting agent. For example, the first spacer group intermediate can include a first reactive group at one end that is complementary to a reactive group on the targeting agent (e.g., an alkyne group that is complementary to an azide group and can react together to form a triazole group); and a second reactive group that is complementary to a reactive group on the second spacer group (e.g., a tetrazine-containing group that reacts complementary to a trans-cyclooctene-containing group). The second spacer group intermediate may, for example, comprise a third reactive group at one end that reacts complementarily with a reactive group on the dendrimer (e.g., a carboxylic acid group that reacts complementarily with an amine group); and a fourth reactive group at the other end that reacts complementarily with a reactive group on the first spacer group intermediate (e.g., a trans-cyclooctene group that reacts complementarily with a tetrazine group). For example, the first spacer group and the second spacer group may be attached via a group that is generated by the reaction of a trans-cyclooctene group and a tetrazine group and comprises the following structure:

[0206]

[0207] In some embodiments, the targeting moiety can be linked to a dendrimer via a spacer group, which is 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 reaction of a tetrazine moiety attached to the dendrimer with a strained olefin group (e.g., trans-cyclooctene) at the other end of the spacer group.

[0208] The targeting agent precursor is reacted with the spacer group precursor to form the attachment of the targeting agent and the spacer group. The means by which the spacer group precursor is attached to the targeting agent precursor is known in the art. For example, the site for covalent attachment to the targeting agent precursor includes but is not limited to cysteine ​​residues, lysine residues, C-terminal amino acid residues, N-terminal amine, tyrosine residues, carbohydrates, suitable non-natural amino acid residues or transaminase or recognition sequence. For covalent attachment to the binding site of protein (such as targeting agent precursor) is known in the art (for example, Milla P. et al., 2012). For example, the spacer group precursor can be made to react with the targeting agent precursor at the C-terminal of the targeting agent so that the spacer group is attached to the targeting agent via the C-terminal. In some embodiments, the targeting agent is attached to the spacer group via the C-terminal of the targeting agent. In one embodiment, the targeting agent is covalently attached to or connected to the spacer group via the C-terminal of the targeting agent.

[0209] Therapeutic agents

[0210] As used herein, the term "therapeutic agent" refers to an agent that exerts an in vivo therapeutic effect, for example, against HER-2 expressing cancer cells. Examples of therapeutic agents include, but are not limited to, anticancer agents, also known as chemotherapeutic agents, anti-neoplastic agents, and cytotoxic agents. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent. As used herein, the term "cytotoxic agent" refers to an agent that exhibits the properties of a chemotherapeutic agent. Examples of anticancer agents include, but are not limited to, DNA alkylating agents and cross-linking agents (e.g., platinum-containing agents such as cisplatin, oxaliplatin), DNA damaging agents such as 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, nemorubicin), 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), pemetrexed, and antimitotic agents. Such anticancer agents can be used to treat cancer with curative intent (i.e., to eliminate the cancer patient) or in addition with the intent to alleviate / relieve symptoms in order to provide the patient with a better quality and longer life. Anticancer agents can be used in conjunction with other non-chemotherapeutic treatments of cancer (i.e., radiation therapy, surgery / biopsy).

[0211] In one embodiment, the cytotoxic agent is nemorubicin. The structure of nemorubicin is as follows:

[0212]

[0213] In one embodiment, the cytotoxic agent is cabazitaxel. The structure of cabazitaxel is as follows:

[0214] In one embodiment, the cytotoxic agent is docetaxel. The structure of docetaxel is as follows:

[0215] In one embodiment, the cytotoxic agent is SN-38. The structure of SN-38 is as follows:

[0216]

[0217] In one embodiment, the cytotoxic agent is irinotecan. The structure of irinotecan is as follows:

[0218]

[0219] In one embodiment, the cytotoxic agent is topotecan. The structure of topotecan is as follows:

[0220]

[0221] In one embodiment, the cytotoxic agent is gemcitabine. The structure of gemcitabine is as follows:

[0222]

[0223] In some embodiments, the therapeutic agent is a super cytotoxic agent. As used herein, the term "super cytotoxic agent" refers to an agent that exhibits highly effective chemotherapeutic properties, but which is itself too toxic to be administered alone as an anticancer agent. That is, super cytotoxic agents, despite exhibiting chemotherapeutic properties, generally cannot be safely administered to a subject because the adverse toxic side effects outweigh the chemotherapeutic benefits.

[0224] Super cytotoxic agents include, for example, nemorubicin, dolastatin (e.g., dolastatin-10, dolastatin-15), auristatin (e.g., monomethyl auristatin-E, monomethyl auristatin-F), maytansinoid alkaloids (e.g., maytansine, mertansine / maytansine (DM1, ravtansine (DM4)), calicheamicin (e.g., calicheamicin γ1), esperamicin (e.g., esperamicin A1), and pyrrolobenzodiazepine (PDB), among others.

[0225] In some embodiments, the therapeutic agent is auristatin. In some embodiments, the therapeutic agent is monomethyl auristatin. In one embodiment, the therapeutic agent is monomethyl auristatin E (MMAE). In one embodiment, the therapeutic agent is monomethyl auristatin F (MMAF). Both MMAE and MMAF are understood to inhibit cell division by blocking the polymerization of tubulin.

[0226] The chemical structure of MMAE is as follows:

[0227]

[0228] The chemical structure of MMAF is as follows:

[0229]

[0230] 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 maytansine / mertansine (DM1). In one embodiment, the super cytotoxic agent is raftansine (DM4). Maytansinoids are understood to inhibit the assembly of microtubules by binding to tubulin.

[0231] The chemical structure of maytansine is as follows:

[0232]

[0233] The chemical structure of Maytansine / Mertansine is as follows:

[0234]

[0235] In some embodiments, the super cytotoxic agent has an in vitro IC against cancer cell lines (e.g., SKBR3 and / or HEK293 cells and / or MCF7 cells) of 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.50 The in vitro activities of MMAE and maytansine are discussed in Abdollahpour-Alitappeh et al. (2017) and Oroudjev et al. (2010), respectively.

[0236] It will be appreciated that the dendrimer-targeting agent conjugate may have a distribution of therapeutic agents / targeting agents. For example, it is possible that the dendrimer-targeting agent conjugate may have up to about 8 therapeutic agents / targeting agents or even more.

[0237] Thus, it should be understood that the therapeutic agent to targeting agent ratio (DTR) of each dendrimer-targeting agent conjugate substance may vary in a population. Generally, it is desirable to produce a dendrimer-targeting agent conjugate with a high DTR. Generally speaking, this results in a more effective therapy. However, a DTR that is too high may lead to undesirable toxicity, for example when the therapeutic agent is a super cytotoxic agent.

[0238] 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.

[0239] In some embodiments, the dendrimer-targeting agent conjugate has a DTR between about 1 and about 32. In some embodiments, the dendrimer-targeting agent conjugate has a DTR between about 7 and about 32. In some embodiments, the dendrimer-targeting agent conjugate is a G5 dendrimer and has a DTR between about 26 and about 32. In some embodiments, the dendrimer-targeting agent conjugate is a G4 dendrimer and has a DTR between about 14 and about 16. In some embodiments, the dendrimer-targeting agent conjugate is a G3 dendrimer and has a DTR between about 6 and about 8.

[0240] In the case where more than one targeting agent is conjugated to a dendron, the DTR will be reduced relative to a dendron-targeting agent conjugate with a single targeting agent. In some embodiments, the dendron-targeting agent conjugate has a DTR between about 2 and about 16. In some embodiments, the dendron-targeting agent conjugate has a DTR between about 8 and about 16. In some embodiments, the dendron-targeting agent conjugate is a G5 dendron and has a DTR between about 4 and about 16. In some embodiments, the dendron-targeting agent conjugate is a G5 dendron and has a DTR between about 6 and about 16. In some embodiments, the dendron-targeting agent conjugate is a G4 dendron and has a DTR between about 4 and about 8. In some embodiments, the dendron-targeting agent conjugate is a G4 dendron and has a DTR between about 4 and about 8. In some embodiments, the dendron-targeting agent conjugate is a G3 dendron and has a DTR between about 2 and about 4.

[0241] Due to the ability of dendrimer-therapeutics to carry more than one therapeutic agent, the need for super-cytotoxic agents may be avoided. For example, the ability to target delivery of multiple cytotoxic agents (such as, for example, docetaxel) on a single dendrimer may be equally or more effective therapeutically when compared to the delivery of a single super-cytotoxic agent (such as, for example, MMAE). Avoidance of super-cytotoxic agents may be an advantage.

[0242] Connectors

[0243] In some embodiments, the therapeutic agent is attached to the dendrimer via a linker. The linker should preferably retain the favorable properties of the therapeutic agent and remain substantially intact and non-toxic in the systemic circulation. The linker group can be used, for example, to provide a suitable group for attaching the pharmaceutically active agent to the dendrimer, for example, where the available functionality in the pharmaceutically active agent is not suitable for direct attachment to the structural unit. The linker group can also or alternatively be used to promote the controlled release of the pharmaceutically active agent from the dendritic scaffold, thereby providing a therapeutically effective concentration of the pharmaceutically active agent and a desired pharmacokinetic profile for a suitable (e.g., extended) period of time.

[0244] 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 can be, for example, to a surface moiety of the dendrimer.

[0245] Those skilled in the art will appreciate that any of a variety of suitable linkers may be used. The linker should provide adequate stability during systemic circulation, but allow rapid and efficient release of the cytotoxic drug in active form at its site of action (e.g., once internalized into a cancer cell).

[0246] The linker can be a non-cleavable or cleavable linker. In one embodiment, the linker is a non-cleavable linker. In one embodiment, the linker is a cleavable linker. The cleavable linker (either by itself or in conjunction with its connection to the pharmaceutically active agent) comprises one or more of the following cleavable moieties: an ester group, a hydrazone group, an oxime group, an imine group, or a disulfide group. In some embodiments, the linker is tumor environment cleavable, acid labile, reducing environment labile, hydrolytically labile, or protease sensitive.

[0247] Chemically unstable joints include, but are not limited to, acid-labile joints (i.e., hydrazones) and disulfide joints. Enzymatically cleavable joints include, but are not limited to, peptide joints and β-glucuronide joints. Examples of peptide joints 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. Advantageously, peptide joints and their peptide bonds are expected to have good serum stability because lysosomal proteolytic enzymes have low activity in blood. Val-Ala, Val-Cit, Phe-Lys, Phe-Arg, Phe-Cit, Val-Arg, Val-Cit, Ala-Arg, and Ala-Cit joints are rapidly hydrolyzed by cathepsin B. In some embodiments, the joint is an enzymatically cleavable joint. For example, in some embodiments, the joint comprises an amino acid residue that can be recognized and cleaved by an enzyme.

[0248] In some embodiments, the linker comprises a peptide group. In some embodiments, the linker comprises a valine-citrulline-p-aminobenzyl alcohol group (Val-Cit-PAB), for example having the following structure:

[0249]

[0250] For example, the PAB group can be covalently attached to an amine group present on the therapeutic agent moiety via the carbonyl group, thereby forming a carbamate bond, and can be attached to an amine group present on the external building block via a diacyl linker that forms an amide bond with the valine amino group and the amine group present on the external building block.

[0251] In some embodiments, the linker comprises or consists of a glutaric acid-valine-citrulline-p-aminobenzyl alcohol group, for example having the following structure:

[0252]

[0253] In some embodiments, the linker comprises or consists of a valine-alanine-p-aminobenzyl alcohol group, for example having the following structure:

[0254]

[0255] 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. This method allows attachment to the linker via an ester group, and it has been found that such ester groups can be cleaved in vivo, thereby releasing the pharmaceutically active agent at a desired rate.

[0256] In some embodiments, the core unit is formed from a core unit precursor comprising an amino group, the building block is a lysine residue or an analog thereof, the pharmaceutically active agent comprises a hydroxyl group, the residue of the pharmaceutically active agent is attached via the oxygen atom of the hydroxyl group, such that there is an ester bond between the residue of the pharmaceutically active agent and the linker, and there is an amide bond between the linker and the 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 via the oxygen atom of the hydroxyl group, and the cleavable linker is a diacyl linker group having the formula:

[0257] Where A is C2-C with O, S, SS, NH or N(Me) inserted 10 An alkylene group, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine.

[0258] As used herein, the term "alkyl" refers to a monovalent straight-chain (ie, linear) or branched saturated hydrocarbon group. In one example, the alkyl group contains 1 to 10 carbon atoms (ie, C 1-10 In one example, the alkyl group contains 1 to 6 carbon atoms (i.e., C 1-6 Examples of alkyl groups include methyl, ethyl, propyl (eg, n-propyl, isopropyl), butyl (eg, n-butyl, sec-butyl, tert-butyl), pentyl, and hexyl groups.

[0259] As used herein, the term "alkylene" refers to a divalent straight chain (ie, linear) or branched saturated hydrocarbon group. In one example, an alkylene group contains 2 to 10 carbon atoms (ie, C 2-10 In one example, the alkylene group contains 2 to 6 carbon atoms (i.e., C 2-6 Examples of alkylene groups include, for example, -CH2CH2-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2-, and the like.

[0260] Such linkers are particularly suitable for attachment to the therapeutic agent via hydroxyl groups present in the therapeutic agent. In such cases, the conjugate contains an ester bond between the linker and the therapeutic agent portion, and contains an amide bond between the linker and the dendrimer portion.

[0261] In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, the residue of the pharmaceutically active agent is attached via the oxygen atom of the hydroxyl group, and the cleavable linker is a diacyl linker group having the formula:

[0262] Where A is C2-C with O, S, NH or N(Me) inserted 10 Alkylene group.

[0263] In some embodiments, the pharmaceutically active agent comprises a hydroxyl group, the residue of the pharmaceutically active agent is attached via the oxygen atom of the hydroxyl group, and the diacyl linker is

[0264]

[0265] A specific type of cleavable linker is a cleavable linker containing a disulfide moiety. Such linkers are susceptible to cleavage by glutathione. For example, a linker of this type may comprise two acyl groups connected via an alkyl chain interposed with a disulfide moiety.

[0266] In some embodiments, the linker comprises an alkyl chain inserted with a disulfide moiety, wherein one or two carbon atoms next to the disulfide group are substituted by one or more methyl groups. For example, one carbon atom next to the disulfide group can be substituted by a geminal dimethyl group, for example, the linker can comprise the following groups:

[0267]

[0268] Another specific type of cleavable linker is a cleavable linker containing a diphosphate moiety. Such linkers are susceptible to hydrolysis by lysosomal acid pyrophosphatases and acid phosphatases. In some embodiments, the linker comprises an alkyl chain inserted with a diphosphate moiety (also referred to as a pyrophosphate moiety):

[0269]

[0270] Another specific type of cleavable linker is a cleavable linker containing a carbamate moiety. Such linkers have high hydrolytic stability before activation and efficient cleavage by different intramolecular mechanisms. In some embodiments, the linker comprises an alkyl chain inserted with a carbamate moiety:

[0271]

[0272] Another specific type of cleavable linker is one that contains an orthoester moiety:

[0273]

[0274] A non-cleavable linker is a linking group that is inert or substantially inert to cleavage for a desired period of time when exposed to in vivo conditions. A non-cleavable linker is not cleaved under biological conditions.

[0275] Examples include alkylene or cycloalkylene groups (e.g., C 1-10 Alkylene group or C 3-10 The invention also provides a diacyl linker bridged by a cycloalkylene group). Another example of a non-cleavable linker includes a thioether linker. A specific example of a non-cleavable linker is a non-cleavable linker formed by using SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). SMCC can react with a thiol group present on or attached to the therapeutic agent moiety via the maleimide functionality to form a thioester bond. The carboxylic acid functionality can be used to react with an amino group present on an external building block.

[0276] 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.

[0277] In one embodiment, the linker comprises a Val-Cit-PAB group, the therapeutic agent moiety is MMAE, and the Val-Cit-PAB group is attached to the therapeutic agent moiety (i.e., Val-Cit-PAB-MMAE) as follows:

[0278]

[0279] 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 (i.e., Val-Cit-PAB-MMAF) as follows:

[0280]

[0281] In one embodiment, the linker comprises a glutarate-Val-Cit-PAB group, the therapeutic agent moiety is MMAE, and the glutarate-Val-Cit-PAB group is attached to the therapeutic agent moiety (i.e., glutarate-Val-Cit-PAB-MMAE) as follows:

[0282]

[0283] In one embodiment, the linker is a DGA (diglycolic acid) or glutaric acid group and the therapeutic moiety is MMAF or an ester thereof, for example:

[0284]

[0285] In some embodiments, the therapeutic agent is covalently attached to the surface structural unit of the dendron through a linker group. For example, the therapeutic agent can be covalently attached to the surface structural unit of the dendron through a linker group other than a linker group. The linker group allows the therapeutic agent to be tethered at different lengths relative to the surface of the dendron. For example, the linker group can be used to extend the distance of the therapeutic agent relative to the surface of the dendron. This can eliminate any potential problems associated with steric hindrance, which may prevent access to the cleavable linker and may reduce the ability of the therapeutic agent to interact with the target. In some embodiments, the dendrimer-targeting agent conjugate comprises a therapeutic agent covalently attached to the surface of the dendrimer through a linker group and a linker group. In some embodiments, the connection order is dendrimer-linker-linker-therapeutic agent. In some embodiments, the connection order is dendrimer-linker-linker-therapeutic agent.

[0286] The linker group may comprise any chemical moiety for extending the distance of the therapeutic agent relative to the surface of the dendrimer. In some embodiments, the linker group may comprise one or more hydrophilic polymer groups. In some embodiments, the linker group comprises between about 1 and about 50 hydrophilic polymer groups. In some embodiments, the linker group comprises a PEG group. In some embodiments, the linker group comprises a PEOX group. In some embodiments, the linker group comprises a polysarcosine group. In some embodiments, the linker group comprises more than one, more than two, more than four, more than 10, more than 20, more than 30, more than 40, more than 50 PEG moieties. In some embodiments, the linker group comprises between about 1 and about 50 PEG moieties. In some embodiments, the linker group comprises four PEG moieties. In some embodiments, the linker group comprises 12 PEG moieties. In some embodiments, the linker group comprises 24 PEG moieties. In some embodiments, the linker group comprises 48 PEG moieties.

[0287] In some embodiments, the linker comprises a linker group and is (PEG) X -Val-Arg-PAB-P-trigger-moiety. X can be a number between about 1 and about 50 as described above.

[0288] In one embodiment, the linker comprises a linker group and is a (PEG)9-Val-Arg-PAB-P-trigger-NHCO moiety:

[0289]

[0290] In one embodiment, the linker comprises a linker group and is a (PEG)9-Val-Arg-PAB-P-Trigger-N(CH3)CO moiety:

[0291]

[0292] In some embodiments, the linker comprises a linker group and is (PEG) x -OP(O)OH-OP(O)OH-O- moiety. Likewise, X can be a number between about 1 and about 50 as described above.

[0293] In one embodiment, the linker comprises a linker group and is (PEG) x -OP(O)OH-OP(O)OH-O-part:

[0294]

[0295] In some embodiments, the linker comprises a linker group and is (PEG) X -Val-Ala-PAB-P-trigger-moiety. X can be a number between about 1 and about 50 as described above. In one embodiment, the linker comprises a linker group and is a (PEG)9-Val-Ala-PAB-P-trigger-NHCO moiety.

[0296] Specific linkers, such as (PEG) X -Val-Arg-PAB-P-trigger-or (PEG) X The use of -OP(O)OH-OP(O)OH-O- can provide release of therapeutic agents.

[0297] In example (PEG) X -Val-Arg-PAB-P-trigger-NHCO, (PEG) X In the case of -Val-Arg-PAB-P-trigger-N(CH3)CO, the linker is initially activated by cleavage of the dipeptide portion by a cellular enzyme such as cathepsin B, thereby displaying a self-cleaving 2-(aminomethyl)pyrrolidine linker. After intramolecular cyclization, elimination of the hydroxyl-containing therapeutic agent occurs. Exemplary hydroxyl-containing therapeutic agents include cabazitaxel, docetaxel, and SN-38.

[0298] In (PEG) xIn the case of the -OP(O)OH-OP(O)OH-O- moiety, the linker is cleaved specifically and cleanly to provide hydroxyl-containing drugs such as cabazitaxel, docetaxel, and SN-38, but has the added advantage of providing monophosphate drugs, which are the active species of nucleotide inhibitors such as gemcitabine.

[0299] Pharmacokinetic Modifiers

[0300] The conjugate comprises a plurality of hydrophilic polymer groups covalently attached to the surface building blocks of the dendrimer, which can modify the pharmacokinetic properties of the conjugate. The hydrophilic polymer groups can, for example, provide improved pharmacokinetic properties.

[0301] In some embodiments, the dendrimer comprises a plurality of hydrophilic polymer groups covalently attached to the surface structural units of the dendrimer. The term "hydrophilic polymer group" generally refers to a polymer group having a solubility in water at 25°C of at least 25 mg / ml, more preferably at least 50 mg / ml and still more preferably at least 100 mg / ml.

[0302] In some embodiments, the hydrophilic polymer group comprises repeating units of amino acids, alkoxy groups or alkyl (acyl) amino groups. In some embodiments, the hydrophilic polymer group comprises repeating units of amino acids (such as sarcosine). In some embodiments, the hydrophilic polymer group comprises repeating units of alkoxy groups (e.g., the hydrophilic polymer is a PEG group). In some embodiments, the hydrophilic polymer comprises repeating units of alkyl (acyl) amino groups (e.g., the hydrophilic polymer is a PEOX group).

[0303] In some embodiments, the hydrophilic polymer group comprises at least 10 monomeric units. In some embodiments, the hydrophilic polymer group comprises up to 100 monomeric units. In some embodiments, the hydrophilic polymer group comprises 10 to 100 or 10 to 50 monomeric units.

[0304] In some embodiments, the hydrophilic polymer group is a PEG group. A PEG group is a polyethylene glycol group, i.e., a group comprising repeating units having the formula -CH2CH2O-. The PEG material used to produce the dendrimers of the present disclosure typically contains a mixture of PEGs with some variation in molecular weight (i.e., ±10%), and thus, where a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEG composition. For example, the term "PEG ～2100 ” refers to polyethylene glycol (PEG) having an average molecular weight of about 2100 Daltons (i.e. ± about 10%) 1890 To PEG 2310 ). The term "PEG ～2300” refers to polyethylene glycol (PEG) having an average molecular weight of about 2300 Daltons (i.e. ± about 10%) 2070 To PEG 2530 ). Three methods are commonly used to calculate the MW averages: number average molecular weight, weight average molecular weight, and z average molecular weight. As used herein, the phrase "molecular weight" is intended to refer to the 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.

[0305] In some embodiments, the PEG group has an average molecular weight in the range of 500 to 2500 Daltons. In some embodiments, the PEG group has an average molecular weight in the range of 800 to 1200 Daltons. In some embodiments, the PEG group has an average molecular weight in the range of 900 to 1100 Daltons. In some embodiments, the PEG group has an average molecular weight in the range of 1500 to 2500 Daltons. In some embodiments, the PEG group has an average molecular weight in the range of 1900 to 2300 Daltons. In some embodiments, the PEG group has an average molecular weight in the range of 2100 to 2500 Daltons.

[0306] 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.

[0307] In some embodiments, the PEG group has a polydispersity index (PDI) between about 1.00 and about 1.50, between about 1.00 and about 1.25, or between about 1.00 and about 1.10. In some embodiments, the PEG group has a polydispersity index (PDI) of about 1.05. The term "polydispersity index" refers to a measure of the distribution of molecular weights in a given polymer sample. The polydispersity index (PDI) is equal to the weight average molecular weight (M). w ) divided by the number average molecular weight (M n ), and indicates the distribution of individual molecular masses in a batch of polymers. The polydispersity index (PDI) has a value equal to or greater than 1, but will be closer to 1 as the polymer approaches uniform length and average molecular weight.

[0308] The PEG group can be straight or branched. If desired, an end-capped PEG group can be used. In some embodiments, the PEG group is a methoxy-end-capped PEG.

[0309] In some embodiments, the hydrophilic polymer group is a PEOX group. In some embodiments, the dendrimer comprises a plurality of PEOX groups covalently attached to the surface structural unit of the dendrimer portion. The PEOX group is a polyethyloxazoline group, i.e., a group comprising a repeating unit having the formula:

[0310]

[0311] PEOX groups are so named because they can be produced by the polymerization of ethyl oxazoline. The PEOX materials used to produce dendrons of the present disclosure generally contain a mixture of PEOX with some variations (i.e., ± 10%) of molecular weight, and therefore, in the case of a specified molecular weight, it is generally an approximation of the mean molecular weight of the PEOX composition. In some embodiments, the second end group comprises a PEOX group with a mean 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 with a mean molecular weight in the range of 750 daltons to 2500 daltons, or 1000 daltons to 2000 daltons. If desired, end-capped PEOX groups can be used. In some embodiments, the PEOX group is a methoxy-end-capped PEOX.

[0312] In some embodiments, the hydrophilic polymer group comprises a polysarcosine group. In some embodiments, the polysarcosine group has an average molecular weight of at least 750 Daltons, at least 1000 Daltons, or at least 1500 Daltons. In some embodiments, the hydrophilic polymer group comprises a polysarcosine group having an average molecular weight in the range of 750 Daltons to 2500 Daltons, or 1000 Daltons to 2000 Daltons.

[0313] The hydrophilic polymer group may be attached to the external building block via any suitable means. In some embodiments, a linking group is used to attach the hydrophilic polymer group to the external building block.

[0314] Hydrophilic polymer groups are typically attached through the use of precursors containing reactive groups that react with amine groups, such as reactive acyl groups (which can form amide bonds), or aldehydes (which can form amine groups under reductive amination conditions).

[0315] In some embodiments, the PEG group is covalently attached to the PEG linking group (L1) via an ether bond 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 the building block via an amide bond formed between a nitrogen atom present in the building block and a carbon atom of an acyl group present in the PEG linking group. In some embodiments, each of the L1-PEG groups is and wherein the PEG group is a methoxy terminated PEG having an average molecular weight in the range of about 500 to 2500 Daltons.

[0316] In some embodiments, the PEOX group is covalently attached to the PEOX linking group (L1′) via a bond 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 the structural unit via an amide bond formed between a nitrogen atom present in the structural unit and a carbon atom of an acyl group present in the PEOX linking group. In some embodiments, each L1′-PEOX group is

[0317] In some embodiments, the hydrophilic polymer group comprises a polysarcosine group, ie, a group comprising repeating units having the formula:

[0318]

[0319] In some embodiments, the polysarcosine group is attached to the structural unit via an amide bond formed between a nitrogen atom present in the structural unit and a carbon atom of an acyl group present in the polysarcosine group. In some embodiments, the hydrophilic polymer group comprises a polysarcosine group having an average molecular weight of at least 750 Daltons, at least 1000 Daltons, or at least 1500 Daltons. In some embodiments, the second terminal group comprises a polysarcosine group having an average molecular weight ranging from 750 Daltons to 2500 Daltons, or from 1000 Daltons to 2000 Daltons.

[0320] In many cases, the population of dendrimers that have been functionalized at the dendrimer surface contains a random stoichiometry and topology of functional groups. For example, reaction of a population of dendrimer-targeting agent conjugates containing, for example, 64 reactive surface groups with one or more reactive functional groups can produce a broad population of functionalized dendrimers, some of which contain a higher number of functional groups than others. In the case where there are multiple different surface groups available for reaction with the reactive functional groups, a broad distribution of dendrimers with different surface topologies can also be obtained.

[0321] The dendrimer-targeting agent conjugates, intermediates and processes of the present invention can allow for high loading of therapeutic agents by covalent attachment to surface building blocks. It is also believed that such dendrimer-targeting agent conjugates promote release of therapeutically effective levels of therapeutic agents over an extended period of time after administration, and thus can reduce the required frequency and / or number of administrations. The dendrimer-targeting agent conjugates of the present invention can also allow for targeted delivery of therapeutic agents to their site of action. Thus, such dendrimer-targeting moiety agents can reduce off-target activities, such as cytotoxic activity.

[0322] Internalization of dendrimer-targeting agent conjugates

[0323] Dendrimer-targeting agent conjugates provide targeted anti-cancer therapy. The beneficial properties of the conjugates are understood to be associated, at least in part, with the observed rapid internalization of exemplary conjugates containing MMAE therapeutic agents into target cells. In some embodiments, the dendrimer-targeting agent conjugates are internalized in HER2 expressing cells.

[0324] As used herein, the term "internalization" refers to the internalization of the dendrimer-targeting agent conjugate into a cell. The ability of the dendrimer-targeting agent conjugate to specifically seek out and bind to the HER2 receptor on the target HER2-expressing cancer cells and to rapidly internalize means that a reduced amount of the conjugate is circulating in the plasma. Therefore, as a result, the chance of the therapeutic agent (such as a super cytotoxic moiety like MMAE) being subjected to undesirable cleavage from the dendrimer-targeting agent conjugate at sites that do not overexpress HER2 (e.g., tumor cells) should be reduced, and the likelihood of side effects or toxicity associated with non-specific release of the therapeutic agent in a subject should be reduced. In contrast, administration of an equivalent dose of a therapeutic agent (such as a super cytotoxic moiety) may cause extreme toxicity to a 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, it is believed that the dendrimer-targeting agent conjugate provides a safer means for delivering therapeutic agents, such as super cytotoxic agents (including MMAE and MMAF).

[0325] In some embodiments, administration of the conjugate provides reduced side effects compared to administration of an equivalent dose of free therapeutic agent. In some embodiments, administration of the conjugate provides at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduction in plasma concentration of the therapeutic agent (e.g., Cmax, or concentration at a specified time point after administration (e.g., 1 h, 6 h, 12 h, 24 h, 48 h)).

[0326] In some embodiments, the therapeutic agent is a super-cytotoxic agent (e.g., MMAE, MMAF), and administration of the dendrimer-targeting agent conjugate provides 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% reduction in plasma concentration of the therapeutic agent (e.g., Cmax, or concentration at a specified time point (e.g., 1 h, 6 h, 12 h, 24 h, 48 h) after administration) compared to administration of an equivalent dose of the free therapeutic agent.

[0327] Therefore, a method for killing HER2-expressing cells is also provided, comprising: contacting a conjugate as defined herein with a HER2-expressing cell so that the conjugate is internalized in the cell, whereby the therapeutic agent kills the HER2-expressing cell. In some embodiments, the therapeutic agent is a super cytotoxic agent (e.g., MMAE, MMAF). In some embodiments, the conjugate (e.g., a super cytotoxic agent conjugate) is internalized into the cell by endocytosis.

[0328] The conjugates of the present disclosure have been found to be effective in in vivo cancer studies, i.e., xenograft studies. It is believed that the relatively small size (e.g., the low molecular weight of the entire conjugate) aids in penetration into tumor tissue and contributes to the effectiveness of the therapy. Thus, in some embodiments, the molecular weight of the conjugate is in the range of up to about 120 kDa, or up to about 100 kDa, or up to about 80 kDa, or up to about 60 kDa, or up to about 50 kDa, or up to about 45 kDa, or up to about 40 kDa.

[0329] Composition

[0330] In some embodiments, the dendrimer-targeting agent conjugate is presented as a composition, preferably a pharmaceutical composition.

[0331] It is understood that as a result of the nature of the synthetic methods used to generate the dendrimers, there will be some variation in molecular composition between the dendrimer-targeting agent conjugates present in a given composition. For example, as discussed above, one or more of the synthetic steps used to generate the dendrimer-targeting agent conjugates may not be carried through to completion, which may result in the presence of dendrimer-targeting agent conjugates that do not all contain the same number of therapeutic agent moieties, targeting agents, or contain incomplete generations of building blocks.

[0332] 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 attached to the dendrimer is 1. 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 1.2. 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. 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. 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 pharmaceutically 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.

[0333] The present disclosure also provides pharmaceutical formulations or compositions for veterinary and human medical use, which comprise the conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredients, stabilizers, and the like.

[0334] Accordingly, there is also provided a composition for pharmaceutical use comprising i) a conjugate as defined herein; and ii) a pharmaceutically acceptable excipient.

[0335] The excipient must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation / composition and not unduly deleterious to the recipient thereof.

[0336] In some embodiments, the composition comprises a pharmaceutically acceptable solvent, such as water for injection and / or a pharmaceutically acceptable organic solvent.

[0337] The compositions of the present disclosure may, for example, comprise polymeric excipients / additives or carriers, for example, polyvinyl pyrrolidone, derivatized celluloses (such as hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl methyl cellulose), Ficoll (a polymer sugar), hydroxyethyl starch (HES), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin and sulfobutyl ether-β-cyclodextrin), polyethylene glycol, and pectin.

[0338] The composition may further comprise diluents, buffers, citrates, trehalose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants), inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc and other such suitable cations). Other pharmaceutical excipients and / or additives suitable for use in the compositions according to the present disclosure are listed in "Remington: The Science & Practice of Pharmacy", 19th edition, Williams & Williams, (1995) and the "Physician's Desk Reference", 52nd edition, Medical Economics, Montvale, NJ (1998) and "Handbook of Pharmaceutical Excipients", 3rd edition, AH Kibbe, ed., Pharmaceutical Press, 2000.

[0339] For example, the dendrimer-targeting agent conjugates of the present disclosure may be formulated in compositions, including those suitable for administration by aerosol inhalation to the lungs, or parenteral administration, including intraperitoneal, intravenous, subcutaneous or intramuscular injection. The compositions may be conveniently presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art. All methods include the step of combining the dendrimer-targeting agent conjugate with a carrier constituting one or more auxiliary ingredients. Generally, the composition is prepared by combining the dendrimer-targeting agent conjugate with a liquid carrier to form a solution or suspension; or alternatively, combining the dendrimer-targeting agent conjugate with formulation components suitable for forming a solid, optionally particulate product, and then, if necessary, shaping the product into the desired delivery form. When particulate, the solid formulations of the present disclosure will generally contain particles ranging in size from about 1 nanometer to about 500 microns. Generally, for solid formulations intended for intravenous administration, the diameter of the particles will generally be in the range of about 1 nm to about 10 microns. The composition may contain a dendrimer-targeting moiety conjugate of the present disclosure, which is a nanoparticle having a particle diameter of less than 1000 nm, for example between 5 and 1000 nm, especially between 5 and 500 nm, more especially between 5 and 400 nm, such as between 5 and 50 nm and especially between 5 and 20 nm. In one example, the composition contains a dendrimer-targeting moiety conjugate having an average size between 5 and 20 nm. In some embodiments, the dendrimer-targeting moiety conjugate is polydisperse in the composition, wherein the PDI is between 1.01 and 1.8, especially between 1.01 and 1.5, and more especially between 1.01 and 1.2. In one example, the dendrimer-targeting agent conjugate is monodisperse in the composition.

[0340] In some preferred embodiments, the composition is formulated for parenteral delivery. For example, in one embodiment, the formulation can be a lyophilized composition suitable for reconstitution in an aqueous vehicle prior to injection.

[0341] 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.

[0342] In some embodiments, the composition is formulated for parenteral infusion as part of a chemotherapy regimen.

[0343] In some embodiments, the composition is formulated for intraperitoneal delivery. Any suitable means of delivery can be used. For example, in some embodiments, delivery can be performed by lavage or aerosol. In one embodiment, the composition is formulated for intraperitoneal delivery and is used to treat cancer in the peritoneal cavity, including malignant epithelial tumors (e.g., ovarian cancer) and peritoneal carcinomatosis (e.g., gastrointestinal cancer, especially colorectal cancer, gastric cancer, gynecological cancer, and primary peritoneal neoplasms).

[0344] Also provided are pharmaceutical formulations suitable for administration by inhalation as an aerosol. These formulations include a solution or suspension of the desired dendrimer or its salt. The desired formulation can be placed in a small chamber and atomized. Atomization can be accomplished by compressed air or by ultrasonic energy to form a plurality of droplets or solid particles comprising the dendrimer or its salt.

[0345] As discussed below, the dendrimer-targeting agent conjugates of the present disclosure may be administered, for example, in combination with one or more additional pharmaceutically active agents. For example, the dendrimer-targeting agent conjugates may be administered together with the additional pharmaceutically active agents as a composition.

[0346] Examples of additional pharmaceutically active agents include chemotherapeutic and cytotoxic agents, small molecule cytotoxic agents, tyrosine kinase inhibitors, checkpoint inhibitors, EGFR inhibitors, antibody therapies, taxanes, and aromatase inhibitors.

[0347] The dendrimer-targeting agent conjugates of the present disclosure can be administered not only together with other chemotherapeutic drugs but also in combination with appropriate other drug treatments.

[0348] How to use

[0349] 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.

[0350] 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 treating cancer.

[0351] 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 treating cancer.

[0352] In some embodiments, the dendrimer-targeting agent conjugate is used to treat or prevent cancer, for example, in a method of inhibiting the growth of a tumor. In some embodiments, the dendrimer-targeting agent conjugate is for use in treating cancer.

[0353] 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.

[0354] In some embodiments, the cancer is characterized by abnormal expression or overexpression of HER2 (also referred to as ERBB2). Such abnormal expression or overexpression of HER2 is known to occur in, for example, the following cancers: breast cancer, testicular cancer, ovarian cancer, stomach cancer, adenocarcinoma of the lung, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., uterine serous endometrial cancer). In some embodiments, the cancer is selected from the group consisting of: breast cancer, testicular cancer, ovarian cancer, stomach cancer, adenocarcinoma of the lung, gastric cancer, pancreatic cancer, salivary duct cancer, esophageal cancer, and uterine cancer (e.g., uterine serous 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 adenocarcinoma of the lung. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is salivary duct cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is uterine cancer.

[0355] Thus, also provided are methods, uses, dendrimer-targeting agent conjugates or compositions for use thereof, wherein the cancer is selected from the group consisting of ovarian cancer, breast cancer, gastric cancer, uterine cancer or another cancer characterized by aberrant expression or overexpression of HER2 (ie, ERBB2).

[0356] combination

[0357] Especially during chemotherapy, drugs are often administered in combination with other drugs. 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 anticancer agents / drugs). The dendrimer-targeting agent conjugate and the one or more additional pharmaceutically active agents may be administered simultaneously, subsequently, 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, for example, be anticancer agents for the treatment of colorectal cancer, gastric cancer, pancreatic cancer, prostate cancer, or breast cancer. Examples of additional pharmaceutically active agents include chemotherapeutic and cytotoxic agents, small molecule cytotoxic agents, tyrosine kinase inhibitors, checkpoint inhibitors, EGFR inhibitors, antibody therapies, taxanes, and aromatase inhibitors.

[0358] dose

[0359] It should be understood that the term "therapeutically effective amount" refers to an amount of the dendrimer-targeting agent conjugate administered that is sufficient to alleviate or prevent to some extent one or more symptoms of the disorder or condition being treated. The therapeutically effective amount of the dendrimer-targeting agent conjugate can be referred to, for example, based on the amount of the dendrimer-targeting agent conjugate administered. Alternatively, it can be determined based on the amount of therapeutic agent that the dendrimer-targeting agent conjugate can theoretically deliver, for example, based on the loading amount of the therapeutic agent on the dendrimer-targeting agent conjugate.

[0360] The dendrimer-targeting agent conjugate can be administered by any suitable route, including, for example, the dendrimer-targeting agent conjugate can be administered intravenously. In some embodiments, the dendrimer-targeting agent conjugate is delivered as an IV bolus. In some embodiments, the dendrimer-targeting agent conjugate is administered IV over a period of time ranging from 0.5 to 60 minutes, or ranging from 0.5 to 15 minutes, or ranging from 0.5 to 5 minutes. In another example, the dendrimer-targeting agent conjugate can be administered intraperitoneally. The route of administration can, for example, be targeted to a disease or disorder that the subject suffers from. For example, in some embodiments, the disease or disorder can be an intra-abdominal malignancy, such as a gynecological or gastrointestinal cancer, and the dendrimer-targeting agent conjugate can be administered intraperitoneally. In some embodiments, the dendrimer-targeting agent conjugates can be used to treat cancers of the peritoneal cavity, such as malignant epithelial tumors (e.g., ovarian cancer) or peritoneal carcinomatosis (e.g., gastrointestinal cancer, especially colorectal cancer, gastric cancer, gynecological cancers, and primary peritoneal neoplasms), and the dendrimer-targeting moiety conjugate is administered intraperitoneally.

[0361] In some embodiments, the amount of dendrimer-targeting agent conjugate administered is sufficient to deliver between 2 and 100 mg of active agent / m 2 , between 2 and 50 mg active agent / m2 , between 2 and 40 mg active agent / m 2 , between 2 and 30 mg active agent / m 2 , between 2 and 25 mg active agent / m 2 , between 2 and 20 mg active agent / m 2 , between 5 and 50 mg active agent / m 2 , between 10 and 40 mg active agent / m 2 , between 15 and 35 mg active agent / m 2 , between 10 and 20 mg / m 2 Between 20 and 30 mg / m 2 or between 25 and 35 mg active agent / m 2 A dose of 10 mg / kg of active agent in mice should be approximately equivalent to 30 mg / m 2 Human dose (FDA Guidance 2005). (To convert human mg / kg doses to mg / m 2 , you can multiply the number by 37, FDA Guidance 2005).

[0362] In some embodiments, a therapeutically effective amount of a dendrimer-targeting agent conjugate is administered to a subject in need thereof at a predetermined frequency. In some embodiments, a dendrimer-targeting agent conjugate is administered to a subject in need thereof according to a dosage regimen in which the dendrimer-targeting agent conjugate is administered once every one to four weeks. In some embodiments, a dendrimer-targeting agent conjugate is administered to a subject in need thereof according to a dosage regimen in which the dendrimer-targeting agent conjugate is administered once every three to four weeks.

[0363] The targeted conjugates disclosed herein provide therapeutic agents in a controlled manner. By controlling the release of the therapeutic agent from the conjugate, the level of circulating therapeutic agents (e.g., super cytotoxic agents) can also be controlled. For example, the maximum plasma concentration of the therapeutic agent can be much lower than the maximum plasma concentration produced by administering an equivalent amount of free therapeutic agent. In some embodiments, the administration of the conjugate results in 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% reduction in the maximum plasma concentration of the released therapeutic agent compared to the administration of an equivalent dose of free therapeutic agent.

[0364] Preparation of conjugates

[0365] Dendrimer-targeting agents can be prepared using any suitable synthetic route, such as those described in the Examples.

[0366] In some embodiments, the dendrimer-targeting agent conjugate is produced by a method comprising:

[0367] reacting the first intermediate with the second intermediate,

[0368] The first intermediate comprises a HER2 targeting agent, which is a peptide portion having a molecular weight of up to about 80 kDa and comprising an antigen binding site, the targeting agent is covalently attached to a spacer precursor group, and the spacer precursor group comprises a first reactive group

[0369] The second intermediate is a dendrimer, and the dendrimer comprises

[0370] i) Core Unit (C); and

[0371] ii) building blocks (BU), each building block being a lysine residue or an analog thereof;

[0372] wherein the core unit is covalently attached to at least two structural units via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a structural unit;

[0373] a therapeutic agent covalently attached to a surface moiety of the dendrimer; and

[0374] A hydrophilic polymer group covalently attached to the surface structural unit of the dendrimer, the second intermediate comprising a second reactive group

[0375] wherein the first reactive group and the second reactive group are complementary such that they are suitable for reacting with each other in order to covalently attach the HER2 targeting agent to the dendrimer.

[0376] The present disclosure also provides intermediates that can be used to synthesize dendrimer-targeting agent conjugates. Thus, intermediates for preparing dendrimer-targeting agent conjugates are also provided, the intermediates comprising a HER2 targeting agent, the HER2 targeting agent being a peptide portion having a molecular weight of up to about 80 kDa and comprising an antigen binding site, the targeting agent being covalently attached to a spacer precursor group, and the spacer precursor group comprising a reactive group suitable for reacting with a complementary reactive group present on the dendrimer intermediate.

[0377] In some embodiments, the targeting agent is covalently attached to the spacer precursor group via a non-natural amino acid residue. In some embodiments, the targeting agent is covalently attached to the spacer precursor group via a triazole moiety (e.g., generated by the reaction of an azide-containing non-natural amino acid such as azidophenylalanine with an alkyne-containing group such as DBCO [dibenzylcyclooctyne]).

[0378] In some embodiments, the spacer precursor group comprises one or more oligomer or polymer groups, such as PEG, PEOX or polyamino acid (e.g. polysarcosine) groups. For example, it can contain a PEG group, such as a PEG group of 2 to 100 -CH2CH2O- units.

[0379] In some embodiments, the reactive group present on the spacer precursor group is an olefin group, such as a reactive olefin suitable for reaction with a tetrazine-containing group. In some embodiments, the reactive group is a trans-cyclooctene group.

[0380] This disclosure also relates to the following numbered clauses:

[0381] 1. A dendrimer-targeting agent conjugate comprising:

[0382] a) a dendrimer, said dendrimer comprising

[0383] i) Core Unit (C); and

[0384] ii) building blocks (BU), each building block being a lysine residue or an analog thereof;

[0385] wherein the core unit is covalently attached to at least two structural units via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a structural unit;

[0386] 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 via a spacer group;

[0387] c) a therapeutic agent covalently attached to a surface moiety of the dendrimer; and

[0388] d) PEG or PEOX groups, which are covalently attached to the surface building blocks of the dendrimer.

[0389] 2. The conjugate according to clause 1, wherein the peptide portion is selected from: a heavy chain antibody, Fab, Fv, scFv or a single domain antibody.

[0390] 3. The conjugate according to clause 1 or clause 2, wherein the peptide portion comprises a heavy chain variable (V H ) domain or consist of it.

[0391] 4. The conjugate according to any one of clauses 1 to 3, wherein the peptide portion comprises a light chain variable (V L ) domain or consist of it.

[0392] 5. The conjugate according to any one of clauses 1 to 4, wherein the targeting agent has a molecular weight of about 5 kDa to about 30 kDa.

[0393] 6. The conjugate according to clause 5, wherein the targeting agent has a molecular weight of about 5 kDa to about 15 kDa.

[0394] 7. The conjugate according to clause 6, wherein the targeting agent has a molecular weight of about 10 kDa to about 16 kDa.

[0395] 8. The conjugate according to any one of clauses 1 to 7, wherein the targeting agent comprises less than 120 amino acid residues.

[0396] 9. The conjugate according to any one of clauses 1 to 8, wherein the targeting agent comprises or consists of any one of the amino acid sequences as defined herein.

[0397] 10. The conjugate according to any one of clauses 1 to 9, wherein the covalent bond between the targeting agent and the spacer group has been formed by reaction between complementary reactive functional groups present on a targeting agent precursor and a spacer group precursor.

[0398] 11. The conjugate according to clause 10, wherein the targeting agent precursor comprises a non-natural amino acid residue having a side chain comprising a reactive functional group.

[0399] 12. The conjugate according to clause 11, wherein the non-natural amino acid residue is a 4-azidophenylalanine residue.

[0400] 13. The conjugate according to any one of clauses 1 to 12, wherein the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting agent.

[0401] 14. The conjugate according to any one of clauses 10 to 13, wherein the spacer group precursor comprises a reactive functional group which is an alkyne group.

[0402] 15. The conjugate according to clause 14, wherein the alkyne group is a dibenzocyclooctyne group.

[0403] 16. The conjugate according to any one of clauses 1 to 15, wherein the therapeutic agent is a super cytotoxic agent.

[0404] 17. The conjugate according to clause 16, wherein the therapeutic agent is an auristatin or a maytansinoid.

[0405] 18. The conjugate according to clause 17, wherein the therapeutic agent is monomethyl auristatin E.

[0406] 19. The conjugate according to clause 17, wherein the therapeutic agent is monomethyl auristatin F.

[0407] 20. The conjugate according to any one of clauses 1 to 19, wherein the therapeutic agent is covalently attached to the surface building block of the dendrimer via a linker.

[0408] 21. The conjugate according to any one of clauses 1 to 20, wherein the therapeutic agent is covalently attached to the surface building block of the dendrimer via a cleavable linker.

[0409] 22. The conjugate according to clause 21, wherein the cleavable linker comprises a Val-Cit-PAB group.

[0410] 23. The conjugate according to any one of clauses 1 to 22, wherein the conjugate comprises a PEG group covalently linked to a surface building block of the dendrimer.

[0411] 24. The conjugate according to clause 23, wherein the PEG group has an average molecular weight in the range of about 500 to about 2500 g / mol.

[0412] 25. The conjugate according to any one of clauses 1 to 24, wherein the spacer group comprises a PEG group.

[0413] 26. The conjugate according to any one of clauses 1 to 25, wherein the core unit comprises the following structure:

[0414]

[0415] 27. The conjugate according to any one of clauses 1 to 26, wherein the dendrimer has structural units of one to five generations.

[0416] 28. The conjugate according to clause 27, wherein the dendrimer has three generations of structural units.

[0417] 29. The conjugate according to any one of clauses 1 to 28, wherein each of the structural units is:

[0418]

[0419] 30. The conjugate according to any one of clauses 1 to 29, for administration in combination with an additional active agent.

[0420] 31. The conjugate according to any one of clauses 1 to 30, wherein the conjugate is internalized in a HER2 expressing cell.

[0421] 32. The conjugate according to 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 free therapeutic agent.

[0422] 33. The conjugate according to any one of clauses 1 to 32, wherein administration of the conjugate results in at least a 50% reduction in maximum plasma concentration of the released therapeutic agent compared to administration of an equivalent dose of free therapeutic agent.

[0423] 34. A composition comprising a plurality of conjugates as defined in any one of clauses 1 to 33.

[0424] 35. A pharmaceutical composition comprising:

[0425] i) a conjugate according to any one of clauses 1 to 34; and

[0426] ii) a pharmaceutically acceptable excipient.

[0427] 36. A composition according to clause 34 or 35, wherein the composition is formulated for parenteral delivery.

[0428] 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 treating cancer.

[0429] 38. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate according to any one of clauses 1 to 33 or a composition according to any one of clauses 34 to 37.

[0430] 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 treating cancer.

[0431] 40. The method, use, conjugate or composition for 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 characterised by aberrant expression of the ERBB2 gene.

[0432] 41. A method for killing HER2-expressing cells, comprising:

[0433] The conjugate according to any one of clauses 1 to 33 is contacted with a HER2-expressing cell such that the conjugate is internalized within the cell, whereby the therapeutic agent kills the HER2-expressing cell.

[0434] The present disclosure is further described by the following examples. It should be understood that the following description is only for the purpose of describing specific embodiments and is not intended to be limiting of the above description.

[0435] Example

[0436] The following nomenclature is used herein with respect to dendrimer conjugate synthesis:

[0437]

[0438]

[0439]

[0440]

[0441] HPLC MS (mass spectrometry) and NMR equipment details:

[0442] HPLC–Waters 2795 with 2996 diode array detector (DAD)

[0443] MS—Waters ZQ4000 with ESI probe, inlet flow split to produce approximately 50 μL / min for MS.

[0444] Mass spectrometry data were acquired in positive or negative electrospray ionization mode as indicated. Raw data were deconvoluted using the maximum entropy method (MaxEnt) as implemented in MassLynx software v4.0 provided by Waters Corporation. The data reported in the experimental details correspond to the observed values ​​after deconvolution to the theoretical zero charge state.

[0445] NMR-300MHz Bruker.

[0446] The preparation of carboxyl-reactive dendrimer scaffolds has been previously described, inter alia, in WO2007 / 082331, WO2008 / 017125, WO2012 / 167309 and WO2015 / 184510. Those skilled in the art can use or adapt these methods to prepare the various dendrimers outlined herein.

[0447] In the following examples, [Lys] in the formula refers to a lysine structural unit on the surface layer of a dendrimer.

[0448] Example 1

[0449] General Procedure A. Boc Deprotection

[0450] To an ice-cooled stirred suspension of the Boc compound (1.0 eq.) in water was added TFA (40-200 eq. / Boc group). After 5 minutes, the ice bath was removed and the reaction mixture was stirred overnight at room temperature. The volatiles were removed in vacuo, and the remaining aqueous solution was further diluted with water and lyophilized to produce the deprotected product in quantitative yield.

[0451] General Procedure B. Addition of a lysine layer on the dendrimer surface.

[0452] To a stirred solution of TFA dendrimer (1.0 eq.) in DMF was added TEA (6.0 eq. / NH2) followed by DBL-oPNP (2.0 eq. / NH2) under nitrogen atmosphere. The reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo and the resulting crude material was purified using standard methods.

[0453] General Procedure C. Use of HO-Lys-(α-NHBoc)(ε-NHPEG 1100 ) wedges for PEGylation of the dendrimer surface.

[0454] To a stirred solution of TFA dendrimer (1.0 eq.) in DMF under nitrogen atmosphere were added PyBOP (2.0 eq. / NH2) and DIPEA (8.0 eq. / NH2). After 10 min, HO-Lys-(α-NHBoc)(ε-NHPEG 1100 A solution of ) (1.35 equiv. / NH2) in DMF was added and the subsequent reaction mixture was stirred at room temperature overnight. The volatiles were removed in vacuo and the resulting crude material was purified using standard methods.

[0455] General Procedure D. Capping of dendrimer surface with HO-Lys-(α-NHBoc)(ε-NHFmoc) wedges followed by Fmoc deprotection

[0456] Step 1: To a stirred solution of HO-Lys-(α-NHBoc)(ε-NHFmoc) (1.5 eq / NH) and NMM (2.5 eq / NH) in DMF was added PyBOP (1.4 eq / NH). The subsequent reaction mixture was stirred at room temperature for 15 min, then a solution of TFA-dendrimer (1.0 eq) and NMM (2.5 eq / NH) in DMF was added. The subsequent reaction mixture was stirred at room temperature for 1 hour, then slowly added to ice-cold MeCN and stirred for 15 min. The resulting solid was collected by filtration and washed with MeCN (3x), then lyophilized.

[0457] Step 2: To a solution of Fmoc / Boc dendrimer (1.0 eq.) in DMF was added piperidine (21 eq. / Fmoc). The solution was stirred at room temperature for 90 min and then slowly added to ice-cold Et2O. After 15 min, the precipitated solid was collected by filtration, washed with Et2O, dissolved in H2O and lyophilized.

[0458] General Procedure E. Capping of the Dendrimer Surface with DGA-Doxorubicin (Dox) or DGA-Nemorubicin (Nemo)

[0459] DGA-3'-NH-doxorubicin

[0460] Step 1: A solution of DMAP (2.5 eq.) in DMF was added to a stirred solution of Dox.HCl (1.0 eq.) in DMF at room temperature. After 5 minutes, a solution of DGA (0.9 eq.) in DMF was added. The reaction was complete within 3 hours.

[0461] Step 2: PyBOP (2.0 equiv / dendrimer NH2) was added to the above reaction mixture followed by TFA-dendrimer (azido-PEG 24 -CO[N(PN)2[Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )]4 or azido-PEG 24 -CO[N(PN)2[Lys]8[(α-NH2.TFA)(ε-NHPEG 1100 )]8) (1.0 eq.) and DIPEA (6.0 eq. / dendrimer NH2) in DMF. The reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo and the resulting crude material was purified by SEC.

[0462] DGA-14-O-nemorubicin

[0463] Step 1: A solution of DMAP (2.5 eq.) in DMF was added to a stirred solution of nemorubicin (1.0 eq.) in DMF at room temperature. After 5 minutes, a solution of DGA (0.9 eq.) in DMF was added. The reaction was complete within 3 hours.

[0464] Step 2: PyBOP (2.0 equiv / dendrimer NH2) was added to the above reaction mixture followed by TFA-dendrimer (azido-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 eq.) and DIPEA (6.0 eq. / dendrimer NH2) in DMF. The reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo and the resulting crude material was purified by SEC.

[0465] General Procedure F. Capping of Dendrimer Surfaces with Glu-vc-PAB-MMAE or DGA-MMAF(OMe)

[0466] At room temperature, azido-PEG 24 -CO[N(PN)2[Lys]8[(α-NH2.TFA)(ε-NHPEG 570 / 1100 / 2000 )]8 (1.0 eq.) was dissolved in a mixture of DMF and NMM (5.0 eq. / NH2). The solution was added to HO-Glu-vc-PAB-MMAE or DGA-MMAF(OMe) (1.2 eq. / NH2) and PyBOP (2.0 eq. / NH2) and allowed to stand at room temperature. The resulting crude material was purified by SEC.

[0467] General Procedure G. Conjugation of Affibody to Dox / Pt(IV)-Acetate / MMAE / MMAF Dendrimers

[0468] Step 1: A solution of affibody protein (1.0 mg / mL PBS) was treated with TCEP (50 mM, 39.0 eq.) and the reaction mixture was shaken at 650 rpm for 2 hours at room temperature. The resulting solution was purified by SEC.

[0469] Step 2: The collected permeate was treated with a solution of maleimide-bicyclo[6.1.0]nonyne (Mal-BCN) (20.0 equiv.) in DMSO. The subsequent reaction mixture was shaken at 650 rpm for 2 hours at room temperature. The resulting solution was purified by SEC.

[0470] Step 3: Add the Affibody-BCN solution to

[0471] -PEG 24 -CO[N(PN)2[Lys]4[(α-DGA-3′-NH-Dox)(ε-NHPEG 1100 )]4 in PBS (858 μM) or azido-PEG 24 -CO[N(PN)2[Lys]8[(α-DGA-3′-NH-Dox)(ε-NHPEG 1100)]8 (580 μM in PBS) or azido-PEG 24 -CO[N(PN)2][Lys]8[(α-Pt(IV)-acetate)(ε-NHPEG 1100 )]8 (903 μM in PBS) or azido-PEG 24 -CO[N(PN)2[Lys]8[(α-Glu-vc-PAB-MMAE)(ε-NHPEG 570 / 1100 / 2000 )]8 (240 μM in PBS) or azido-PEG 24 -CO[N(PN)2[Lys]8[(α-DGA-MMAF(OMe))(ε-NHPEG 570 / 1100 / 2000 )]8 (365 μM in PBS) (1.3 eq. Affibody-BCN / dendrimer). The subsequent reaction mixture was shaken at 650 rpm overnight at room temperature and then treated with a 9.38 mM (30% EtOH / water) solution of DBCO agarose (5.0 eq. / dendrimer). The subsequent suspension was shaken at 1200 rpm overnight at room temperature. The suspension was purified using SEC.

[0472] General Procedure H. Conjugation of Nanobodies to MMAE Dendrimers

[0473] Step 1: Prepare the linker (BCN-PEG2NH-Glu-NHPEG) by dissolving the linker (1 mg) in 20:80 (DMSO / 10 mM PBS, 1 mL). 24 CO-NHPEG3-TCO or DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO).

[0474] Step 2: Add the TCO-linker solution (1 eq) to a solution of tetrazine functionalized dendrimer (e.g. Tz-MMAE-dendrimer or BHA-Tz-MMAE-dendrimer) (1.0 eq, 8 mg / mL) in PBS (1). Allow the reaction mixture to stand at room temperature for 30 min. Completion of the reaction is indicated by the disappearance of the pink tetrazine color. The reaction is monitored by HPLC.

[0475] Step 3: Once the reaction is complete, the contents are diluted with PBS (to a final volume of 0.5 mL). A portion of the BCN / DBCO-MMAE-dendrimer (1.0 eq) is added to a solution of Nanobody-azide (1.0 eq, 9.2 mg / ml) in Tris buffer (20 mM, 1 mL). The resulting solution is allowed to stand at RT for 7 h and then at 4°C overnight. Purification of the Nanobody-dendrimer constructs is performed by anion exchange chromatography followed by SEC.

[0476] Example 1a Synthesis of intermediate

[0477] 1.1 Azide-PEG 24 -CO[N(PNBoc)2]Compound 1

[0478] Azido-PEG 24 To a stirred solution of 1-acid (2.00 g, 1.71 mmol) and PyBOP (1.33 g, 2.56 mmol) in DMF (20 mL) was added NMM (563 μL, 5.12 mmol). After 10 min, a solution of N(PNBoc)2 (622 mg, 1.88 mmol) in DMF (5 mL) was added and the reaction mixture was then stirred 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, R t 47-50 min) to give a light yellow oily solid (1.37 g, 54%). 1 H-NMR (300MHz, CD3OD) δ (ppm): 1.44 (m, 18H); 1.65-1.84 (m, 4H); 2.63 (t, J 6.3Hz, 2H); 3.05 (dt, J 6.9 and 14.7Hz, 4H); 3.36-3.41 (m, 6H); 3.60-3.78 (m, 98H). LCMS (philic method, formic acid buffer) R t =9.32min. ESIMS(+ve)1486.3[M] + ; C 67 H 132 N6O 29 [M] + Calculated for m / z: 1486.8.

[0479] 1.2 Azido-PEG 24 -CO[N(PNH2.TFA)2], compound 2

[0480] Azido-PEG was used according to the general procedure A. 24-CO[N(PNBoc)2] (1.37 g, 922 μmol) was prepared. The lyophilized product was obtained as a light yellow oil (1.67 g, 119%). 1 H-NMR (300MHz, D2O) δ (ppm): 1.88-2.06 (m, 4H); 2.74 (t, J 6.0Hz, 2H); 2.96 (t, J 7.2Hz, 2H); 3.04 (apparent t, J 7.5Hz, 2H); 3.42-3.52 (m, 6H);

[0481] 3.67-3.95 (m, 93H). LCMS (affinity method, TFA buffer) R t =8.47min,ESIMS(+ve)1286.0[M] + ; C 57 H 116 N6O 25 [M] + Calculated for m / z = 1286.6.

[0482] 1.3 Azide-PEG 24 -CO[N(PN)2][Lys]2[Boc]4, G1, compound 3

[0483] Azido-PEG was used according to the general procedure B. 24 -CO[N(PNH2.TFA)2] (186 mg, 145 μmol) was prepared. The crude material was dissolved in MeCN and purified by preparative HPLC (30%-80% MeCN, R t 33.5-36 min) to give a light yellow oil (224 mg, 80%). 1 H-NMR (300MHz, CD3OD) δ (ppm): 1.22-1.87 (m, 56H); 2.64 (t, J 6.0Hz, 2H); 3.03 (t, J 6.6Hz, 4H); 3.13-3.23 (m, 4H), 3.36-3.45 (m, 6H); 3.60-3.69 (m, 100H); 3.77 (t, J 6.0Hz, 2H); 3.85-3.88 (m, 1H); 3.92-4.02 (m, 2H). LCMS (phobic method, formic acid buffer) R t =6.74min;ESIMS(+ve)1942.4,[M] + ; C 89 H 172 N 10 O 35 + [M+H] +Calculated for m / z = 1942.4.

[0484] 1.4 Azide-PEG 24 -CO[N(PN)2][Lys]2[NH2.TFA]4, G1, compound 4

[0485] Azido-PEG was used according to the general procedure A. 24 -CO[N(PN)2[Lys]2[Boc]4 (220 mg, 113 μmol) was prepared. The lyophilized product was obtained as a light yellow oil (251 mg, 111%). LCMS (affinity method, formic acid buffer) R t =6.49min,ESIMS(+ve)1542.1[M] + ; C 69 H 140 N 10 O 27 [M] + Calculated for m / z = 1541.90.

[0486] 1.5 Azido-PEG 24 -CO[N(PN)2][Lys]4[(α-Boc)(ε-NHPEG 1100 )]4, G2, compound 5

[0487] Azido-PEG was used according to the general procedure C. 24 -CO[N(PN)2[Lys]2[NH2.TFA]4 (120 mg, 60.1 μmol) was prepared. The crude material was dissolved in MeCN / H2O (1:1) and purified by preparative HPLC (20%-70% MeCN, R t 31-32.5 min) to give the product as a light 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 (affinity method, formic acid buffer) R t =8.70min;ESIMS(+ve)1714.0[M+4H] 4+ / 4,1371.7[M+5H] 5+ / 5;1143.0

[0488] [M+6H] 6+ / 6,980.0[M+7H] 7+ / 7. Converts to 6852.

[0489] 1.6 Azido-PEG 24 -CO[N(PN)2][Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )]4, G2, compound 6

[0490] Azido-PEG was used according to the general procedure A. 24 -CO[N(PN)2[Lys]4[(α-Boc)(ε-NHPEG 1100 )]4 (244 mg, 35.6 μmol) was prepared. The crude lyophilized material 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 light 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 (broad t, J 6.0Hz, 2H); 3.16-3.30 (m, 16H); 3.40 (s, 12H); 3.46-3.53 (m, 4H); 3.62-3.97 (m, 490H); 4.03 (t, J 6.6Hz, 2H); 4.24-4.29 (m, 2H). LCMS (affinity method, TFA buffer) R t =9.85min;ESIMS(+ve)1614.1[M+4H] 4+ / 4,1291.6[M+5H] 5+ / 5; 1076.5[M+6H] 6+ / 6. Converts to 6452.

[0491] 1.7 Azido-PEG 24 -CO[N(PN)2][Lys]4[Boc]8, G2, compound 7

[0492] Azido-PEG was used according to the general procedure B. 24 -CO[N(PN)2][Lys]2[NH2.TFA]4 (117 mg, 58.6 μmol) was prepared. The crude material was obtained as a light yellow oil (167 mg, 100%). LCMS (hydrophobicity method 4.1a, formic acid buffer) R t =8.35min;ESIMS(+ve)1328.6[M+2H] 2+ / 2–Boc;C 133 H 252 N 18 O 47 [M] + Calculated for m / z = 2855.6.

[0493] 1.8 Azido-PEG 24 -CO[N(PN)2][Lys]4[NH2.TFA]8, G2, compound 8

[0494] Azido-PEG was used according to the general procedure A. 24 -CO[N(PN)2][Lys]4[Boc]4 (167 mg, 58.6 μmol) was prepared. The crude aqueous solution was purified by preparative HPLC (10%-60% MeCN, 0.1% TFA buffer; R t 27-29 min) to give the product as a very pale yellow sticky solid (124 mg, 71% over 2 steps). 1 H-NMR (300MHz, D2O) δ (ppm): 1.30-1.96 (m, 42H); 2.71 (t, J 6.0Hz, 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, J 6.6Hz, 2H); 4.04 (t, J 6.6Hz, 2H); 4.25 (t, J 7.2Hz, 2H). LCMS (affinity method, TFA buffer) R t =7.76min, ESIMS(+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 31 3+ [M+3H] 3+ Calculated m / z for / 3: 685.9.

[0495] 1.9 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG1100)]8, G3, Compound 9

[0496] Azido-PEG was used according to the general procedure C. 24 -CO[N(PN)2][Lys]4[NH2.TFA]8 (123 mg, 41.5 μmol) was prepared to give a crude substance as a brown oil. LCMS (affinity method, formic acid buffer) R t =11.52min,ESIMS(+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.

[0497] 1.10 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-TFA)(ε-NHPEG 1100 )]8, G3, compound 10

[0498] Azido-PEG was used according to the general procedure A. 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 1100 )]8 (526 mg, 41.5 μmol) was prepared. The crude aqueous solution was purified by preparative HPLC (3%-60% MeCN, 0.1% TFA buffer; R t 38-39 min) to give the product as a light yellow sticky solid (359 mg, 68% over 2 steps). LCMS (affinity method, TFA buffer) R t =10.27min. Converted to 11 880.

[0499] 1.11 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-Fmoc)]8, G3, compound 11

[0500] Azido-PEG was used according to the general procedure D. 24 -CO[N(PN)2][Lys]4[NH2.TFA]8 (105 mg, 35.4 μmol) was prepared. 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).

[0501] 1.12 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NH2)]8, G3, compound 12

[0502] Azido-PEG was used according to the general procedure D. 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-Fmoc)]8 (169 mg, 29.9 μmol) was prepared 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); 3.0 9-3.19(m,18H);3.38-3.41(m,8H);3.55-3.78(m,96H),3.89-4.33(m,14H).

[0503] 1.13 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 570 )]8, G3, compound 13 and azido-PEG 24 -CO[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NHPEG 570 )]8, G3, compound 14

[0504] mPEG 570 To a solution of -CO2H (205 mg, 348 μmol), NMM (60 μL, 546 μmol) and PyBOP (171 mg, 329 μmol) in DMF (1.5 mL) was added azido-PEG in DMF (0.5 mL). 24-CO[N(PN)2[Lys]8[(α-Boc)(ε-NH2)]8. The reaction mixture was then stirred at room temperature overnight, then concentrated in vacuo, dissolved in water, treated with TFA and stirred at room temperature overnight. The mixture was concentrated and dissolved in water, then purified using a Millipore centrifugal filtration unit (3K MWCO regenerated cellulose) and freeze-dried to give the product as an off-white fluffy mass (68% over 2 steps). 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 (affinity method, formic acid buffer) R t =7.50min.

[0505] 1.14 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 2000 )]8, G3, compound 15

[0506] Azido-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 the addition of mPEG 2000 -NHS (720 mg, 313 μmol). The reaction mixture was then stirred at room temperature overnight. The crude residue was dissolved in water and purified by ultrafiltration (5K, Pall PES membrane). The retentate was collected and freeze-dried to produce 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 (affinity method, formic acid buffer) R t =11.24min.

[0507] 1.15 Azido-PEG 24 -CO[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NHPEG 2000 )]8, G3, compound 16

[0508] Azido-PEG was used according to the general procedure A. 24 -CO[N(PN)2][Lys]8[(α-Boc)(ε-NHPEG 2000 )]8 (40.0 mg, 1.87 μmol) was prepared to give the product as an off-white fluffy substance (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 (affinity method, formic acid buffer) R t =9.12min.

[0509] 1.16(MeTzPh)-PEG 24 -CO[N(PNBoc)2]Compound 46

[0510] Under N2 atmosphere, (MeTzPh)-PEG 24 To a stirred solution of -CO2H (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)2 (0.147 g, 0.444 mmol). The reaction mixture was then stirred at room temperature overnight. Volatiles were removed in vacuo, and the resulting oil was purified on silica gel chromatography (5% to 10% MeOH / DCM) to yield the desired product compound 106 (0.474 g, 95%) as a red residue. 1 HNMR (300MHz, CD3OD) δ (ppm): 1.43-1.44 (m, 18H); 1.64-1.80 (m, 4H); 2.63 (t, J 6.0Hz, 2H); 3.00 (s, 3H); 3.02-3.09 (m, 4H); 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 = 6.20min. ESIMS (+ve) 1631.0 [M+H] +; C 76 H 140 N7O 30 [M+H] + Calculated for m / z: 1631.0.

[0511] 1.17(MeTzPh)-PEG 24 -CO[N(PNH2.HCl)2]Compound 47

[0512] To (MeTzPh)-PEG in an ice / water bath 24 -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 subsequent reaction mixture was stirred at room temperature overnight. The volatiles were removed in vacuo to give the product Compound 107 (0.388 g, 100%) as a red residue. 1 HNMR (300MHz, CD3OD) δ (ppm): 1.91-2.07 (m, 4H); 2.68 (t, J 6.0Hz, 2H); 2.94-3.09 (m, 7H); 3.53-3.80 (m, 108H); 3.88-3.91 (m, 2H); 4.25-4.28 (m, 2H); 7.16-7.20 (m, 2H); 8.47-8.51 (m, 2H). LCMS (affinity method, formic acid buffer) RT = 8.12min. ESIMS (+ve) 1430.9 [M+H] + ;

[0513] C 66 H 124 N7O 26 [M+H] + Calculated m / z: 1430.8.

[0514] 1.18(MeTzPh)-PEG4CO-NH-PEG 24 -CO[N(PNH2.HCl)2]Compound 48

[0515] H2N-PEG 24-CO[N(PNBoc) ] (0.418 g, 0.286 mmol) was added (MeTzPh)-PEG-CO H (0.15 g, 0.344 mmol), PyBOP (0.178 g, 0.342 mmol) and NMM (80 μ L, 0.727 mmol) in a stirred solution in DMF (2.0 mL). The reaction mixture was then stirred at room temperature overnight. Volatiles were removed in vacuo, and the resulting oil was cooled in an ice / water bath, then 1.25 M HCl / MeOH solution (10.0 mL, 12.5 mmol) was slowly added. After 5 min, the ice bath was removed and the reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo and the resulting oil was dissolved in MeCN / H2O (8 mL, 1:1) and purified by preparative HPLC (10%-50% MeCN, 0.1% formic acid buffer, RT 35 min) to give red solid compound 108 (1.37 g, 54%). 1 HNMR (300MHz, CD3OD) δ (ppm): 1.91-2.04 (m, 4H); 2.43 (t, J 6.0Hz, 2H); 2.68 (t, J 6.0Hz, 2H); 2.94-3.07 (m, 4H); 3.00 (s, 3H); 3.35 (t, J 6.0Hz, 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 (affinity method, formic acid buffer) RT = 8.15min. ESIMS (+ve) 1677.9 [M+H] + ; C 77 H 145 N8O 31 [M+H] + Calculated for m / z: 1678.0.

[0516] 1.19(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NHBoc]4G1 Compound 49

[0517] Under N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24-CO[N(PNH2.HCl)2] Compound 48 (0.195 g, 0.111 mmol) and DBL-oPNP (0.146 g, 0.312 mmol) were stirred in DMF (3.0 mL) and NMM (125 μL, 0.864 mmol) was added. The reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo, and the resulting oily residue was purified by silica gel column chromatography (5%-10%-15% MeOH / DCM) to give the desired product Compound 109 (0.186 g, 72%) as a red oil. 1 HNMR (300MHz, CD3OD) δ (ppm): 1.28-1.87 (m, 56H); 2.43 (t, J6.0Hz, 2H); 2.63 (t, J 6.0Hz,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; ESIMS (+ve) 2335.3, [M+H] + ; C 109 H 201 N 12 O41+[M+H] + Calculated for m / z = 2335.4.

[0518] 1.20(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NH2.HCl]4G1 Compound 50

[0519] To ice-cooled (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NHBoc]4 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 reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo to produce the product Compound 110 (0.208 g, 108%) as a red oil. %. 1HNMR (300MHz, CD3OD) δ (ppm): 1.51-1.99 (m, 20H); 2.47 (t, J 6.0Hz, 2H); 2.67 (t, J 6.0Hz, 4H); 2.90-3.04 (m, 7H); 3.35-3.41 (m, 7H); 3.51-3.78 (m, 106H); 4.25-4.28 (m, 2H); 7.17-7.20 (m, 2H); 8.47-8.51 (m, 2H); LCMS (affinity method, formic acid buffer) RT = 7.28min, ESIMS (+ve) 1934.9 [M+H] + ; C 89 H 169 N 12 O 33 [M] + Calculated for m / z = 1935.2.

[0520] 1.21(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]4[NHBoc]8G2 Compound 51

[0521] Under N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2[Lys]2[NH2.HCl]4Compound 50 (0.192 g, 0.0822 mmol) and DBL-oPNP (reference 1) (0.215 g, 0.460 mmol) were added to a stirred solution in DMF (3.0 mL) with NMM (215 μL, 0.1.96 mmol). The subsequent reaction mixture was then stirred at room temperature overnight. Volatiles were removed in vacuo, and the resulting oily residue was purified on silica gel chromatography (5%-10%-15% MeOH / DCM) to give the desired product compound 51 (0.231 g, 87%) as a red oil. 1HNMR (300MHz, CD3OD) δ (ppm): 1.28-1.87 (m, 140H); 2.44 (t, J 6.0Hz, 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 = 8.15min; ESIMS (+ve) [M+2] + =1624.5;[(M-3Boc)+3]=1016.6;C 153 H 281 N 20 O 53 + [M+H] + Calculated for m / z = 3247.99.

[0522] 1.22(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]4[NH2.HCl]8G2 Compound 52

[0523] To ice-cooled (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]4[NHBoc]8Compound 51 (0.231 g, 0.0711 mmol) was slowly added a solution of 1.25M HCl / MeOH (9.0 mL, 11.3 mmol). After 5 min, the ice bath was removed and the subsequent reaction mixture was stirred at room temperature overnight. The volatiles were removed in vacuo to give the product Compound 51 (0.226 g, 100%) as a red oil. 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 (affinity method, formic acid buffer) RT = 6.38min, ESIMS (+ve) 2447.6 [M+H] + ; C113 H 217 N 20 O 37 [M+H] + Calculated for m / z = 2447.6.

[0524] 1.23(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]8[(α-NH2.HCl)(ε-NH-COPEG 1100 )]8G3 Compound 53

[0525] HO-Lys(α-NHBoc)(ε-NH-COPEG 1100 To a stirred solution of (MeTzPh)-PEG4CO-NHPEG in DMF (1 mL) was added NMM (225 μL, 2.96 mmol). After 10 min, this solution was added to a solution of (MeTzPh)-PEG4CO-NHPEG in DMF (1 mL). 24 -CO[N(PN)2][Lys]4[NH2.HCl]8Compound 52 (0.109 g, 0.0398 mmol). The reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo, and the resulting crude was cooled in an ice / water bath. 1.25 M (8.0 mL, 10 mmol) was slowly added to the cooled residue. After 10 min, the cold bath was removed, and the reactant was stirred at room temperature overnight. The volatiles were removed in vacuo and then dissolved in H2O (16 mL). The solution was purified by centrifugation using a Millipore Amicon Ultra-15 centrifugal filter unit (4 units x 10 kDa MWCO units). The retentate was freeze-dried overnight to produce 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 (affinity method, TFA buffer)RT=10.28min.

[0526] *[The cell was pre-rinsed with H2O (5 mL) and spun at 4000 rpm for 5 min. This process was repeated. The crude solution was filtered through a 0.45 μm syringe filter and placed in a centrifuge cell. The cell was then spun at 4000 rpm for 15 min. The retentate was then diluted with H2O (4 mL) and spun at 4000 rpm for another 15 min. This process was repeated 8 times.]

[0527] 1.24(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]8[NH2.HCl] 16 G3 Compound 54

[0528] Under N2 atmosphere, (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys]4[NH2.HCl]8Compound 52 (0.098g, 0.0358mmol) and DBL-oPNP (0.207g, 0.443mmol) were added to a stirred solution in DMF (2.0mL) with NMM (190 μL, 1.973mmol). The reaction mixture was then stirred at room temperature overnight. Volatiles were removed in vacuo, and the resulting oily residue was purified on silica gel chromatography (5%-10%-15% MeOH / DCM) to produce a Boc protected product (0.128g, 70%) in a red oil. The purified material was cooled in an ice / water bath, and a solution of 1.25M HCl / MeOH (6.5mL, 8.5mmol) was then slowly added. After 5min, the ice bath was removed and the reaction mixture was then stirred at room temperature overnight. Volatiles were removed in vacuo to produce a product compound 54 (0.107g, 100%) in a red oil. LCMS (affinity method, TFA buffer) RT = 7.48 min, ESIMS (+ve) 3471 [M+H] + ; Calculate m / z [M+H] + =3472.

[0529] 1.25(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 16 [(α-NH2.HCl)(ε-NH-COPEG 1100 )] 16 G4 Compound 55

[0530] HO-Lys(α-NHBoc)(ε-NH-COPEG 1100To 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, this solution was added to the (MeTzPh)-PEG4-PEG 24 -CO[N(PN)2][Lys]8[NH2.HCl] 16 Compound 54 (0.0221g, 0.00545mmol). The reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo, and the resulting crude product was cooled in an ice / water bath. 1.25M HCl / MeOH (1.5mL, 1.88mmol) was slowly added to the cooled residue. After 10min, the cold bath was removed, and the reactant was stirred at room temperature overnight. The volatiles were removed in vacuo and then dissolved in H2O (5mL). The solution was purified by centrifugation using Millipore Amicon Ultra-15 centrifugal filter units (10kDaMWCO). The retentate was freeze-dried overnight, and further purified on Gilson [single gradient 60min, 10>60% ACN (0.1% TFA buffer, RT 36min] to produce the product compound 55 (0.021g, 25%) as a yellow 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 (m 60H); 3.34-3.40 (m, 63H); 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 (affinity method, TFA buffer) RT = 8.96min. ESIMS (+ve) 2095 [M + 7H] + ; 1834[M+8H] + ; 1630[M+9H] + .

[0531] 1.26(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 16 [NH2.TFA] 32 G4 Compound 56

[0532] Prepared according to compound 54 and deprotected using TFA / AcOH, compound 56 (0.078 g, 78%) was obtained as a red solid. LCMS (affinity method, TFA buffer) RT = 7.74 min. ESIMS (+ve) 2095 [M + 7H] + ; 1834[M+8H] + ; 1630[M+9H] + .

[0533] 1.27(MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 32 [(α-NH2.TFA)(ε-NH-COPEG 1100 )] 32 G5 Compound 57

[0534] 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, this solution was added to the (MeTzPh)-PEG4CO-NHPEG 24 -CO[N(PN)2][Lys] 16 [NH2.TFA] 32 Compound 56 (0.078 g, 0.00850 mmol). The reaction mixture was then stirred at room temperature overnight. The volatiles were removed in vacuo, and the resulting crude product was dissolved in H2O (1.0 mL) and TFA (1.0 mL) was slowly added. The reactant was stirred at room temperature overnight. The reactant was diluted with H2O (12 mL), and purified by centrifugation using a Millipore Amicon Ultra-15 centrifugal filter unit (10 kDa MWCO). The retentate was freeze-dried overnight to produce the product 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) 3.48-3.98 (m, 2960H); 4.27-4.39 (m, 32H); 7.30-7.33 (m, 2H); 8.46-8.49 (m, 2H); HPLC analysis (affinity method, ammonium formate as buffer) RT = 8.60 min. 1.28

[0536] (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NHCy5)1(α-NHAc)7)(ε-NH-COPEG 1100 )8], G3, compound 37

[0537] 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 a solution containing (MeTzPh)PEG4CO-NHPEG in DMF (0.5 mL). 24 CO-[N(PN)2][Lys]8[((α-NH2.HCl)8)(ε-NH-COPEG 1100 )8], a vial of compound 53 (20 mg, 1.59 μmol). To this solution was added NMM (10 μL, 91.0 μmol), and the reaction mixture was then protected from light and stirred at RT. After 3.5 h, acetic anhydride (20 μL, 212 μmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure, then dissolved in MQ water (5 mL) and divided into two portions for purification by two (pre-equilibrated) PD10 columns. Once the sample entered the column bed, the sample was eluted with 3.5 mL MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-dried to produce the title product (19.3 mg (93%)) as a bright blue powder. HPLC (C8 1.29

[0539] (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 16 [((α-NHCy5)1(α-NHAc) 15 )(ε-NH-COPEG 1100 ) 16 ], G4, compound 38

[0540] A solution of Cy5-NHS ester (520 μL of a 1 mg / mL solution in DMF; 0.52 mg, 844 nmol) was added to a solution containing (MeTzPh)PEG4CO-NHPEG in DMF (1.0 mL). 24 CO-[N(PN)2][Lys] 16 [((α-NH2.HCl) 16 )(ε-NH-COPEG 1100 ) 16 ], a vial of compound 55 (20 mg, 844 nmol). NMM (10 μL, 94.5 μmol) was added to the solution, and the reaction mixture was then protected from light and stirred at RT. After 3.5 h, acetic anhydride (20 μL, 230 μmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure, then dissolved in MQ water (5 mL) and divided into two portions for purification by two (pre-equilibrium) PD10 columns. Once the sample enters the column bed, the sample is eluted with 3.5 mL MQ water, and the filtrate is collected. The filtrates are combined and freeze-dried overnight. Lyophilization produces the title product (19.9 mg (98%)) as a bright blue powder. HPLC (C8 1.30

[0542] (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys] 32 [((α-NHCy5)1(α-NHAc) 31 )(ε-NH-COPEG 1100 ) 32 ], G5, compound 39

[0543] A solution of Cy5-NHS ester (300 μL of a 1 mg / mL solution in DMF; 0.30 mg, 487 nmol) was added to a solution containing (MeTzPh)PEG4CO-NHPEG in DMF (1.2 mL). 24 CO-[N(PN)2][Lys] 32 [(α-NH2.HCl) 32 (ε-NH-COPEG 1100 ) 32], a vial of compound 57 (20 mg, 435 nmol). NMM (11 μL, 97.5 μmol) was added to the solution, and the 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 stirred overnight. The reaction mixture was concentrated under reduced pressure, then dissolved in MQ water (5 mL) and divided into two portions for purification by two (pre-equilibrated) PD10 columns. Once the sample enters the column bed, the sample is eluted with 3.5 mL MQ water, and the filtrate is collected. The filtrates are combined and freeze-dried overnight. Freeze-dried to produce the title product (18.7 mg (91%)) as a bright blue powder. HPLC (C8

[0544] 1.31 Cabazitaxel (CTX)-2'-OCOO-oPNP ester, compound 58

[0545] 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 obtained residue was purified using silica gel column chromatography (using a gradient of 0:100 to 50:50 (ethyl acetate:hexane)) to produce the title compound (28 mg, 21%) as a white solid. LCMS (affinity method, TFA buffer) Rt = 6.55 min. ESIMS (+ve) 1001 [M] +; C 52 H 60 N2O 18 Calculated m / z for [M]+: 1001

[0546] 1.32Fmoc-Val-Ala-PAB-P-trigger-(NMeBoc), compound 59

[0547] The compound can be obtained as described in Dal Corso et al. Angew.Chem Int.Ed.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 dissolved in a round-bottom flask, followed by addition of THF (5 mL). The reaction mixture was stirred at room temperature for 2 h under an inert atmosphere. LCMS analysis of the reaction mixture showed the formation of the title compound. The solvent was removed under reduced pressure, and the obtained product was used in the next reaction without purification. LCMS (affinity method, TFA buffer), Rt = 6.60 min. ESIMS (+ve) 756 [M] + 1; C 42 H 53 Calculated m / z for N5O8[M]+1: 756]

[0548] 1.33TFA.NH2-Val-Ala-PAB-P-trigger-(NMeBoc), compound 60

[0549] 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 obtained residue was dissolved in acetonitrile and purified using preparative HPLC (40%-90% MeCN, 0.05% TFA buffer, RT about 45 min) to produce the title compound (63.0 mg, 52%) as a colorless liquid after lyophilization. LCMS (affinity method, TFA buffer) ESIMS (+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).

[0550] 1.34COOH-PEG9-Val-Ala-PAB-P-trigger-(NMeBoc), compound 61

[0551] To a stirred solution of compound 60 (52.0 mg, 0.081 mmol) in acetonitrile (2 mL) was added DIPEA (87.2 μL, 0.50 mmol), 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 showed the formation of the product LCMS (affinity method, TFA buffer; 5%-60% acetonitrile in 15 min), Rt=11.32 min. ESIMS (+ve) 1048 [M] + H2O; C 49 H 83 N5O 18 [M] + calculated m / z for H2O: 1048]. The solvent was removed under reduced pressure, and the obtained product was used in the next reaction without purification.

[0552] 1.35COOH-PEG9-Val-Ala--PAB-P-trigger-(NMe.TFA), compound 62

[0553] To a stirred solution of compound 61 (84.0 mg, 0.081 mmol) in dichloromethane (2 mL) was added TFA (0.5 mL, 6. mmol) at 0°C, and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the obtained residue was dissolved in acetonitrile and purified using preparative HPLC (5%-70% acetonitrile, 0.05% TFA buffer, RT 32-33 min) to produce the title compound (87.0 mg, 100%) as a thick colorless liquid after lyophilization. LCMS (affinity method, FA buffer) ESIMS (+ve) 930 [M] + 1; C 45 H 77 N5O 15 Calculated m / z for [M]+1: 930; 1H-NMR (300MHz, MeOD) δ (ppm): 7.64 (d, J = 9.0 Hz, 2H), 7.37 (d, J = 9.0 Hz, 2H), 5.13 (d, J = 9.0 Hz, 1H), 4.61–4.40 (m, 2H), 4.19 (d, J = 6.0 Hz, 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),2.8 5(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).

[0554] 1.36COOH-PEG9-Val-Ala-PAB-P-trigger-NMeCO-CTX, compound 63

[0555] To a solution of compound 46 (28.0 mg, 0.027 mmol) in DMF (3 mL) was added DIPEA (9.40 μL, 0.54 mmol) and compound 51 (27.3 mg, 0.027 mmol). The mixture was stirred at room temperature for 16 h. The solvent was removed under reduced pressure, and the obtained residue was dissolved in acetonitrile (2 mL). The solution was purified by preparative HPLC (5%-60% ACN, Rt 32.2-34.3 min) to produce the title product (26 mg, 56%) as a white solid after lyophilization. LCMS (affinity method, TFA buffer) Rt = 5.57 min. ESIMS (+ve) 1792 [M] +; C 90 H 130 N6O 31 Calculated m / z for [M]+: 1792

[0556] Example 1b Synthesis of BCN and DBCO Linkers

[0557] 1.37BCN-PEG2-Glu-CO-NHPEG 24 CO2H, compound 31

[0558] NH2-PEG 24-COOH (93.7mg, 0.082mmol) in a solution of water / THF mixture (1: 1, 4mL) was added with sodium bicarbonate (15.2mg, 0.181mmol). The mixture was stirred at room temperature for 5min, and then a solution of BCN-PEG2-NHS ester (50mg, 0.093mmol) in THF (2mL) was added. The resulting reaction mixture was stirred at RT for 15h. The volatiles were then removed under reduced pressure, and ACN (2.5mL) was added to the resulting aqueous suspension. The solution was purified by preparative HPLC (5%-60% ACN, Rf 32.2-34.3min), producing the title product (42mg, 33%) as a white solid 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 = 9.0 Hz, 2H); 3.21-3.31 (m, 6H); 3.35-3.85 (m, 106H); 4.10 (d, J = 9.0 Hz, 2H). LCMS (affinity method, formic acid buffer) Rt = 10.28 min; ESIMS (+ve) m / z 1566.7 [M] +.

[0559] 1.38BCN-PEG2-Glu-CO-NHPEG 24 CO-NHPEG3-TCO, compound 32

[0560] BCN-PEG2-Glu-CO-NHPEG 24 -COOH compound 31 (10.0 mg, 0.006 mmol) was added to a stirred solution in DMF (3.0 mL) with PyBOP (3.64 mg, 0.007 mmol), NMM (1.31 μL, 0.012 mmol), followed by TCO-PEG3-NH2 (2.41 mg, 0.007 mmol). The reaction mixture was then stirred at room temperature overnight. The solvent was then removed under reduced pressure, and the resulting residue was dissolved in ACN (2 mL) and then 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 ACN. The remaining aqueous solution was lyophilized overnight to produce the title product (4.7 mg, 39%) as a white solid. 1H-NMR (300 MHz, 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 = 6.0 Hz), 3.35-3.44 (m, 6H), 3.47-3.79 (m, 102H), 3.83-3.92 (m, 1H), 4.16 (d, 2H, J = 6.0 Hz), 4.26-4.41 (m, 1H), 4.58 (bs, 10H), 5.39-5.74 (m, 3H). LCMS (affinity method, formic acid buffer) Rt = 11.0 min. ESIMS (+ve) 1894.0 [M] +;

[0561] Calculated m / z for C90H165N5O36[M]+: 1894.2

[0562] 1.39DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO, compound 33

[0563] DBCO-Glu-NHPEG 24 To a stirred solution of COOPFP (50.0 mg, 0.031 mmol) in DMF (3 mL) was added TCO-PEG3-NH2 (10.7 mg, 0.031 mmol), followed by NMM (4.08 μL, 0.037 mmol). The reaction mixture was then stirred at room temperature for 3 h and then concentrated under reduced pressure. The resulting residue was dissolved in ACN (2 mL), filtered through a 0.45 μm filter and the filtrate was purified by preparative HPLC (40%-70% ACN Rt 27-29 min). The product fractions were concentrated under reduced pressure to remove ACN, and the remaining aqueous solution was lyophilized overnight to produce the title product (25.0 mg, 42%) as a viscous colorless liquid. 1H-NMR (300 MHz, 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 = 6.0 Hz), 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 (affinity method, formic acid buffer) Rt = 7.0 min. ESIMS (+ve) 1775.0.0 [M] +; Calculated m / z for C88H148N4O32 [M] +: 1775.12

[0564] 1.40MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys]8[(α-Cy5)1(α-NHAc)7(ε-NHPEG 1100 )8], G3, compound 37

[0565] 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 a solution containing MeTzPh-PEG4PEG in DMF (0.5 mL). 24 -CO[N(PN)2][Lys]8(α-NH2)8(ε-NHPEG 1100 )8] (20 mg, 1.59 μmol) vial. NMM (10 μL, 91.0 μmol) was added to the solution, and the reaction mixture was then protected from light and stirred at RT. After 3.5 h, acetic anhydride (20 μL, 212 μmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure, then dissolved in MQ water (5 mL) and divided into two portions for purification by two (pre-equilibrated) PD10 columns. Once the sample entered the column bed, the sample was eluted with 3.5 mL MQ water, and the filtrate was collected. The filtrates were combined and freeze-dried overnight. Freeze-drying produced the title product (19.3 mg (93%)) as a bright blue powder. HPLC (C8

[0566] 1.41MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 16 [(α-Cy5)1(α-NHAc) 15 (ε-NHPEG 1100 ) 16 ], G4, compound 38

[0567] A solution of Cy5-NHS ester (520 μL of a 1 mg / mL solution in DMF; 0.52 mg, 844 nmol) was added to a solution containing MeTzPh-PEG4PEG in DMF (1.0 mL). 24 -CO[N(PN)2][Lys] 16 [(α-NH2) 16 (ε-NHPEG 1100 ) 16 ] (20mg, 844nmol) of vial. NMM (10 μL, 94.5 μmol) was added to the solution, and the subsequent reaction mixture was protected from light and stirred at RT. After 3.5h, acetic anhydride (20 μL, 230 μmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure, then dissolved in MQ water (5mL) and divided into two parts for purification by two (pre-equilibrium) PD10 columns. Once the sample enters the column bed, the sample is eluted with 3.5mL MQ water, and the filtrate is collected. The filtrates are combined and freeze-dried overnight. Freeze-dried to produce the title product (19.9mg (98%)) as a bright blue powder. HPLC (C8XBridge, 3x 100mm) gradient: 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 214nm, 0.4mL / min, Rt (min) = 8.40.

[0568] 1.42MeTzPh-PEG4PEG 24 -CO[N(PN)2][Lys] 32 [(α-Cy5)1(α-NHAc) 31 (ε-NHPEG 1100 ) 32 ], G5, compound 39

[0569] A solution of Cy5-NHS ester (300 μL of a 1 mg / mL solution in DMF; 0.30 mg, 487 nmol) was added to a solution containing MeTzPh-PEG4PEG in DMF (1.2 mL). 24-CO[N(PN)2][Lys] 32 [(α-NH2) 32 (ε-NHPEG 1100 ) 32 ] (20mg, 435nmol) of vial. NMM (11 μL, 97.5 μmol) was added to the solution, and the reaction mixture was then protected from light and stirred at RT. After 3.5h, acetic anhydride (22 μL, 237 μmol) was added, and the reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure, then dissolved in MQ water (5mL) and divided into two portions for purification by two (pre-equilibrium) PD10 columns. Once the sample enters the column bed, the sample is eluted with 3.5mL MQ water, and the filtrate is collected. The filtrates are combined and freeze-dried overnight. Freeze-dried to produce the title product (18.7mg (91%)) as a bright blue powder. HPLC (C8XBridge, 3x 100mm) gradient: 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 214nm, 0.4mL / min, Rt (min) = 8.50. 1.43

[0571] 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

[0572] Use according to general procedure C

[0573] BHA[Lys]4[(α-NH-COPEG 24 NH-COPEG4(PhTzMe) 1-3 (α-NH2) 1-3 )(ε-NH-COPEG 1000 )4], G2, compound 107 (3.0 mg, 0.414 μmol) and HO-Lys[(α-Cy5)(ε-DFO)] compound 108 (0.56 mg, 0.414 μmol) were prepared and purified by spin column (10 kDa MW cutoff, washed with 10x 450 μL MQ water) to give the desired product compound 99 (final concentration of 3.56 mg in 300 μL MQ water). 1.44

[0575] BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe) 1-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 3-6 )(ε-NH-COPEG 412 )8], G3, compound 100

[0576] Use according to general procedure C

[0577] 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 and purified by spin column (10 kDa MW cutoff, washed with 10x 450 μL MQ water) to give the product compound 100 (final concentration of 3.60 mg in 300 μL MQ water). 1.45

[0579] 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

[0580] Use according to general procedure C

[0581] 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 and purified by spin column (10 kDa MW cutoff, washed with 10x 450 μL MQ water) and purified by spin column (10 kDa MW cutoff, washed with 10x 450 μL MQ water) to give the product compound 27 (final concentration of 3.45 mg in 300 μL MQ water). 1.46

[0583] 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

[0584] Use according to general procedure C

[0585] 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)(ε-NHDFO) compound 108 (0.62 mg, 0.454 μmol) were prepared and purified by spin column (10 kDa MW cutoff, washed with 10 x 450 μL MQ water) to give 9.3 mg of compound 28 as a blue solid (after lyophilization). 1.47

[0587] 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

[0588] Use according to general procedure C

[0589] BHA[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe) 1-4 (α-NH2) 27-30 )(ε-NH-COPEG 1000 ) 32 ], G4, compound 112 (1.9 mg, 0.271 μmol) and HO-Lys(α-Cy5)(ε-DFO) compound 108 (0.37 mg, 0.271 μmol) were prepared and purified by spin column (10 kDa MW cutoff, washed with 10 x 450 μL MQ water) to give 8.4 mg of product compound 103 as a blue solid after lyophilization. 1.48

[0591] BHA[Lys]4[((α-Lys(α-NHCy5)(α-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 105

[0592] To BHA[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24NH-COPEG4(PhTzMe)4], G2, compound 113 (11.0 mg, 0.0015 mmol) were added 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) in a stirred solution in DMF (3 mL) at RT. After 18 h, the volatiles were removed under reduced pressure, and the blue solid residue was dissolved in MQ water, and the solution was filtered (0.45 μm acrodisc syringe filter). The filtrate was concentrated by spin column (Amicon Ultra, 0.5 mL, 3 kDa MW cutoff), and the retentate was washed with MQ water (10× 450 μL) and repeated washing to produce compound 24 (concentration 10 mg / mL in MQ water; 1.3 mL. LCMS (affinity method, TFA buffer), gradient: 20% to 90% acetonitrile in 8 min; Rt = 5.64 min; 1H NMR (300 MHz, D2O) δ (ppm): 8.52 (d, 8H, J = 9.0 Hz), 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 = 9.9 Hz), 7. .15-7.08(m,1H),7.06-7.00(m,1H),6.70-6.60(m,2H),6.21-6.12(m,2 H),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).

[0593] 1.49BHA[Lys]4[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)3)(ε-NH-COPEG 1000 )4], G2, compound 107

[0594] BHA[Lys]4[(α-NH2.TFA)4(ε-NH-COPEG 1000)4](50.0mg, 0.007mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (click chemistry tool, 13.39 mg, 0.010 mmol) was prepared except that the reaction vessel was wrapped in foil to block light and the residue was purified by SEC (Sephadex TM LH-20) (using methanol as the eluent) to give the product compound 107 (44.00 mg, 77%); 1 HNMR (300 MHz, 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, 3x 100mm) gradient (formate buffer): 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 214nm, 0.4mL / min, Rt (min) = 8.08-9.01.

[0595] 1.50BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NH-COPEG 412 )8], G3, compound 109

[0596] BHA[Lys]8[(α-NH 2. TFA)8(ε-NH-COPEG 412 )8] (50.0 mg, 0.008 mmol) and HOOC-PEG 24 NH-COPEG4(PhTzMe) (click chemistry tool; 11.2 mg, 0.008 mmol) was prepared except that the reaction vessel was wrapped in foil to block light and the residue was purified by SEC (Sephadex TM LH-20) (using methanol as the eluent) to give the product compound 109 (29 mg, 55%); 1HNMR (300 MHz, 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 (C8 XBridge, 3x100mm) gradient (formate buffer): 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN(13-15min),214nm,0.4mL / min,R t =8.16-8.93min.

[0597] 1.51BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2)7)(ε-NH-COPEG 1000 )8], G3, compound 110

[0598] BHA[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1000 )8](100.0mg, 0.007mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (click chemistry tool; 15.97 mg, 0.010 mmol) was prepared except that the reaction vessel was wrapped in foil to block light and the residue was purified by SEC (Sephadex TM LH-20) (using methanol as eluent) to give the product compound 19 (SPL-9248) (69 mg, 66%); 1HNMR (300 MHz, 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, 3x 100mm) gradient (formate buffer): 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN(13-15min),214nm,0.4mL / min,R t =8.21-9.15min.

[0599] 1.52BHA[Lys] 16 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe))1(α-NH2) 15 )(ε-NH-COPEG 1000 ) 16 ], G4, compound 111

[0600] BHALys[Lys]2[Lys]4[Lys]8[Lys] was used according to the general procedure C. 16 [(α-NH2.TFA) 16 (ε-NH-COPEG 1000 ) 16 ](Reference 1) (100.0 mg, 0.004 mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (click chemistry tool; 6.74 mg, 0.005 mmol) was prepared except that the reaction vessel was wrapped in foil to block light and the residue was purified by SEC (Sephadex TM LH-20) (using methanol as the eluent) to give the product compound 111 (63 mg, 65%); 1HNMR (300 MHz, 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 (C8 XBridge, 3x 100mm) gradient (formate buffer): 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN(13-15min),243nm,0.4mL / min,R t =8.51-9.02min. 1.53

[0602] BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhTzMe) 1-4 (α-NH2) 28-31 )(ε-NH-COPEG 1000 ) 32 ], G5, compound 112

[0603] BHA[Lys] was used according to General Procedure C. 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1000 ) 32 ](100.0 mg, 0.002 mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (click chemistry tool; 3.38 mg, 0.005 mmol) was prepared except that the reaction vessel was wrapped in foil to block light and the residue was purified by SEC (Sephadex TM LH-20) (using methanol as the eluent) to give the product compound 21 (68 mg, 72%); 1HNMR (300 MHz, 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 (C8 XBridge, 3x100mm) gradient (formate buffer): 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN(13-15min),214nm,0.4mL / min,R t =8.77min.

[0604] 1.54BHA[Lys]4[(α-NHBoc)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 113

[0605] To a stirred solution of BHA[Lys]4[(α-NHBoc)4(ε-NH2)4] (46.0 mg, 0.031 mmol) in DMF at RT were added NMM (68.0 μL, 0.620 mmol), HOOCPEG 24 NH-COPEG4(PhTzMe) (click chemistry tool; 43.0 mg, 0.155 mmol) and PyBOP (81.0 mg, 0.155 mmol). 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 filter). The filtrate was purified by preparative HPLC (30%-80% ACN in 60 min, mobile phase: MQ water and acetonitrile, R t 33.0-36.0 min) to give compound 22 (52 mg (22%)) as a pink solid. LCMS (affinity method, TFA buffer) R t =5.66min; 1HNMR (300MHz, D2O) δ (ppm): 8.52 (d, 8H, J = 9.0Hz), 7.40-7.26 (m, 10H), 7.20 (d, 8H, J = 9.0Hz), 6.23-6.17 (m, 1H), 4.36-4.21 (m, 10H), 3. 99-3.83(m,13H),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).

[0606] 1.55BHA[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, compound 114

[0607] To BHA[Lys]4[(α-NHBoc)4(ε-NH-COPEG 24 NH-COPEG4(PhTzMe)4], G2, a stirred solution of compound 113 (48.0 mg) in methanol (2 mL) was added with a 3M solution of HCl in methanol (2 mL), and the reaction mixture was stirred at room temperature for 20 h. The volatiles were removed under reduced pressure to yield BHALys[Lys]2[Lys]4[(α-NH2.HCl)4(ε-NH-COPEG4(PhTzMe)4] as a pink solid. 24 NH-COPEG4(PhTzMe)4], G2, compound 23 (41.0 mg (91%)). LCMS (affinity method, TFA buffer), gradient: 20% to 90% acetonitrile in 8 min; R t =5.27min.

[0608] 1.56HO-Lys[(α-NHCy5)(ε-NHDFO)], compound 108

[0609] DIPEA (32 μL, 0.224 mmol) was added to a stirred solution of compound 115 (20.0 mg, 0.032 mmol) in DMSO (5 mL), followed by p-SCN-deferoxamine (Macrocyclics, 24.0 mg, 0.032 mmol). The reaction mixture was stirred at room temperature for 4 h, and then concentrated by blowing a stream of nitrogen over the solution for several hours. The obtained residue was then purified by preparative HPLC (30%-60% ACN (60 min, Rt 39-42 min) in MQ water+0.1% TFA) to produce product compound 108 (15 mg (34%)) as a blue solid. LCMS (affinity method, TFA buffer) R t =5.93min;ESIMS(+ve)1364[M] + ; C 71 H 103 N 12 O 11 S2[M] + Calculated for m / z = 1364 1 HNMR (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).

[0610] 1.57HO-Lys[(α-NHCy5)(ε-NH2)], compound 115

[0611] 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 in MQ water+0.1% formic acid (60 min, Rt 32.0-33.0 min)) to give the product compound 59 (12 mg (44%)) as a blue solid. LCMS (affinity method, TFA buffer) R t =7.65min; ESIMS (+ve) 611 [M] + ; C 38 H51 N4O3[M] + Calculated for m / z=611.

[0612] 1.58 Synthesis of HO-Lys[(α-NHCy5)(ε-NHDFO)] wedge, compound 116

[0613] 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 5NHS ester (Lumiprobes, 40.0 mg, 0.059 mmol). The reaction mixture was stirred at room temperature overnight, 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 in MQ water + 0.1% formic acid (60 min, R t 39.0-42.0 min)) to give Compound 58 (33 mg (67%)) as a blue solid. LCMS (affinity method, TFA buffer) R t =10.54min;ESIMS(+ve)833[M] + ; C 53 H 61 N4O5[M] + Calculated for m / z = 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).

[0614] Example 2 Synthesis of dendrimer drug conjugates

[0615] 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

[0616] Azido-PEG was used according to the general procedure E. 24 -CO[N(PN)2][Lys]4[(α-NH2.TFA)(ε-NHPEG 1100 )]4 (50.0 mg, 7.24 μmol) was prepared 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 (affinity method, formic acid buffer) R t =11.23min. ESIMS (+ve) conversion: 9016.

[0617] 2.2 Azido-PEG 24 CO-[N(PN)2][Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4, G2, compound 64

[0618] Azido-PEG was used according to the general procedure E. 24 CO-[N(PN)2][Lys]4[(α-NH2.TFA)(ε-NH-COPEG 1100 )]4 (50.0 mg, 7.24 μmol) was prepared 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 (affinity method, formic acid buffer) R t =11.23min. ESIMS (+ve) conversion: 9016.

[0619] 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

[0620] Azido-PEG was used according to the general procedure E. 24 -CO[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NHPEG 1100 )]8 (50.0 mg, 3.91 μmol) was prepared 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 (affinity method, formic acid buffer) R t =11.30min

[0621] 2.4 Azide-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]8, G3, compound 65

[0622] Azido-PEG was used according to the general procedure E. 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1100 )8] (50.0 mg, 3.91 μmol) was prepared to produce 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 (affinity method, formic acid buffer) R t =11.30min

[0623] 2.5 Azide

[0624] -PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG 570 )8], G3, compound 19

[0625] Azido-PEG was used according to the general procedure F. 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 570 )8] (7.2 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol) were prepared to produce a product concentration of 14.6 mg / 3.5 mL (240 μM).

[0626] 2.6 Azide

[0627] -PEG 24 CO-[N(PN)2][Lys]8[(α-NH-Glu-Val-Cit-PAB-MMAE)8(ε-NH-COPEG 1100 )8], G3, compound 20

[0628] Azido-PEG was used according to the general procedure F. 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1100 )8] (10.8 mg, 842 nmol) and HO-Glu-vc-PAB-MMAE (10.0 mg, 8.08 μmol) were prepared to produce a product concentration of 18 mg / 3.5 mL (240 μM).

[0629] 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

[0630] Use azido group according to general procedure F

[0631] -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) were prepared to produce a product concentration of 25.5 mg / 3.5 mL (240 μM).

[0632] 2.8 Azide

[0633] -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

[0634] Use azido group according to general procedure F

[0635] -PEG 24 CO-[N(PN)2][Lys]2[Lys]4[Lys]8[(α-NH2.TFA)(ε-NH0-COPEG 1100 )]8 (16.3 mg, 1.28 μmol) and DGA-MMAF(OMe) (10.6 mg, 12.3 μmol) were prepared to give a product concentration of 23.8 mg / 3.5 mL (365 μM).

[0636] 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

[0637] Azido-PEG 24 CO-[N(PN)2][Lys]8[(α-NH2.TFA)(ε-NH-COPEG 1100 )]8 (SPL 19 and SPL 32) (22.4 mg, 1.75 μmol) in a stirred solution 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 acetate-diglycolic acid-1R, 2R-cyclohexane-1,2-diamine-oxalate platinum (IV) (10.4 mg, 17.7 μmol) and PyBOP (8.74 mg, 16.8 μmol) in DMF (0.5 mL). The reaction mixture was then protected from light and stirred at room temperature overnight and then purified by SEC to yield 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 (affinity method, TFA buffer) R t = 10.63 min. ICP-OES: Pt% determined 9.0%, indicating the presence of 7 Pt-containing moieties on the macromolecule. The actual molecular weight of the conjugate was determined to be 16.1 kDa.

[0638] 2.10BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz) 1-4 (α-NH2) 4-7 )(ε-NH-COPEG 1000 )8], G3, compound 34

[0639] Preparation of BHA[Lys]8[(αNH2.TFA)8(ε-NH-COPEG 1000 )8] (100 mg, 0.00786 mmol, 1.0 equiv) in DMF (300 μL). To this stirred solution was added (MeTzPh)PEG4CO-NHPEG 24 CO2H (click chemistry tool; 16mg, 0.01mmol, 1.3 equivalents), PyBOP (8mg, 0.013mmol, 1.6 equivalents) and DMF (200μL). The reaction mixture was stirred for 3min, then NMM (40mg, 50μL, 0.38mmol, 48 equivalents) was added. The contents were protected from light and stirred overnight at RT. The reaction mixture was diluted with MQ water and lyophilized overnight. The lyophilized material was dissolved in MeOH (1mL) and purified by SEC (400 drops / tube, MeOH sephadex LH20, 35 drops / min). The product-containing fractions were checked by HPLC and collected in 2 different fractions. Each fraction was concentrated under reduced pressure, and then the resulting residue was dissolved in MQ water, filtered (0.45μm acrodisc filter) and freeze-dried to produce the title product (69mg, 66%) as a pink solid.

[0640] HPLC (C8 Xbridge, 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, 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 (br s,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).

[0641] 2.11BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG2-BCN) 1-4 (α-NH2) 4-7 )(ε-NH-COPEG 1000 )8], G3, compound 35

[0642] Preparation of BCN-PEG2CO-NHPEG at RT 24 A stirred solution of -CO2H compound 31 (9.6 mg, 0.006 mmol, 1.3 eq) in DMF (200 μL) was added. PyBOP (4 mg, 0.008 mmol, 1.6 eq) and NMM (23 mg, 25 μL, 0.226 mmol, 48 eq) were added to the stirred solution and after 5 min, BHA[Lys]8[(α-NH2.TFA)8(ε-NH-COPEG 1000 )8] (60mg, 0.006mmol, 1.0 equivalent), DMF (200μL) was added afterwards. The contents were protected from light and stirred overnight at RT. The reaction mixture was diluted with ACN (10mL), then purified by SEC (400 drops / tube, ACN sephadex LH20, 35 drops / min). The product fractions were checked by HPLC, collected, filtered (0.45μm acrodisc filter), concentrated under reduced pressure and freeze-dried overnight to produce the title compound (58mg, 92% yield) as a light yellow solid.

[0643] HPLC (C8 Xbridge, 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, 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 (br s,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(br s,7H),6.18(s,0.9H),7.12-7.48(m,7H). 2.12

[0645] (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH-Cy5) 1-4 (α-NH-Glu-Val-Cit-PAB-MMAE) 4-7 )(ε-NH-COPEG 1100 )8], G3, compound 36

[0646] A solution of Cy5-NHS (214 μL of a 4.2 mg / mL solution in DMF; 1.43 μmol, 1.0 equiv) was added to neat (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 )8] Compound 8 (18 mg, 1.43 μmol). Once completely dissolved, NMM (10 μL, 91.0 μmol) was added, and the subsequent 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 a solution of Glu-VC-PAB-MMAE in DMF (290 μL of a 60 mg / mL solution, 14.0 μmol, 9.8 equiv). The subsequent reaction mixture was protected from light and stirred overnight at RT.

[0647] The reaction mixture was diluted with PBS (2.0 mL) to produce a final volume of 2.5 mL. The diluted solution was then passed through a PD10 desalting column (pre-equilibrated with PBS). Once the entire solution had entered the column bed, PBS (3.5 mL) was added to elute the product (which appeared as a blue band). The final theoretical concentration of the construct in PBS = 8.7 mg / mL. The material was stored frozen at -80 ° C. HPLC (C8 Xbridge, 3x 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) = 95-10 min (broad peak); 83% conjugate-related peaks with 17% MMAE / MMAE-joints. 2.13

[0649] (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

[0650] Towards

[0651] (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]2[ 3 H-Lys]4[Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100 )8] (prepared using the method for the synthesis of compound 53) (36.8 mg, 2.93 μmol) was added to a solution of solid PyBOP (24.7 mg, 47.5 μmol) in NMM / DMF (7.8 μL / 0.5 mL). Once completely dissolved, the dendrimer solution was added to a solution of HO-Glu-VC-PAB-MMAE (40 mg, 32.3 μmol) in NMM / DMF (7.8 μL / 0.5 mL). The reaction mixture was then protected from light and stirred overnight at RT.

[0652] The reaction mixture was diluted with PBS (4.0 mL) to produce a final volume of 5 mL. The diluted solution was then passed through two PD10 desalting columns (pre-equilibrated with PBS, 2.5 mL was passed through each column). Once the entire solution had entered the column bed, PBS (3.5 mL) was added to each column to elute the product. The resulting crude mixture was further purified using a regenerated cellulose Amicon Ultra-0.5 mL centrifugal unit (10K MWCO). The final theoretical concentration of the construct in PBS = 29-30 mg / mL. The material was stored frozen at -20 °C. HPLC (C8 Xbridge, 3x 100mm) gradient: 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 214nm, 0.4mL / min, Rt(min)=9.5-12min (broad peak); 91.4% conjugate-related peak with 8.6% MMAE / MMAE-linker-related peak. 2.14

[0654] (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

[0655] A stirred solution of p-SCN-deferoxamine (2.1 mg, 2.79 μmol) in DMSO (100 μL) was prepared at RT. To this stirred solution was added (MeTzPh)PEG4CO-NHPEG in DMF (200 μL). 24 CO[N(PN)2][Lys]8[(α-NH2.HCl)8(ε-NH-COPEG 1100)8] Compound 53 (17.0 mg, 1.35 μmol). The reaction mixture was then stirred for 3 min, and then NMM (10 μL, 91.0 μmol) was added. The resulting solution was protected from light and stirred for 4 h at RT. PyBOP (7.0 mg, 13.5 μmol) was added, and after 5 min, the reaction mixture was added to pure HO-Glu-Val-Cit-PAB-MMAE (9.76 mg, 7.89 μmol). The reaction mixture was then left to stand overnight. The reaction mixture was diluted with PBS buffer (4.5 mL) and divided across 4 Amicon ultracentrifugal filters (10K MWCO), and centrifuged (14K rcf, 15 min) filter. The retentate was diafiltered in PBS (400 μL, 14K rcf, 15 min x 10 times). The retentates were combined to produce a pink solution with an approximate concentration of 16 mg compound 67 in 2 mL. HPLC (C8 XBridge, 3x 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) = 8.7-9.8 min (broad peak). 2.15

[0657] BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMA E)5)(ε-NH-COPEG 1100 )8], G3, compound 67

[0658] A stirred solution of p-SCN-deferoxamine (2.0 mg, 2.66 μmol) in DMSO (100 μL) was prepared at RT. To this stirred solution was added BHALys[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz))1(α-NH2)7)(ε-NHPEG 1100)8] Compound 34 (17.0 mg, 1.27 μmol). The reaction mixture was then stirred for 3 min, and then NMM (10 μL, 91.0 μmol) was added. The resulting solution was protected from light and stirred for 4 h at RT. PyBOP (7.0 mg, 13.5 μmol) was added, and after 5 min, the reaction mixture was added to pure HO-Glu-Val-Cit-PAB-MMAE (9.17 mg, 7.41 μmol). The reaction mixture was then left to stand overnight. The reaction mixture was diluted with PBS buffer (4.5 mL) and divided across 4 Amicon ultracentrifugal filters (10K MWCO), and centrifuged (14K rcf, 15 min) filter. The retentate was diafiltered in PBS (400 μL, 14K rcf, 15 min x 10 times). The retentate was combined to produce a pink solution with an approximate concentration of 16 mg compound 68 in 2 mL. HPLC (C8 XBridge, 3x 100mm) 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 (broad peak). 2.16

[0660] (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH-Cy5)1(α-NH-Glu-SN38)7)(ε-NH-COPEG 1100 )8], G3 compound 68

[0661] To (MeTzPh)PEG4CO-NHPEG 24 CO-[N(PN)2][Lys]8[((α-NH2.HCl)8(ε-NH-COPEG 1100)8] Compound 53 (10 mg, 0.75 μmol) was added to a stirred solution of DMF (2 mL) with NMM (4.0 μL, 36.0 μmmol) and sulfoCy5 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 μmmol) and PyBOP (4.05 mg, 7.8 μmmol) were added to the reaction mixture, and the reaction mixture was stirred for another 20 h at room temperature. The solvent was removed under reduced pressure, and the obtained residue was purified using size exclusion chromatography (Sephadex LH 20) (using acetonitrile as mobile phase) to produce the title compound (6.0 mg) as a blue solid. UPLC-TOF analysis of the reaction mixture confirmed the random addition of Cy5 NHS ester and SN-38-Glu-COOH to the dendrimer. UPLC-TOF (affinity method, TFA buffer) Rt = 84.80-5.80min. 2.17

[0663] BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) 1-4 (α-NH2) 28-31 (ε-NH-COPEG 1100 ) 32 ], G5, compound 69

[0664] BHALys[Lys] was used according to the general procedure C. 32 [(α-NH2.TFA) 32 (ε-NH-COPEG 1000 ) 32 ](100.0 mg, 0.002 mmol) and HOOCPEG 24 NH-COPEG4(PhTzMe) (click chemistry tool; 3.38 mg, 0.005 mmol) was prepared except that the reaction vessel was wrapped in foil to block light and the residue was purified by SEC (Sephadex TM LH-20) (using methanol as the eluent) to give the product compound 69 (68 mg, 72%); 1HNMR (300 MHz, 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 (C8 XBridge, 3x100mm) gradient (formate buffer): 5% ACN / H2O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN(13-15min),214nm,0.4mL / min,R t =8.77min. 2.18

[0666] BHA[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 )(α-NH2) 5-8 (ε-NH-COPEG 1100 ) 32 ], G5, compound 70

[0667] To BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) 1-4 ((α-NH2) 28-31 (ε-NH-COPEG 1100 ) 32] Compound 69 (20.0 mg, 0.4 μmol) was added to NMM (8.0 μL, 72 μmol) in a stirred solution in DMF, followed by addition of 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, and the mixture was stirred for another 20 h at room temperature. The solvent was removed under reduced pressure, and the crude residue was dissolved in acetonitrile (2 mL), and the obtained solution was filtered through a 0.45 μM filter. The filtrate was collected and purified using size exclusion chromatography (Sephadex LH20) (using acetonitrile as solvent) to produce the title compound (27 mg; 75%) as a blue solid. 1 HNMR (300 MHz, MeOD) δ (ppm): 8.52 (d, 2H, J = 9.0 Hz), 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 (C8 XBridge, 3x 100mm) gradient (formate buffer): 75% ACN / H2O (0-1min), 75-90% ACN (1-7min), 90% ACN (7-12min), 90-75% ACN (12-13min), 75% ACN (13-15min), 214nm, 0.4mL / min, Rt (min) = 8.90-9.10.

[0668] Example 3 Synthesis of Targeted Dendrimer Conjugates

[0669] 3.1 Affibody-MPED-BCN-triazol

[0670] -PEG 24 -CO[N(PN)2][Lys]4[(α-NH-DGA-3'NH-Dox) 1-4 (ε-NH-COPEG 1100 )]4, G2, compound 24

[0671] Affibody-BCN (900 μL) and azido were used according to general procedure G.

[0672] -PEG 24 -CO[N(PN)2][Lys]4[(α-NH-DGA-3'-NH-Dox) 1-4 (ε-NH-COPEG1100 )]4 (300 μL of 858 μM solution) (Affibody-BCN:DGA-Dox dendrimer molar ratio; 1:3). SDS-PAGE analysis showed a band of about 19 kDa corresponding to the affibody-dendrimer conjugate (700 nm).

[0673] 3.2 Affibody

[0674] -BCN / N3-PEG 24 CO-[N(PN)2][Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4, G2, compound 71

[0675] Affibody-BCN (900 μL) and azido were used according to general procedure G.

[0676] -PEG 24 CO-[N(PN)2][Lys]4[(α-NH-14-O-DGA-Nemo)(ε-NH-COPEG 1100 )]4 (300 μL of 858 μM solution) (Affibody-BCN:DGA-Nemo dendrimer molar ratio; 1:3). SDS-PAGE analysis showed a band of about 19 kDa corresponding to the affibody-dendrimer conjugate (700 nm).

[0677] 3.3 Affibody-MPED-BCN-triazol

[0678] -PEG 24 -CO[N(PN)2][Lys]8[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100 )]8, G3, compound 25

[0679] Affibody-BCN (105 μL) and azido were used according to general procedure G.

[0680] -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) (Affibody-BCN:DGA-Dox dendrimer molar ratio; 1:1). SDS-PAGE analysis showed a band of approximately 27 kDa corresponding to the Affibody-dendrimer conjugate (700 nm). The conjugate yield was estimated to be 80%, which was calculated by adding the Azide IR dye 800CW and measuring the fluorescence at 800 nm.

[0681] 3.4 Affibody

[0682] -BCN / N3-PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]8, G3, compound 72

[0683] Affibody-BCN (105 μL) and azido were used according to general procedure G.

[0684] -PEG 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]8 (35 μL of a 580 μM solution) (Affibody-BCN:DGA-Nemo dendrimer molar ratio; 1:1). SDS-PAGE analysis showed a band of approximately 27 kDa corresponding to the Affibody-dendrimer conjugate (700 nm). The conjugate yield was estimated to be 80%, which was calculated by adding the Azide IR dye 800CW and measuring the fluorescence at 800 nm.

[0685] 3.5 Affibody

[0686] -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

[0687] Affibody BCN (2.0 mg, 286 nmol in 1.0 mg / mL PBS) and azido-PEG were used according to General Procedure G. 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). Lyophilization of the purified material yielded a white fluffy powder (2.19 mg, 31%). SDS-PAGE analysis showed a band of approximately 30 kDa corresponding to the affibody-dendrimer conjugate (700 nm).

[0688] 3.6 Affibody

[0689] -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 in SPL 40)

[0690] Affibody BCN (1.0 mg, 143 nmol in 1.0 mg / mL PBS) and azido-PEG were used according to General Procedure G. 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). SDS-PAGE analysis showed a band of approximately 40 kDa corresponding to the affibody-dendrimer conjugate (700 nm).

[0691] 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

[0692] Affibody BCN (5.0 mg, 715 nmol in 1.0 mg / mL PBS) and azido-PEG were used according to General Procedure G. 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). Lyophilization of the purified material yielded a white fluffy powder (5.90 mg, 47%). SDS-PAGE analysis showed a band of approximately 30 kDa corresponding to the affibody-dendrimer conjugate (700 nm).

[0693] 3.8Fab-triazol DBCO-PEG 24 -CO[N(PN)2[Lys]4[(α-DGA-3′-NH-Dox)(ε-NHPEG 1100 )]4G2-Dox / PEG1100 dendrimer, compound 29

[0694] Azide-PEG 24 -CO[N(PN)2[Lys]4[(α-NH-DGA-3'-NH-Dox)(ε-NH-COPEG 1100 )]4 solution (10 μL of 500 μM solution in PBS) was added to a solution of Fab-DBCO* (40 μL of 13.4 μM solution in HEPES buffer). The reaction mixture was then shaken at room temperature (650 rpm) overnight to produce Fab-azido-PEG 24-CO[N(PN)2[Lys]4[(α-NH-DGA-3'NH-Dox)(ε-NH-COPEG 1100 )] 4. SDS-PAGE analysis was performed and scanned at 700 nm and 800 nm using an Odyssey scanner. SDS-PAGE when scanned at 700 nm showed a band corresponding to DBCO-Fab at 50 kDa and the expected Fab-[Dendrimer-Dox / PEG 1100 ]2, compound 30.

[0695] 3.9Fab-DBCO / N3-PEG 24 CO-[N(PN)2[Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4, compound 73

[0696] Azide-PEG 24 CO-[N(PN)2[Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )]4 solution (10 μL of 500 μM solution in PBS) was added to a solution of Fab-DBCO* (40 μL of 13.4 μM solution in HEPES buffer). The reaction mixture was then shaken at room temperature (650 rpm) overnight to produce Fab-DBCO / N3-PEG 24 CO-[N(PN)2[Lys]4[(α-NH-DGA-14-O-Nemo)(ε-NH-COPEG 1100 )] 4. SDS-PAGE analysis was performed and scanned at 700 nm and 800 nm using an Odyssey scanner. SDS-PAGE when scanned at 700 nm showed a band corresponding to DBCO-Fab at 50 kDa and the expected Fab-[Dendrimer-Nemo / PEG 1100 ]2, compound 30.

[0697] 3.10 Affibody

[0698] -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

[0699] Affibody BCN (2.0 mg, 286 nmol in 1.0 mg / mL PBS) and azido-PEG were used according to General Procedure G. 24 CO-[N(PN)2][Lys]8[(α-NH-DGA-MMAF(OMe))(ε-NH-COPEG 1100 )]8 (600 μL of a 365 μM solution). Lyophilization of the purified material yielded a white fluffy powder (2.06 mg, 33%). SDS-PAGE analysis showed a band of approximately 30 kDa corresponding to the affibody-dendrimer conjugate (700 nm).

[0700] 3.11 Nanobodies

[0701] -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

[0702] DBCO-PEG was used according to Example 4 below 12 -TCO and nanobody 2D3 N-terminal tag, TEV, and C-terminal azide were used to prepare nanobody constructs.

[0703] 3.12 Nanobodies

[0704] -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

[0705] DBCO-PEG was used according to Example 4 below 12 -TCO and nanobody 2D3 N-terminal tag, TEV, and C-terminal azide were used to prepare nanobody constructs.

[0706] 3.13 Nanobodies

[0707] -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

[0708] DBCO-PEG was used according to Example 4 below 12 -TCO and Nanobody 2D3 N-terminal tag, TEV, C-terminal azide to prepare nanobody constructs

[0709] 3.14 Nanobodies

[0710] -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

[0711] DBCO-PEG was used according to Example 4 below 12 -TCO and Nanobody 2D3 N-terminal tag, TEV, C-terminal azide to prepare nanobody constructs

[0712] 3.15 Nanoantibodies

[0713] -PEG 12 -TCO-MeTzPh-PEG4PEG 24 -CO[N(PN)2][3H-Lys]4[Lys(α-NH-Glu-VC-PAB-MMAE)8(ε-NH-COPEG 1100 )8], compound 45

[0714] DBCO-PEG was used according to Example 4 below 12 -TCO and Nanobody 2D3 N-terminal tag, TEV, C-terminal azide to prepare nanobody constructs

[0715] 3.16 Nanobodies

[0716] -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

[0717] Use DBCO-Glu-NHPEG using the general procedure HO 24CO-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 eq.) and Nanobody-N3 ("Nanobody-N3-C-terminal tag") (1 eq.) were prepared and SDS-PAGE analysis showed a band of about 37 kDa corresponding to the Nanobody-dendrimer conjugate ( Figure 1 ).

[0718] 3.17 Nanobodies

[0719] -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

[0720] Use BCN-NHPEG2-Glu-NHPEG using the general procedure HO. 24 CONHPEG3-TCO linker compound 32 (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 eq.) and Nanobody-N3 ("Nanobody-N3-C-terminal tag") (1 eq.) were prepared and SDS-PAGE analysis showed a band of about 37 kDa corresponding to the Nanobody-dendrimer conjugate ( Figure 1 ). 3.18

[0722] BHA[Lys]8[((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24NH-Glu-DBCO / N3-Nanobody)1(α-NH-DFO)2(α-NH-Glu-Val-Cit-PAB-MMAE)5)(ε-NH-COPEG 1100 )8], G3, compound 76

[0723] Use DBCO-Glu-NHPEG using the general procedure HO 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazine dendrimer

[0724] 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 eq.) and Nanobody-N3 ("Nanobody-N3-C-terminal tag") (1 eq.) were prepared and SDS-PAGE analysis showed a band of approximately 37 kDa corresponding to the Nanobody-dendrimer conjugate ( Figure 1 ).

[0725] 3.19 Nanoantibodies

[0726] -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

[0727] Use DBCO-Glu-NHPEG using the general procedure HO 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazine dendrimer (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 68 (1 eq.) and Nanobody-N3 ("Nanobody-N3-C-terminal tag") (1 eq.) were prepared. SDS-PAGE analysis showed a band of approximately 37 kDa corresponding to the Nanobody-dendrimer conjugate ( Figure 2 ). 3.20

[0729] 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

[0730] Use DBCO-Glu-NHPEG using the general procedure HO 24 CO-NHPEG3-TCO linker compound 33 (1 equivalent), tetrazine dendrimer

[0731] 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 eq.) and Nanobody-N3 ("Nanobody-N3-C-terminal tag") (1 eq.) were prepared. SDS-PAGE analysis showed a band of approximately 95 kDa corresponding to the Nanobody-dendrimer conjugate ( Figure 2 ) 3.21

[0733] 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 24NH-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

[0734] Use according to general procedure H

[0735] 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, prepared from compound 99, except that:

[0736] Step 1: Prepare TCO linker solution 80 in pure DMSO

[0737] Step 2: The dendrimers were dissolved in Tris buffer at pH 8, and after the reaction of the TCO linker with the dendrimers was completed (UPLC or LCMS), the reaction mixture was diluted with Tris buffer so that the final concentration of DMSO was ≤5% (v / v) and purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO).

[0738] The purification method was the same as that used to purify compounds 74 and 76, yielding Nanobody-dendrimer conjugate compound 86 (232 μg; 0.45 μg / μL in HEPES buffer at pH 8) and Nanobody-dendrimer conjugate compound 87 (60 μg; 0.30 μg / μL in HEPES buffer at pH 8. 3.22

[0740] 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 24NH-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 412 )8], G3 compound 88a

[0741] Use according to general procedure H

[0742] 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, prepared from compound 100, except that:

[0743] Step 1: Prepare a solution of TCO linker compound 80 in pure DMSO

[0744] Step 2: The dendrimers were dissolved in Tris buffer at pH 8, and after the reaction of the TCO linker and the dendrimers was completed (UPLC or LCMS), the reaction mixture was diluted with Tris buffer so that the final concentration of DMSO was ≤ 5% (v / v) and purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO)

[0745] The purification method was identical to that used to purify compounds 74 and 76, yielding the Nanobody-dendrimer conjugate compound 88 (140 μg; 1.48 μg / μL in HEPES buffer at pH 8) and compound 88a (impure). 3.23

[0747] 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-COPEG24 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

[0748] Use according to general procedure H

[0749] 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, prepared from compound 101, except that:

[0750] Step 1: Prepare a solution of TCO linker (Compound 80) in pure DMSO

[0751] Step 2: The dendrimers were dissolved in Tris buffer at pH 8, and after the reaction of the TCO linker and the dendrimers was completed (UPLC or LCMS), the reaction mixture was diluted with Tris buffer so that the final concentration of DMSO was ≤ 5% (v / v) and purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO)

[0752] The purification method was identical to that used to purify compounds 74 and 76, yielding the Nanobody-dendrimer conjugate compound 89 (249 μg; 2.68 μg / μL in HEPES buffer at pH 8) and compound 89a (impure). 3.24

[0754] 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 24NH-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

[0755] Use according to general procedure H

[0756] 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, prepared from compound 102, except that:

[0757] Step 1: Prepare a solution of TCO linker (Compound 80) in pure DMSO

[0758] Step 2: The dendrimers were dissolved in Tris buffer at pH 8, and after the reaction of the TCO linker and the dendrimers was completed (UPLC or LCMS), the reaction mixture was diluted with Tris buffer so that the final concentration of DMSO was ≤ 5% (v / v) and purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO)

[0759] The purification method was the same as that used to purify compounds 74 and 76, yielding Nanobody-dendrimer conjugates compound 90 (203 μg; 0.975 μg / μL and 0.556 μg / μL in HEPES buffer at pH 8) and compound 91 (101 μg; 0.34 μg / μL in HEPES buffer at pH 8). 3.25

[0761] BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody)1(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 30 (ε-NH-COPEG 1000 )32 ], G5, compound 92 and BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-Nanobody) 2-4 [α-Lys(α-NHCy5)(ε-NHDFO)]1(α-NH2) 27-29 (ε-NH-COPEG 1000 ) 32 ], G5, compound 93

[0762] Use according to general procedure H

[0763] BHA[Lys] 32 [((α-NH-COPEG 24 NH-COPEG4(PhMeTz) 1-4 (α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2) 27-30 )(ε-NH-COPEG 1000 ) 32 ], G5, prepared from compound 103, except that:

[0764] Step 1: Prepare a solution of TCO linker compound 80 in pure DMSO

[0765] Step 2: The dendrimers were dissolved in Tris buffer at pH 8, and after the reaction of the TCO linker and the dendrimers was completed (UPLC or LCMS), the reaction mixture was diluted with Tris buffer so that the final concentration of DMSO was ≤ 5% (v / v) and purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO)

[0766] The purification method was the same as that used to purify compounds 74 and 76, yielding the Nanobody-dendrimer conjugate compound 92 (370 μg; 3.66 μg / μL in HEPES buffer at pH 8) and the product compound 93 (177 μg; 1.72 μg / μL in HEPES buffer at pH 8). 3.26

[0768] BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhMeTz))2)(ε-NH-COPEG 24NH-COPEG4(PhMeTz)) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanoantibody)2], G2, compound 94a,

[0769] BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhMeTz))1(ε-NH-COPEG 24 NH-COPEG4(PhMeTz) / TCO-PEG3NH-COPEG 24 NH-Glu-DBCO / N3-nanobody)3], G2, compound 94 and

[0770] 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

[0771] Use according to general procedure H

[0772] BHA[Lys]4[(α-Lys(α-NHCy5)(ε-NHDFO))1(α-NH2)3)(ε-NH-COPEG 24 NH-COPEG4(PhMeTz))4], G2 dendrimer, prepared from compound 24, except that:

[0773] Step 1: Prepare a solution of TCO linker compound 51 in water

[0774] Step 2: Dissolve the dendrimers in MQ water.

[0775] The purification method was the same as that used to purify compounds 74 and 76, yielding the Nanobody-dendrimer conjugate compound 95 and compound 94 as an inseparable mixture (final concentration 294 ug in HEPES buffer at pH 8).

[0776] It was also prepared by: DBCO-Glu-NHPEG 24A solution of CO-NHPEG3-TCO compound 80 (57 μL of a 10 mg / mL solution in 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 Tris buffer at pH 8). The reaction mixture was allowed to stand at RT for 2 d, and then the reaction mixture was purified by centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO; 10 x 450 μL) using Tris buffer (pH 8). To this solution was added a solution of compound 105 (230.0 μg in 500 μL MQ water; 0.026 umol), and the reaction mixture was allowed to stand at RT overnight. The purification method was the same as that used to purify compounds 74 and 76, resulting in Nanobody-dendrimer compound 95, compound 94a and compound 94 (final concentration of pH 8) as an inseparable mixture. 124 μg in HEPES buffer at 8 °C.

[0777] 3.27BHA[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

[0778] A solution of dibromomaleimide linker compound 106 was prepared by dissolving 2.0 mg in 1 mL DMSO (1.0 mL). A solution of TCEP (0.5 M in PBS pH 7) (2.3 μL, 1.174 μmmol) was added to a solution of Nanobody ("Nanobody-N3-C-terminal tag") (Compound 79) (1.0 mg, 0.058 μmmol in 715 μL Tris buffer at pH 8). 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 μmmol) in DMSO (47.0 μL). After 1.5 h, the reaction mixture was cooled to RT and centrifuged, then a solution of compound 99 (100 μg, 17 μL, 1.78 mg in 300 μL MQ water) was added to the precipitate, and the solution was left standing overnight at 4° C. The purification method was the same as that used to purify compounds 74 and 76, resulting in compound 97 (29 μg) as a solution in Tris buffer at pH 8 (final concentration = 1.78 mg / mL).

[0779] 3.28BHA[Lys]4[((α-NH-COPEG 24 -NH-COPEG4(PhTzMe) / TCO-PEG3-Nanobody) 1-3 (α-Lys(α-NHCy5)1(ε-NHDFO)1)1(α-NH2) 0-2 )(ε-NH-COPEG 1000 )4], G2, compound 98

[0780] A solution of TCO-PEG3-aldehyde (Conju-Probe) linker solution (0.7 mg, 1.46 μmol) in DMSO (50 μL) was added to a solution of nanobody ("Nanobody-N3-C terminal tag") (compound 79) (931 μL 1.07 mg / mL stock solution in PBS buffer at pH 6.5), followed by a solution of NaBH3CN (0.09 mg, 1.5 μmol) in water (19 μL). The reaction mixture was cooled to 4°C and the reaction was monitored by UPLC analysis. After 16 h, the reaction mixture was diluted to a total volume of 2.0 mL with PBS buffer (pH 6.5) and then purified by UPLC using centrifugal ultrafiltration (Amicon, 0.5 mL regenerated cellulose membrane, 10 kDa MWCO; 14 x 450 μL) with PBS buffer (pH 6.5): 5-20-30 ACN% in 15 min, using 0.01% TFA buffer; Nanobody (Compound 79) Rt = 8.46 min with m / z 13802; Product: 9.99 min with 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 76, yielding compound 98 (12 μg) as a solution in Tris buffer at pH 8 (final concentration = 575 μg / mL).

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787] Example 4: Synthesis of HER2-targeted dendrimer conjugates

[0788] a) Nanobody sequence information

[0789] Nanobody 2D3 sequence compound 79

[0790] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS

[0791] Nanobody 2D3 N-terminal tag, TEV, C-terminal azide ("N-terminal tag-Nanobody-N3")

[0792] GGSHHHHHHGMASMTGGQQMGRDLYENLYFQGEVQLVESGGSLVQPGGSLRLSCAA SGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNANNTLYLQM NSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS#

[0793] Nanobody 2D3 with C-terminal tag, TEV, azide ("Nanobody-N3-C-terminal tag")

[0794] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTH TDYADSVKGRFTISRNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQ GTQVTVSS#ENLYFQGHHHHHH

[0795] Where # = unnatural amino acid.

[0796] b) Nanobody-N3 expression plasmid

[0797] The coding sequence of anti-HER2 nanobody clone 2D3 (US20110028695A1SEQ ID NO: 1986) was inserted into the expression plasmid pET-His6-TEV-1B (Addgene plasmid 29653) using standard molecular biology techniques. The DNA sequence optimized by Escherichia coli K12 codons was synthesized and then cloned into the plasmid pET-His6-TEV-1B.

[0798] To incorporate unnatural amino acids into recombinant proteins, an amber stop codon (TAG) was inserted in-frame at the last position of the Nanobody coding sequence, followed by an ochre stop codon (TAA) or an opal stop codon (TGA) for translation termination. In the case of using a C-terminal his6 tag, an amber stop codon (TAG) was inserted at the last position of the Nanobody coding sequence, and then a sequence encoding a TEV protease cleavage site and a 6his tag followed by an ochre stop codon (TAA) or an opal stop codon (TGA) for translation termination was appended to the sequence.

[0799] c) Expression and purification of Nanobody-N3

[0800] The anti-HER2 nanobody 2D3 was expressed in E. coli strain B95 (DE3) (Mukai et al., Scientific Reports (2015), 5, 9699), which was transformed with a nanobody expression plasmid and an orthogonal pair expression plasmid pEVOL-pAcFRS.2.t1 (Amiram et al., Nat. Biotechnol (2015), 33 (12). The cells were cultured in a baffled shake flask at 37°C in a premium broth (25 g / L tryptone, 30 g / L yeast and 5 g / L glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4) until an OD of 1.0 was reached. 600The cell density of 0.7-1.0. Recombinant protein expression was induced by adding 1.5mM IPTG and 0.05% w / v L-(+)-arabinose and adding 1.5mM p-azido-phenylalanine, and it was carried out at 25°C for 20 hours. The bacteria were harvested by centrifugation and cell lysates were produced by high pressure homogenization, followed by the addition of a protease inhibitor cocktail, lysozyme and DNA enzyme treatment. Insoluble material can be dissolved in refolding buffer (6M guanidine hydrochloride, 50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8). After clarification by high speed centrifugation and filtration through a 0.45μM membrane filter, the his6-tagged nanobody was purified by immobilized metal affinity chromatography (IMAC) using nitrilotriacetic acid agarose loaded with nickel. Refolding on the column was achieved by: first with a column volume of 3M guanidine hydrochloride, 50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8; then with two volumes of 50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH 8 washing the bound protein. The bound protein was eluted with 2 column volumes of 50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8. After IMAC, Nanobody-N3 was further purified by anion exchange chromatography using HiTrap Q HP columns (GE Healthcare, catalog 17115301). Tris buffer (20mM, pH 8) was used for binding and washing steps, and a gradient of 0% to 50% 1M NaCl buffer (1M NaCl, 20mM Tris, pH 8) was used for elution. The relevant Nanobody fractions obtained were buffer exchanged into 20 mM Tris (pH 8) using an Amicon 10k MWCO filter unit (Merck, cat. UFC901008).

[0801] d) Conjugation of Nanobody-N3-C-terminal tag to dendrimers

[0802] Nanobody-N3-C-terminal tag was conjugated to dendrimer compounds 81-85 using bioorthogonal click chemistry according to the procedure described for compounds 74 and 76 to generate compounds 86-95, except that the linker

[0803] DBCO-Glu-NHPEG 24 CO-NHPEG3-TCO (compound 51, click chemistry tool).

[0804] The gel was run for compounds 41 to 45, which showed bands at the appropriate MW for the Nanobody-dendrimer conjugates. The purity of each Nanobody-dendrimer conjugate was confirmed using non-reducing 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 corresponding approximately to the size of the dendrimer plus the Nanobody added mass when imaged using a Typhoon biomolecular imager (GE Healthcare). Subsequent staining with Coomassie Brilliant Blue (CBB) revealed the presence of the Nanobody at the same position. No other bands were revealed by CBB, indicating the presence of unreacted Nanobodies and the purity of the preparation.

[0805] Example 5 SPR Binding Study of Affibody-MMAE Dendrimers and Erb2 ECD

[0806] Direct immobilization of ErbB2-ECD

[0807] ErbB2-ECD was immobilized onto a GLC sensor chip surface (Bio-Rad) at 25°C using a ProteOn XPR36 instrument using HBS-P+ running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween 20). The standard amine coupling method described in the amine coupling kit from Bio-Rad was used for the immobilization. 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. Any remaining activation sites were blocked with 1 M ethanolamine-HCl (pH 8.5). Approximately 590 RU (1 RU = 1 pg protein / mm 2 ) of the protein coupling at the average response level.

[0808] SPR assay analysis

[0809] 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% Tween20, 0.1 mg / ml BSA) as instrument running buffer (RB). After the immobilization procedure, various concentrations of anti-ErbB2 affibody and its MMAE-dendrimer conjugate were injected at 60 μl / min over the immobilized ErbB2 protein and their association was monitored for 90 seconds. Running buffer was then injected over the bound Ab-ErbB2 complex and dissociation was monitored for up to 60 min. Herceptin and affibody controls failed to dissociate from the core surface in running buffer within a moderate time frame (≤ 60 min). All binding measurements were performed in triplicate.

[0810] SPR data processing and analysis

[0811] The collected experimental data were processed using Scrubber-Pro software (www.biologic.com.au). Kinetic parameters (ka = association rate constant and kd = dissociation rate constant) and equilibrium dissociation constant (K) were derived by fitting each set of experimental data to the Langmuir 1:1 binding model. D =k d / k a ). All binding parameters are reported as mean + / - standard deviation.

[0812] SPR results

[0813] The affibody-MMAE-dendrimer conjugates specifically bound to the ErbB2 protein in a process that produced a classical kinetic binding profile. Kinetic binding analysis of the affibody-MMAE-dendrimer conjugates was subsequently performed using a dose response approach. The kinetic rates and affinity parameters derived from the SPR sensorgrams are summarized below. It is noted that the affibody is a dimer and therefore its binding constant is lower than that which would be observed for the monomeric affibody used in the dendrimer conjugates. No binding was observed for compounds 20 and 21. The results clearly indicate nanomolar specific binding of the affibody-dendrimer target, but at reduced amounts compared to the native affibody.

[0814] Table 2 SPR binding studies

[0815]

[0816] Example 6 Cell Growth Inhibition by Affibody-MMAE Dendrimers (SRB Assay)

[0817] HER2 was determined after 72 hours using the sulforhodamine B (SRB) assay [Voigt W. "Sulforhodamine B assay and chemosensitivity" Methods Mol. Med. 2005, 110, 39-48.] for various cancer cell lines. - (ES2) and HER2 + (SKOV3) cell growth inhibition of the cell line, where each experiment was run in duplicate. GI50 is the concentration required to inhibit total cell growth by 50% according to NCI standard protocols.

[0818] All compounds were tested based on equivalent drug loading. The structures show that the PEG 1100 targeted dendrimer conjugate is more effective in inhibiting cell growth than the dendrimer with a PEG 2000 surface. The affibody alone was not effective in this assay in either HER2+ or HER2- cell lines.

[0819] Table 3 HER2 - (ES2) and HER2 + (SKOV3) cell growth inhibition

[0820]

[0821] Example 7 Tolerance of Affibody-MMAE Dendrimers in vivo

[0822] Each group of mice was administered intravenous injections of dendrimers (0.1-0.3 ml solution in PBS) once a week for 3 weeks (days 1, 8, and 15). Mice were weighed daily and observed for signs of toxicity. Animals were monitored for up to 10 days after the final drug dose. Any mouse that exceeded ethical endpoints (≥20% weight loss, poor overall health) was immediately sacrificed and observations recorded.

[0823] Compound 26 at 1 mg / kg was well tolerated in both nude SCID mice and balb / c mice, with no animals needing to be sacrificed due to poor health.

[0824] Example 8: In vitro efficacy of conjugates

[0825] Description of cell lines used:

[0826]

[0827] GI 50 result:

[0828] Evaluation using MTT assay Cytotoxicity of lapatinib, compound 36 (control) and 2D3 nanobody-targeted dendrimers (compound 41) on MDA-MB-231, SKOV-3, SK-Br-3, NCI-N87 and OE-19 cells. Cells were seeded in 96-well plates at a density of 5 x 103 and incubated overnight. The cells were then treated with 1 log unit serial dilutions of the test composition for 72 h (six days for Herceptin). During the final 2 h of incubation, 10% of the culture medium volume of MTT (thiazolyl blue tetrazolium bromide (Merck, catalog number M5655, 5 mg / ml sterile solution) was added. The reduction of MTT in living cells produces an insoluble purple formazan metabolite. After 2 h of incubation, all culture medium was removed from the assay plate, 100 μl of DMSO was added and the plate was immediately read for absorbance at 570 nm. GI 50 The concentration that inhibited cell growth / proliferation by 50% was defined as the concentration that inhibited cell growth / proliferation by 50%. Cell viability and subsequent GI were determined from blank-corrected dose-response curves by 4-parameter nonlinear curve fitting in GraphPad Prism 7.02. 50 The results demonstrate that the exemplary conjugates of the present disclosure have potent cytotoxic effects, particularly against cell lines overexpressing HER2, such as SK-BR-3, NCI-N87, and OE19.

[0829] IC50 results

[0830]

[0831] *Based on estimated weight ADC; #Based on MMAE drug equivalents

[0832] Generation of HER2+ MDA-MB-231 breast cancer cell lines for paired high / low HER2 cell lines

[0833] According to the manufacturer's specification sheets, Lipofectamine 3000 is used by MDA-MB-231 breast cancer cells plasmid HER2WT (Addgene, 16257) transfection. After passage in the presence of 500ug / ml Geneticin (Thermo Fisher, catalog 10131035), the cell with the stable overexpression of HER2 WT is separated by fluorescence activated single cell sorting using MoFloAstrios (Beckman Coulter). The clonal cell colony separated from the process is further passaged in Geneticin, then second round of cell sorting and screening are carried out for transgenic expression. Then the clone selected is further amplified, and the storing solution of the cell line (also referred to as MDA-MB-231 / HER2) is cryopreserved to produce the main storing solution of stable transduced cells.

[0834] IC50 generation of low / high expression MDA-MB-231 model:

[0835] The cytotoxicity of compound 36 (control) and compound 41 (targeted) against MDA-MB-231 and MDA-MB-231 / HER2 transfected cells was evaluated via alamarBlue assay. 3 The density of 100 μg / ml was inoculated in 96-well plates and incubated overnight. The cells were then treated with the test composition of the specified concentration for 48 h. AlamarBlue (Thermo Fisher, DAL1025) was added during the final 4 h of incubation. The reduction of alamarBlue in living cells produces red fluorescent metabolites that can be read on a plate reader (560 nm excitation / 610 nm emission). Cell viability and subsequent IC were determined from blank-corrected dose-response curves by 4-parameter nonlinear curve fitting, variable slope (four parameters) in GraphPad Prism 7.02. 50 The following table shows the IC values ​​of compound 36 (control) and compound 41 (targeted) for MDA-MB-231 and MDA-MB-231 / HER2 cells. 50 IC values ​​of MDA-MB-231 / HER2 for compound 36 (control) and compound 41 (targeted) 50 The values ​​were 2.842 μM and 13.55 nM, respectively.Compound 41 (targeted) showed approximately 140-fold increase in MDA-MB-231 / HER2 cell growth inhibition compared to compound 36 (control).

[0836] The dose response curves and IC values ​​of compound 36 (control) against MDA-MB-231 and MDA-MB-231 / HER2 at different MMAE concentrations are provided below. 50 Values ​​are mean ± standard deviation (sd; n = 4):

[0837]

[0838] Dose response curves and IC of compound 41 (targeted) against MDA-MB-231 and MDA-MB-231 / HER2 at different MMAE concentrations 50 Values ​​are mean ± standard deviation (sd; n = 4):

[0839]

[0840] Example 9: Binding of 2D3-dendrimer conjugates to HER2+ cells

[0841] Nanobody-dendrimer conjugates of different sizes labeled with cyanine 5 (Cy5), compounds 42, 43 and 44, were used to demonstrate binding to HER2+ human cell lines relative to non-targeting compounds 37, 38 and 39 (G3, G4 and G5). HER2-expressing human metastatic cancer cell line MDA-MB-453 (ATCC HTB-131) and HER2-negative epithelial adenocarcinoma cell line MDA-MB-231 (ATCC HTB-26) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and penicillin-streptomycin (100 U / mL) at 37°C in a humidified atmosphere of 5% CO2 and subcultured using trypsin before confluence. Human adenocarcinoma cell line SKOV-3 ( HTB-77) cells were maintained in RPMI medium supplemented with 10% (v / v) FBS and penicillin-streptomycin (100 U / mL) and subcultured before confluence.

[0842] To measure cell association by fluorescence microscopy, cells were plated at 1 x 10 5 Cells / well were seeded in 8-well chamber slides and allowed to attach overnight. The next day, unconjugated dendrimer compounds 37, 38 and 39 (control) or nanobody dendrimer compounds 42, 43 and 44 were added to the culture medium to a final concentration of 0.5ug / ml and incubated on ice for 1 hour. Cells were stained to visualize the plasma membrane (wheat germ agglutinin-alexa fluor 488) and nuclei (Hoechst 33342). Cells were washed three times with 400ul of cooled Fluorobrite medium supplemented with 10% FBS. Cells were imaged using an Olympus IX83 microscope with a 40×0.9NA air objective with a standard “Pinkel” DAPI / FITC / CY5 filter set.

[0843] To measure cell association by flow cytometry, suspended cells were plated at 1 x 10 5 Cells / well were added to a 96-well assay plate. After incubation on ice for 1 hour with Nanobody-Dendrimers or unconjugated dendrimers, cells were washed three times with 200ul of DMEM containing 10% FBS to remove unbound dendrimers. Cells were then resuspended in cooled Fluorobrite medium supplemented with 10% FBS, and Cy5 fluorescence was measured using 642nm excitation and emission collected between 661-691nm. To measure internalization kinetics, Nanobody-Dendrimers were incubated with cells at 37°C for 24, 4, 1, 0.5 and 0.1 hours, then unbound Nanobody-Dendrimers were removed by washing and analyzed by flow cytometry.

[0844] In vitro association studies

[0845] MDA-MB-231, MDA-MB-231 / HER2 (HER2 knock-in) (described above), or SKOV-3 cells were seeded in 24-well plates (1×10 5 500 μL of appropriate growth medium supplemented with 10% (v / v) fetal bovine serum (FBS) in 500 μL of cells / well) and incubated with compound 36 or 41 at 3.33 nM, where the incubation time varied from 1 to 24 h. After incubation, non-bound / non-associated particles were removed from adherent cells by gently washing three times with DPBS (300 μL / well). The cells were washed with phenol red-free TrypLE TM The cells were removed from the plate by treatment with 150 μL / well of Express enzyme (1X) for 5-10 min. The plate was then placed on ice. Cell binding and association of the samples were then determined by flow cytometry by collecting the signal from Cy5.

[0846] Flow cytometry:

[0847] The results show that Nanobody-Dendrimer Compounds 42, 43 and 44 bind to HER2 positive MDA-MB-453 cells. Unconjugated dendrimer compounds 37, 38 and 39 do not bind to MDA-MB-453 cells. The data shown are the mean MFI of cells treated in triplicate wells with standard deviation. ANOVA and Dunnett's multiple comparison test were used for statistical analysis.

[0848] Mean fluorescence intensity (Cy5) of cells incubated with dendrimers:

[0849] Tested compounds MFI ANOVA Cells only 3.047±0.02 - 37 3.097±0.01 NS 38 6.827±0.07 NS 39 4.349±0.08 NS 42 50.83±14.98 <0.0001 43 158.4±6.611 <0.0001 44 176.9±4.041 <0.0001

[0850] Flow cytometry results

[0851] The results clearly indicated minimal binding of compound 36 (control) to all three cell lines within 24 h. Compound 41 (targeted) showed increased binding associated with the HER2 receptor expression level of the cell lines. By 24 h, flow cytometry showed that MDA-MB-231 / HER2 cells treated with compound 41 (146.1 ± 9.6) showed approximately 9 times stronger fluorescence compared to cells treated with compound 36 (16.05 ± 1.89). In addition, SKOV-3 cells treated with compound 41 (277.5 ± 5.9) showed approximately 16 times stronger fluorescence compared to cells treated with compound 36 (16.4 ± 6.86). The results are shown in Figure 4middle.

[0852] Dendrimer sizes G3 to G5:

[0853] 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. Unconjugated control dendrimer compounds 37, 38, and 39 had significantly lower association with MDA-MB-231 (HER2 negative) and MDA-MB-231 / HER2 (HER2 positive). 2D3-conjugated dendrimers 42, 43, and 44 had significant association with MDA-MB-231 / HER2 within 24 h, with compound 44 (G5) having the highest percentage of cell association (73.47% ± 0.92), followed by compound 42 (G3, 60.73 ± 0.21) and compound 43 (G4, 56.27 ± 1.02). Regardless of the dendrimer generation, 2D3 HER2-Nanobody conjugated to dendrimers significantly improved binding to cells overexpressing the HER2 receptor.

[0854] 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 within 24 h. Values ​​are mean ± standard deviation (SD; n=3):

[0855]

[0856]

[0857]

[0858] 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 within 24 h. Values ​​are mean ± standard deviation (SD; n=3):

[0859]

[0860] Average fluorescence intensity values ​​of compound 42 (G3), compound 43 (G4) and compound 44 (G5) with MDA-MB-231 and MDA-MB-231 / HER2 cells within 24 h. Values ​​are mean ± standard deviation (SD; n = 3):

[0861]

[0862] Average fluorescence intensity values ​​of compound 37 (G3), compound 38 (G4) and compound 39 (G5) with MDA-MB-231 and MDA-MB-231 / HER2 cells within 24 h. Values ​​are mean ± standard deviation (SD; n = 3):

[0863]

[0864]

[0865] The flow cytometry results of the conjugates are shown in Figure 5 middle.

[0866] Example 10: Studies demonstrating internalization of the conjugate into HER2 overexpressing cells

[0867] Confocal cell uptake

[0868] MDA-MB-231, MDA-MB-231 / HER2 and SKOV-3 cells were cultured at 1.0×10 4 Cells / well were plated into μ-glass 8-well chamber coverslips (ibidi) and allowed to attach overnight at 37°C, 5% CO2. Compounds 36 and 41 (3.33 nM) were then added and incubated for 1, 3, 6, and 24 hours. The cells were washed and then fixed with 1% paraformaldehyde for 20 min at room temperature. Cell membranes were fixed with Alexa Fluor 488-wheat germ agglutinin (AF488-WGA, 5 μg mL -1 ) for 10 min. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 2 μg mL) in DPBS at room temperature. -1 ) and counterstained for 10 min. Fluorescence images and optical sections were collected using a confocal microscope (Leica SP8). Images were processed using Fiji (ImageJ 1.52n).

[0869] Confocal microscopy images are shown in Figures 6 to 8 Confocal images show a small amount of Figure 6 a) Compound 36 and Figure 6 b) 41 associates with MDA-MB-231. However, the targeting compound 41 was internalized by MDA-MB-231 / HER2 and SKOV-3 cells after 24 h of incubation ( Figure 7 a and 8a), but not compound 36 ( Figure 7 b and 8b).

[0870] Example 11 Tumor Distribution of Tritium-Labeled MMAE-Conjugated Dendrimers (Compounds 40 and 45)

[0871] MDA-MB-231 / HER2 cells (5 x 10 6 10 cells in 50 μL PBS:Matrigel) were subcutaneously transplanted into the fourth mammary fat pad of female NOD / SCID mice (5-7 weeks old). Solid tumors were grown to 100 mm 3 (About 3-4 weeks). Mice were divided into two different groups, each consisting of six mice. Compound 40 (control group) or compound 45 (targeted group) (0.5 μCi in 100 μL, PBS pH 7) was then injected via the tail vein under isoflurane sedation. Mice were anesthetized with isoflurane after 48 h, and blood was collected via cardiac puncture just before cervical dislocation. Selected organs (including tumors, liver, spleen, kidneys, pancreas, lungs, heart and brain) were then removed, weighed and analyzed for tritium ( 3 H) Biodistribution of treatments.

[0872] The results are shown in Fig. 9 Compared to the unconjugated control compound 40 (1.88% dose / g ± 0.28), the targeted dendrimer compound 45 accumulated to a greater extent in HER2-positive tumors (3.53% dose / g ± 0.43), which is equivalent to an increase of approximately 80% in tumor uptake (p < 0.05). The difference in blood concentration was not significant between the targeted dendrimer and the control dendrimer, 4.52% dose / g ± 0.24 and 3.69% dose / g ± 0.31, respectively. Since the difference in pharmacokinetics is unlikely to affect tumor retention, 2D3 HER2-nanobody targeting improves dendrimer retention.

[0873] Example 12 Confocal imaging of efficacy and tumor uptake of Cy5-labeled MMAE-conjugated dendrimers (compounds 36 and 41) in the SKOV3 tumor model

[0874] Female NOD SCID mice (age 8 weeks) were inoculated subcutaneously on the flank with 3x10 6 SKOV3 cells. Grow solid tumors to 200mm 3 (About 3 weeks). The mice were divided into two different groups and then injected with compound 36 (control group) or compound 41 (targeted group) (0.5 mg / kg MMAE equivalent in 250 μL, PBS pH 7) via the tail vein under isoflurane sedation. (40 mg / kg) and (40mg / kg).

[0875] (a) Confocal imaging

[0876] After 48 h, mice were anesthetized with isoflurane, and blood was collected via cardiac puncture just before cervical dislocation (n=2 / group). Tumors were then removed, fixed overnight in 4% paraformaldehyde, washed in PBS and embedded in agarose. Tumors were then vibrating microtome sectioned to 100 μm, where sections were stained for nuclei (DAPI, concentration, time, supplier) and blood vessels (CD31, concentration, time, supplier). Fluorescence images and optical sections were collected using a confocal microscope (Leica SP8). The results are shown in Fig.10 and Fig.11 The targeted dendrimer (compound 41) showed uptake in both the core and peripheral regions of the tumor, whereas compound 36 did not.

[0877] (b) Efficacy of the conjugate

[0878] Tumor measurements (n=6 / group) were obtained at regular intervals until the ethical endpoint was met. The results are shown in Fig.12 (Plot of mean tumor volume over time), Fig.13 (survival over time) and Fig.14 (Average weight change over time). and In contrast, the targeted dendrimer compound 41 showed complete tumor regression.

[0879] Example 13. Kinetics of internalization of multiple nanobody dendrimers over four generations in the MDA-MB-231 / HER2+ cell line

[0880] Compounds 90 and 91 isolated as described above were used to compare binding to HER2+ human cell lines with different numbers of Nanobodies attached per dendrimer using 4th generation, Cy5-labeled Nanobody-dendrimer conjugates with the siderophore-derived chelator deferoxamine (DFO) conjugated to 1 (single Nanobody) or 2 to 4 (multiple Nanobodies) anti-HER2 2D3 Nanobodies to compare binding to HER2+ human cell lines with different numbers of Nanobodies attached per dendrimer.

[0881] The epithelial adenocarcinoma cell line MDA-MB-231 / HER2 with HER2 knock-in (described above) was maintained in a humidified atmosphere of 5% CO2 at 37°C in a medium containing 10% FBS and penicillin-streptomycin (100 U mL -1 ) in Dulbecco's modified Eagle's medium (DMEM).

[0882] To measure cell binding 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, 6, 12 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-cooled and incubated on ice for 6 hours with 30 nM single and multiple Nanobody dendrimers.

[0883] 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 stained with phenol red-free TrypLE TM Express enzyme (1X) (Gibco, 12604013) from the plate. The cells were resuspended in chilled DPBS for analysis by flow cytometry (Stratedigm S1000EON, California), where the presence of dendrimers was measured by collecting the signal from Cy5. To estimate the amount of internalized dendrimers at each concentration and time point, the Cy5 signal of cells incubated with dendrimers on ice was considered as the maximum surface-bound dendrimers assuming no internalization. This signal was subtracted from the signal measured in cells incubated at 37°C (which represents both surface-bound and internalized dendrimers) to isolate the signal from internalized dendrimers only (see Fig.15 ).

[0884] At low concentrations, multiple (compound 91) conjugated anti-HER2 nanobody dendrimers were internalized faster than single (compound 90). At higher concentrations, compound 90 showed a higher internalization level than compound 91 after about 2 hours.

[0885] Example 14. Confocal imaging of conjugates with single and multiple Nanobodies in HER2 high cell lines

[0886] HER2 high SKOV-3 cells were cultured at 1.0×10 4 Cells / well were plated into μ-slide 8-well chamber coverslips (ibidi) and allowed to attach overnight at 37°C, 5% CO2. Compound 91 (multiple 2D3-dendrimer conjugates) and compound 90 (single 2D3-dendrimer conjugate) (3.33 nM) were then added and allowed to incubate for 1, 3, 6, and 24 hours. Cells were washed with DPBS and then fixed with 1% paraformaldehyde for 20 min at room temperature. Cell membranes were stained with Alexa Fluor 500 of wheat germ agglutinin in DPBS at room temperature. Conjugate (AF488-WGA, 5 μg mL -1) for 10 min. The nuclei were stained with Hoechst 33342 (2 μg mL -1 ) were counterstained for 10 min. Fluorescence images and optical sections were collected using a confocal microscope (Leica SP8). Images were processed using Fiji (ImageJ 1.52p).

[0887] Confocal microscopy images are shown in Fig.16 and 17 After 24 h of incubation, both compounds 90 and 91 bound and internalized in the case of SKOV-3 cells. Notably, compound 91 showed a greater degree of binding and internalization at the 3 and 6 h time points compared to compound 90.

[0888] At this concentration multiple (Compound 91) conjugated anti-HER2 Nanobody G4 dendrimers were internalized faster than single Nanobody G4 dendrimers (Compound 90) and the difference could be seen up to 6 hours, but by 24 hours they did not appear different.

[0889] Example 15. Imaging studies of SKOV3 breast cancer xenografts using targeted Zr-containing radionuclide dendrimers

[0890] Accumulation of 89Zr-labeled HER2-targeted and non-targeted dendrimer constructs was studied in the SKOV3 murine xenograft model of ovarian cancer. Biodistribution was measured by PET-CT until 9 days post-injection and verified by ex vivo gamma scintigraphy of resected organs at days 2 and 9 (where available). This study was conducted in 3 parts.

[0891] Tumor initiation and growth

[0892] 5x 10 6 SKOV3 cells (in 50 μL 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 prior to injection of imaging compounds.

[0893] At the time of imaging, all tumors were palpable, with a size of approximately 3-5 mm at the time of imaging experiments.

[0894] Study Compounds

[0895] The compounds in the following table are labeled as follows.

[0896] Table. Study compounds

[0897]

[0898] # = Repeat

[0899] Radiolabeling of Research Compounds with Zr-89 and Radio-TLC Analysis

[0900] All constructs (pretreated to remove iron as needed) were cultured at 37°C:

[0901] http: / / jnm.snmjournals.org / content / 44 / 8 / 1271.long) and in 0.1 M pH 7.4 HEPES buffer for excess dendrimers (for excess see Table 1) 89 Zr were incubated together for 45 minutes. Samples of each solution were obtained and mixed with 50mM DTPA 1: 1. 5uL of each solution was spotted on TLC paper (Agilent iTLC-SG glass microfiber chromatography paper impregnated with silica gel) and run with 50:50H2O: ethanol. The plate was then imaged on an Eckert & Ziegler microscanner and flow counting iTLC reader. If necessary, unbound zirconium was removed by purification using a 7KMWCO Zeba spin column (Thermo Scientific) according to the manufacturer's protocol. All samples showed >95% labeling. For quality control, control experiments were performed to monitor free 89 Zr and DTPA combined 89 The elution behavior of Zr was investigated and samples were also run with and without DTPA to examine the elution behavior of Zr. 89 Any unbound chelator labeled with Zr. Fig.18 Representative radiometric TLC images are shown in 89 Zr is bound to the dendron and not free 89 Zr. The following radioactive TLCs are included:

[0902]

[0903] Study Injection Details

[0904] For in vivo imaging experiments, the study compound was injected into two mice at 100 uL via the tail vein (29G needle; approximately 1.5 to 3.5 MBq) to monitor tumor accumulation and biodistribution at various time points. Ex vivo quantification was performed by gamma counting to determine organ distribution 9 days after injection. For biodistribution experiments, the construct was injected into two mice to monitor ex vivo tumor accumulation and biodistribution at 48 h after injection by gamma counting.

[0905] result

[0906] PET-CT imaging

[0907] Images at 4h, 24h, 48h, 5d, 7d and 9d after injection were acquired using a 30-90 minute static acquisition. PET images were reconstructed using the ordered subset expectation maximization (OSEM2D) algorithm and analyzed using the Inveon Research Workplace software (IRW 4.1) (Siemens), which allows fusion of CT and PET images and definition of regions of interest (ROIs). The CT and PET data sets for each individual animal were compared using IRW software (Siemens) to ensure good overlap of the organs of interest. The activity / voxel was converted to nci / cc using a conversion factor obtained by scanning a cylindrical phantom filled with 89Zr of known activity to account for PET scanner efficiency. The activity concentration was then expressed as the percentage of injected activity / cm corrected for attenuation. 3 tissue, which can be approximated as percent injected dose / g (%ID / g). Fig.19 Shown in FIG. 1 are graphs of the percentage of zirconium dose / gram injected in (a) kidney, (b) liver, and (c) tumor over 9 days for compounds 89, 90, and 92. Representative PET images are shown in FIG. Fig. 20 Middle. Representative maximum intensity projections of radiolabeled conjugates. All PET data are expressed as becquerel / voxel (cm3) and have been thresholded to highlight tumor uptake.

[0908] At 2 and 9 days post-injection, organs were removed and signal intensity was quantified by ex vivo gamma analysis and imaging was performed. In addition, n=2 animals / cohort were selected at 48 hours and biodistribution was evaluated by gamma analysis. The ex vivo biodistribution results are shown in Fig.21 , which provides tumor:organ ratios of ex vivo signals for injected dose / g at days 2 and 9.

[0909] The results showed higher tumor:blood ratios for targeting versus non-targeting, especially in compound 88 (small G3 dendrimers) at day 2. By day 9, tumor:blood ratios for targeting versus non-targeting were better, and especially for compounds 90 and 92 (larger G4 and G5 dendrimers).

[0910] The percentage of injected Zr dose / g in ex vivo tumors and organs is also shown in Fig. 22 in the table.

[0911] The results showed higher in-tumor signals for targeted versus non-targeted, especially at day 2 for compounds 90 and 92 (larger G4 and G5 dendrimers) and at day 9 for compound 89 (G3 dendrimers).

[0912] in conclusion

[0913] 1. The larger the dendron, the better the tumor accumulation. 2D3 showed rapid clearance and low accumulation.

[0914] 2. Targeted therapy resulted in higher tumor accumulation compared to non-targeted dendrimers.

[0915] 3. Compound 88 (G3 1K-Nanobody) showed significantly enhanced signal in tumors compared to other dendrimers.

[0916] 4. Compounds 90 and 92 (G4 and G5 Nanobodies) showed >12% ID / g at 48h, and >4% and >8% at day 9, respectively.

[0917] 5. Small Nanobody-containing dendrimers and Nanobody alone had higher renal retention. No unusual accumulation in clearance organs was observed, with liver and spleen signals showing expected concentration ranges as typically observed for similar systems.

[0918] Example 16: G5, PEG with or without Cabazitaxel 1000 In vitro association studies of dendrimers

[0919] MDA-MB-231, MDA-MB-231 / HER2 (HER2 knock-in), or SKOV-3 cells were seeded in 24-well plates (1×10 5 Cells / well) were added to 500 μL of appropriate growth medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin and incubated with compound 70 (G5, PEG1000, CTX) or 78 (2D3, G5, PEG1000, CTX) at 3.33 nM, with incubation time varying from 1 to 24 h. After incubation, non-bound / non-associated particles were removed from adherent cells by gently washing three times with DPBS (300 μL / well). The cells were washed with phenol red-free TrypLE TM The cells were removed from the plate by treatment with 150 μL / well of Express enzyme (1X) for 5-10 min. The plate was then placed on ice. Cell binding and association of the samples were then determined by flow cytometry by collecting the signal from Cy5.

[0920] Flow cytometry results

[0921] The results clearly indicated minimal binding of Compound 70 (control) to all three cell lines within 24 h. Compound 78 (targeted) showed increased binding associated with the HER2 receptor expression levels of the cell lines. By 24 h, flow cytometry showed that MDA-MB-231 / HER2 cells treated with Compound 78 (targeted) (4,269 ± 322) showed approximately 340 times stronger fluorescence compared to cells treated with Compound 70 (control) (12.4 ± 0.64). In addition, SKOV-3 cells treated with Compound 78 (targeted) (1,817 ± 60.8) showed approximately 160 times stronger fluorescence compared to cells treated with Compound 70 (control) (11.4 ± 0.42). The results are shown in Figure 23-25 middle.

[0922] Mean fluorescence intensity (MFI) values ​​of compound 70 (control) and compound 78 (targeted) with MDA-MB-231, MDA-MB-231 / HER2 and SKOV-3 cells within 24 h. Values ​​are mean ± standard deviation (SD; n=2):

[0923]

[0924] It will be appreciated by those skilled in the art that various changes and / or modifications may be made to the above-described embodiments without departing from the broad overall scope of the present disclosure. Therefore, the embodiments of the present invention are considered in all aspects to be illustrative and not restrictive.

[0925] References

[0926] Abdollahpour-Alitappeh et al (2017) Novelty in Biomedicine 4:145-151.

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[0928] Arezumand et al. (2017) Front Immunol 8:1746.

[0929] Bethesda, Md., 1987 and 1991.

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[0934] Honnegher and Plükthun(2001)J Mol Biol 309:657-670.

[0935] Hussack et al. (2018) BMC Res notes 11(1):866.

[0936] Kabat Sequences of Proteins of Immunological Interest,National Institutes of Health,

[0937] Messerer et al. (2004) Clin. Cancer Res 10(19):6638-6649.

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[0939] Mukai et al. (2015) Scientific Reports 5:9699.

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[0944] Pohlmann et al. (2009) Clin Cancer Research 15(24):7479-7491.

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[0947] Vaneycken et al. (2011) FASEB J 25(7):2433-2446.

[0948] Verel et al. (2003) J Nucl Med 44(8):1271-1281.

[0949] Voigt(2015)Methods Mol Med 110:39-48.

[0950] Wu et al. (2018) Translational Oncology 11(2):366-373. Sequence Listing <110> Star Pharma Pte Ltd <120> Targeted dendrimer conjugates <130> 530387PCT <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 124 <212> PRT <213> Artificial sequence <220> <223> 2D3 Nanobody Sequence <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Trp Ser Gly Thr His Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Asn Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Trp Arg Asp Ala Gly Thr Thr Trp Phe Glu Lys Ser Gly 100 105 110 Ser Ala Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 2 <211> 157 <212> PRT <213> Artificial sequence <220> <223> 2D3 Nanobody N-terminal tag, TEV, C-terminal azide <220> <221> X <222> (157)..(157) <223> Unnatural amino acids <400> 2 Gly Gly Ser His His His His His Gly Met Ala Ser Met Thr Gly 1 5 10 15 Gly Gln Gln Met Gly Arg Asp Leu Tyr Glu Asn Leu Tyr Phe Gln Gly 20 25 30 Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly 35 40 45 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 50 55 60 Ala Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 65 70 75 80 Ser Ser Ile Asn Trp Ser Gly Thr His Thr Asp Tyr Ala Asp Ser Val 85 90 95 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Asn Asn Thr Leu Tyr 100 105 110 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 115 120 125 Ala Lys Asn Trp Arg Asp Ala Gly Thr Thr Trp Phe Glu Lys Ser Gly 130 135 140 Ser Ala Gly Gln Gly Thr Gln Val Thr Val Ser Ser Xaa 145 150 155 <210> 3 <211> 138 <212> PRT <213> Artificial sequence <220> <223> 2D3 Nanobody, with C-terminal tag, TEV, azide <220> <221> X <222> (125)..(125) <223> Unnatural amino acids <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Ser Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asn Trp Ser Gly Thr His Thr Asp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Asn Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asn Trp Arg Asp Ala Gly Thr Thr Trp Phe Glu Lys Ser Gly 100 105 110 Ser Ala Gly Gln Gly Thr Gln Val Thr Val Ser Ser Xaa Glu Asn Leu 115 120 125 Tyr Phe Gln Gly His His His His His His 130 135

Claims

1. A dendrimer-targeting agent conjugate comprising: a) a dendrimer, said dendrimer comprising i) a core unit (C), wherein the core unit comprises the following structure: as well as ii) 3 to 4 generations of building blocks (BU), each building block being a lysine residue; wherein the core unit is covalently attached to at least two structural units via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a structural unit; b) a HER2 targeting agent, wherein the HER2 targeting agent has a heavy chain variable (V H ) domain, a single domain antibody having a molecular weight of 10 kDa to 16 kDa and comprising an antigen binding site, wherein the HER2 targeting agent comprises an amino acid sequence of complementarity determining regions (CDRs) 1, 2 and 3 from the amino acid sequence SEQ ID NO.1, and the HER2 targeting agent is covalently linked to the core of the dendrimer via a spacer group, and the spacer group comprises a polyethylene glycol (PEG) group; c) a therapeutic agent covalently attached to the surface building block of the dendrimer via an enzymatically cleavable linker comprising a peptide group, wherein the therapeutic agent is auristatin; and d) a hydrophilic polymer group covalently attached to the surface structural unit of the dendrimer, wherein the hydrophilic polymer group is a PEG group having an average molecular weight of 900 to 2300 Da.

2. The conjugate of claim 1, wherein the HER2 targeting agent comprises the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.

3.

3. The conjugate of claim 1, wherein the targeting agent precursor comprises a non-natural amino acid residue, The non-natural amino acid residue is the following residue:

4. The conjugate of claim 1, wherein the targeting agent is covalently linked to the spacer group via the C-terminus of the targeting agent.

5. The conjugate of claim 1, wherein the therapeutic agent is monomethyl auristatin E.

6. The conjugate of claim 1, wherein the cleavable linker comprises a Val-Cit-PAB group.

7. The conjugate of claim 1, wherein the dendrimer has three generations of structural units.

8. A pharmaceutical composition comprising: i) the conjugate according to any one of claims 1 to 7; and ii) a pharmaceutically acceptable excipient.

9. Use of the conjugate according to any one of claims 1 to 7 or the composition according to claim 8 in the manufacture of a medicament for treating cancer.

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

Images