KR20240099391A - Targeted linear conjugate containing polyethyleneimine and polyethylene glycol and multicomplex containing the same - Google Patents

Abstract

The present invention relates to a multicomplex comprising a linear conjugate of LPEI and PEG. The LPEI and PEG fragments of the linear conjugate are preferably joined by [3 + 2] cycloaddition between the azide and the alkene or alkyne to form 1,2,3-triazole or 4,5-dihydro-1H-[1 ,2,3]Produces triazole. Preferably, the linear conjugate is further conjugated to a targeting fragment to allow selective interaction with specific cell types. Conjugates can form multicomplexes with therapeutic agents such as nucleic acids and deliver the therapeutic agent to cells.

Description

Targeted linear conjugate containing polyethyleneimine and polyethylene glycol and multicomplex containing the same

The present invention relates to a multicomplex comprising a linear conjugate of LPEI and PEG. The LPEI and PEG fragments of the linear conjugate are preferably joined by a [3 + 2] cycloaddition between the azide and the alkene or alkyne to form 1,2,3-triazole or 4,5-dihydro-1H-[1 ,2,3]Produces triazole. Preferably, the linear conjugate is further conjugated to a targeting fragment to allow selective interaction with specific cell types. Conjugates can form multicomplexes with therapeutic agents such as nucleic acids and deliver the therapeutic agent to cells.

Cancer remains a leading cause of death worldwide. For most solid tumors after surgical removal, chemotherapy remains the key treatment option to manage residual cancer cells. A major reason why chemotherapy fails is ineffective targeting and uptake of chemotherapy agents (Vasir & Labhasetwar, Technology in Cancer Research & Treatment, 4(4): 363-374 (2005)). Poor accessibility to tumors requires higher doses, and the properties of chemotherapeutic agents lead to non-specific uptake and toxicity in healthy cells. Targeted drug delivery strategy involves reversibly binding a therapeutic agent to a targeting ligand and selectively delivering it to the cells to be treated, and is currently being applied to various chemotherapy agents for clinical use. This strategy has shown promise in maximizing the safety and efficacy of a given chemotherapeutic agent because selective delivery into target cells avoids non-specific uptake and associated toxicity to healthy cells, where the maximum tolerated dose may be higher. (Srinivasarao & Low, Chem. Rev., 117: 12133-12164, (2017)).

Cationic polymers are known to form supramolecular multicomplexes with negatively charged nucleic acids in solution. For example, linear polyethyleneimine (LPEI) can be protonated at physiological pH and therefore possesses a net positive charge. When LPEI reacts with a nucleic acid that possesses a net negative charge at physiological pH, the LPEI and the nucleic acid can form a multicomplex that is maintained by electrostatic interactions. These supramolecular multicomplexes can be absorbed by cells in vivo and can deliver nucleic acid sequences into cells. Accordingly, supramolecular multicomplexes containing cationic polymers and nucleic acids can be used as carriers for therapy.

Despite their potential, there have been technical challenges associated with forming uniform and well-characterized cationic polymers. Multicomplexes containing only LPEI may be susceptible to aggregation and interaction with serum proteins, limiting their potential as nucleic acid delivery agents. To overcome this problem, polymer LPEI can be conjugated or copolymerized with polyethylene glycol (PEG). The PEG fragment can help shield LPEI from the surrounding matrix and improve the biocompatibility and blood circulation of the resulting multicomplex.

However, in general, binding of PEG to LPEI occurs by the formation of covalent bonds between the electrophilic PEG fragment(s) and secondary amines embedded within the LPEI fragment backbone, thus forming a branched, heterogeneous conjugate in which the PEG fragments are randomly incorporated. A gate is derived, which is characterized based on the average PEG density involved. In these conjugates, the PEG fragments, which may be single or multiple, are regularly linked to the LPEI fragment and are generally site-specific. This random synthesis and inaccurate characterization of LPEI-PEG conjugates can make it difficult to establish a clear structure-activity relationship (SAR) between the structure of the conjugate and the activity of the resulting supramolecular multicomplex. Therefore, a homogeneous LPEI-PEG conjugate with a well-defined chemical structure is needed.

The present invention encompasses LPEI and PEG fragments that are linked by unambiguous bonds formed through a defined chemoselective reaction rather than through random, uncontrolled binding of the electrophilic PEG fragment to one of the multiple nucleophilic species of the LPEI fragment backbone. Provides a conjugate that The well-defined linkage not only ensures a constant and predictable ratio of LPEI to PEG fragments, but further ensures a defined linear conjugate instead of a random branched conjugate. Accordingly, the LPEI fragment is linked to a single PEG fragment in a linear end-to-end fashion. Chemiselective coupling of LPEI fragments to PEG fragments can occur using any suitable chemical precursor capable of forming a chemoselective bond. In a preferred embodiment, the chemoselective coupling of the LPEI fragment to the PEG fragment occurs by [3 + 2] cycloaddition between the azide and the alkyne or alkene. Alternatively, the chemoselective bond results from a thiol-ene reaction between a thiol and an alkene. When the chemoselective bond is between an azide and an alkyne or alkene, the bond created is 1,2,3-triazole (when an alkyne is bonded) or 4,5-dihydro-1H-[1,2,3] It is a triazole (when an alkene is combined). When the chemoselective bond is between a thiol and an alkene, the resulting bond is a thiotester.

In the case of preferred conjugates of the invention, the PEG fragment is further selectively combined with a targeting fragment to target a specific cell type, thereby targeting the composition, conjugate and/or multicomplex of the invention to said specific cell type, Facilitates their absorption. Accordingly, a preferred embodiment comprises one or more (e.g. one) targeting fragment(s) such as hEGF, HER2 ligand, DUPA or folate etc. specifically linked to the LPEI-PEG biconjugate, which in turn is linked to the LPEI-PEG -Targeting fragment triconjugates can be formed to target the corresponding receptor such as hEGFR, HER2, PSMA or folate receptor on a specific cell type, typically a cancer cell type. In the case of the multicomplexes of the present invention, this triple conjugate is combined with a polyanion, such as a nucleic acid, herein preferably polyinosinic acid:polycytidylic acid (poly(IC)), wherein the polyanion such as poly(IC) is It may act as a cytotoxic and/or immunostimulatory payload that is delivered and absorbed into the body.

Furthermore, advantageously and surprisingly, the present inventors have found that the preferred conjugates and multicomplexes produced according to the invention significantly reduce heterogeneity due to the defined chemoselective binding of the LPEI fragment to the PEG fragment and thus potentially biologically active. The number of conjugates and multicomplexes is significantly reduced to form multicomplexes of suitable size, as well as maintaining or even increasing the overall biological activity, such as potency and selectivity to reduce the survival of targeted cancer cells and induce cell death. The point was confirmed.

Accordingly, in one aspect, the present invention provides a composition comprising a conjugate, wherein the conjugate includes a linear polyethyleneimine (LPEI) fragment comprising an alpha terminus and an omega terminus; and a polyethylene glycol (PEG) fragment, preferably a linear polyethylene glycol (PEG) fragment, comprising a first terminal end and a second terminal end; The omega terminus of the LPEI fragment is connected by a covalent moiety to the first terminal end of the PEG fragment; The covalent moiety is not an amide; Preferably the alpha terminus of the LPEI fragment is bonded to a methyl group or a hydrogen atom, more preferably the alpha terminus of the LPEI fragment is bonded to a hydrogen atom; Preferably the second terminal end of the PEG fragment is linked to the targeting fragment.

In one aspect, the present invention provides a composition comprising a conjugate, wherein the conjugate includes a linear polyethyleneimine fragment comprising an alpha terminus and an omega terminus; and a polyethylene glycol segment comprising a first terminal end and a second terminal end; The alpha terminus of the polyethyleneimine fragment is the initiation residue; The omega terminus of the polyethyleneimine fragment is connected by a covalent moiety to the first terminal end of the polyethylene glycol fragment; The covalent moiety is neither a single bond nor an amide; Preferably the second end of the polyethylene glycol fragment is capable of reacting, and preferably the second end is capable of binding to a targeting fragment.

In one aspect, the present invention provides a composition comprising a conjugate, wherein the conjugate

A linear polyethyleneimine fragment comprising an alpha terminus and an omega terminus; and

A polyethylene glycol fragment comprising a first terminal end and a second terminal end.

Includes;

The alpha terminus of the polyethyleneimine fragment is the initiation residue;

The omega terminus of the polyethyleneimine fragment is connected to the first end of the polyethylene glycol fragment by a covalent bond -ZX ¹ -, where -Z- is not a single bond, -Z- is not an amide, and -X ¹ - is a divalent bond. is a covalent moiety;

The second terminal end of the polyethylene glycol fragment is capable of binding, preferably wherein the polyethylene glycol fragment binds the targeting fragment. In a preferred embodiment of this aspect, the composition consists of the conjugate.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

here

n is any integer from 1 to 1,500;

m is any integer from 1 to 200, preferably m is any integer from 1 to 100;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

X ¹ and X ² are independently divalent covalent moieties;

Z is a divalent covalent moiety, Z is not a single bond and Z is not -NHC(O)-;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor,

Preferably, the composition provides a composition comprising the conjugate.

In another aspect, the present invention provides a conjugate of Formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

here

n is any integer from 1 to 1,500;

m is any integer from 1 to 200, preferably m is any integer from 1 to 100;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

X ¹ and X ² are independently divalent covalent moieties;

Z is a divalent covalent moiety, Z is not a single bond and Z is not -NHC(O)-;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H,

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200, preferably m is any integer from 1 to 100;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ;

R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ,

R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

^{where ​} _​ ^{​ ​ ​} -O-, -S-, -NR ¹³ -, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, wherein each divalent phenyl and divalent heteroaryl are independently selected from one or more R is optionally substituted with ¹³ , and each divalent heterocycle is optionally substituted with one or more R ¹⁴ ; ^R11 , R ¹² and R ¹³ are independently -H or C ₁ -C ₆ alkyl at each occurrence; R ¹⁴ is independently at each occurrence -H, C ₁ -C ₆ alkyl or oxo;

^{and ​} _​ ^{​ ​ ​ ​} , -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, and each divalent phenyl and divalent heteroaryl is independently selected from one or more R ²³ and each divalent heterocycle moiety is optionally substituted with one or more R ²⁴ ; R ²¹ , R ²² and R ²³ are each independently -H, -CO ₂ H or C ₁ -C ₆ alkyl at each occurrence, and each C ₁ -C ₆ alkyl is one or more -OH, oxo, C ₆ -C ₁₀ optionally substituted with aryl or 5- to 8-membered heteroaryl; R ²⁴ is independently at each occurrence -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo;

L is preferably a targeting fragment capable of binding to a cell, and preferably the composition consists of the conjugate.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200, preferably m is any integer from 1 to 100;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ;

R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen, or two R ^A1 together with the atoms to which they are attached form one or more fused C ₆ -C ₁₀ aryl, may form a C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl ring, wherein each fused aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ;

R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

^{where ​} _​ ^{​ ​ ​} -O-, -S-, -NR ¹³ -, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, wherein each divalent phenyl and divalent heteroaryl are independently selected from one or more R is optionally substituted with ¹³ , and each divalent heterocycle is optionally substituted with one or more R ¹⁴ ; ^R11 , R ¹² and R ¹³ are independently -H or C ₁ -C ₆ alkyl at each occurrence; R ¹⁴ is independently at each occurrence -H, C ₁ -C ₆ alkyl or oxo;

X²is the chemical formula -(Y²)_q- is the binding moiety of, q is an integer from 1 to 50, and each Y²In the case of a chemical bond, -CR²¹R²²-, N.R.²³-, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moiety, divalent heterocycle moiety, and divalent heteroaryl moiety, each divalent phenyl and divalent heteroaryl This one or more R²³is optionally substituted with, and each divalent heterocycle moiety is one or more R²⁴is optionally substituted with; R²¹, R²² and R²³In each case, independently -H, -CO₂H or C_One-C₆ alkyl, and each C_One-C₆ Alkyl contains one or more -OH, oxo, C₆-C₁₀ optionally substituted with aryl or 5- to 8-membered heteroaryl; R²⁴In each case independently -H, -CO₂H,C_One-C₆ alkyl or oxo,

L provides a conjugate, preferably a targeting fragment capable of binding to cells.

In a further aspect, the present invention provides a method of synthesizing a composition comprising a conjugate of Formula I, wherein the LPEI fragment comprising an azide is combined with a PEG comprising an alkene or alkyne at a pH of less than about pH 5, preferably less than about pH 4. A method comprising reacting with a fragment is provided. In some preferred embodiments, the LPEI fragment comprises an azide at the omega terminus and the PEG fragment comprises an alkene or alkyne at the first terminus.

In a further aspect, the invention relates to a polycomplex comprising a composition described herein and a polyanion, preferably wherein the polyanion is a nucleic acid, more preferably wherein the nucleic acid is RNA, and even more preferably wherein the polyanion is polyanion. Provided is a multicomplex, inosinic acid:polycytidylic acid (poly(IC)).

In a further aspect, the invention provides a multicomplex comprising a composition described herein and a nucleic acid. In a further aspect, the invention provides a multicomplex comprising a composition described herein and a nucleic acid, wherein the nucleic acid is RNA. In a further aspect, the present invention provides a multicomplex comprising the composition described herein and polyinosinic acid:polycytidylic acid (poly(IC)).

In another aspect, the invention provides a triconjugate as described herein, preferably a conjugate of Formula I ^* or Formula I above, and a polyanion such as a nucleic acid, preferably polyinosinic acid:polycytidylic acid (poly(IC)). Provides a multi-complex that

In one aspect, the invention provides a pharmaceutical composition comprising a triconjugate, preferably a conjugate of Formula (I ^*) or Formula (I) above and/or a multicomplex as described herein and a pharmaceutically acceptable salt thereof. .

In one aspect, the invention provides a multicomplex described herein or a pharmaceutical composition comprising a multicomplex described herein for use in the treatment of a disease or disorder, preferably cancer.

In one aspect, the invention provides use of the multicomplexes described herein for the manufacture of a medicament for the treatment of a disease or disorder, such as cancer.

In another aspect, the invention provides a method of treating a disease or disorder, such as cancer, in a subject in need thereof, comprising administering to the subject an effective amount of a multicomplex described herein.

The compositions and multicomplexes of the invention, including the non-random linear LPEI-PEG biconjugates and resulting triconjugates described herein, not only ensure a constant and predictable ratio of LPEI to PEG fragments, but also typically and preferably further ensure structurally defined linear conjugates of LPEI fragments to PEG fragments. Therefore, they have greater batch-to-batch consistency, are easier to manufacture, and have a more predictable SAR compared to branched LPEI-PEG biconjugates currently prepared using the uncontrolled, random synthesis strategy described above. Grant.

Also advantageously and surprisingly, when the non-random linear conjugates of the invention described herein are combined with polyanions and nucleic acids such as poly(IC) to form polycomplexes and administered to cells, polycomplex XXX is surprisingly random. Not only do they maintain the same anti-tumor activity as multicomplexes made using branched conjugates, but they even increase their activity. Accordingly, a significant reduction in the number and variability in the conjugate structures used and in the possible (bio)active structures, including targeting, expression and subsequent uptake of these targeting fragments on the surface of targeted cells. Despite this, there is no loss in efficacy of the linear LPEI-/-PEG:nucleic acid multicomplex described herein. Quite the opposite, the conjugates and compositions of the present invention can even increase overall biological activity. Additional features and advantages of the present technology will become apparent to those skilled in the art upon reading the detailed description of the invention below, and additional aspects and embodiments of the invention will become apparent as the description continues.

Figure 1A shows the size of triplicate LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplexes in HBG buffer, pH 7.2, 0.125 mg/mL, N/P ratio of 2.4. Indicates DLS back scatter, which measures distribution. The z-average diameter was 306 nm with a polydispersity index (PDI) of 0.35.
Figure 1b shows the size of triplicate LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplexes in HBG buffer, pH 7.2, 0.125 mg/mL, N/P ratio of 4.0. It represents DLS backscattering, which measures the distribution. The z-average diameter was 116 nm with a polydispersity index (PDI) of 0.08.
Figure 1c shows the size of triplicate LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplexes in HBG buffer, pH 7.2, 0.125 mg/mL, N/P ratio of 5.6. It represents DLS backscattering, which measures the distribution. The z-average diameter was 107 nm with a polydispersity index (PDI) of 0.109.
Figure 2 shows triplicate LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplexes obtained in HBG buffer, pH 7.2, 0.1 mg/mL, 1 mL volume, N/ of 4. Shows DLS backscattering, which measures the size distribution and ζ-potential at the P ratio. The z-average diameter was 121 nm with a polydispersity index (PDI) of 0.087. The ζ-potential was 28.7 mV.
Figure 3 shows triplicate LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OMe:poly(IC) multicomplexes in 50 mM acetate buffer, pH 4.3, 5% glucose, 0.1875 mg/mL, 1 mL. DLS backscattering is shown to measure size distribution and ζ-potential at volume, N/P ratio of 4. The z-average diameter was 107 nm with a polydispersity index (PDI) of 0.139. The ζ-potential was 31.4 mV.
Figure 4 shows triplicate LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) multicomplexes in 50 mM acetate buffer, pH 4.3, 5% glucose, 0.1875 mg/mL, 1 mL. DLS backscattering is shown to measure size distribution and ζ-potential at volume, N/P ratio of 4. The z-average diameter was 156 nm with a polydispersity index (PDI) of 0.144. The ζ-potential was 38.3 mV.
Figure 5 shows the size distribution and ζ- obtained from triplicate LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) multicomplexes at 0.1875 mg/mL, 1 mL volume, N/P 4. Indicates DLS backscattering, which measures the potential. The z-average diameter was 120 nm with a polydispersity index (PDI) of 0.125. The ζ-potential was 31.1 mV.
Figure 6A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu) in MCF7 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 6B shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu) in A431 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 7A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu) in MCF7 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 7B shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu) in A431 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 8A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(Glu) in MCF7 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 8B shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(Glu) in A431 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 9A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu) in MCF7 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 9B shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu) in A431 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 10A shows LPEI- l --[N ₃ :DBCO]-PEG ₂₃ -OMe:poly(IC) and LPEI- l --[N ₃ :DBCO]-PEG ₂₃ -OMe:poly(Glu) in MCF7 cells. Cell survival is shown as a function of treatment. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 10b shows LPEI- l --[N ₃ :DBCO]-PEG ₂₃ -OMe:poly(IC) and LPEI- l --[N ₃ :DBCO]-PEG ₂₃ -OMe:poly(Glu) in A431 cells. Cell survival is shown as a function of treatment. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 11A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in LNCaP cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 11B shows the transformation of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in PC-3 cells. Cell survival is shown as a function of treatment. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 11C shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in DU145 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 12A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) in LNCaP cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 12B shows the transformation of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) in PC-3 cells. Cell survival is shown as a function of treatment. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 12C shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) in DU145 cells. indicates cell survival. The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 13 shows LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA:poly(IC); LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA:poly(IC); LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA:poly(IC); and LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA:poly(IC) multicomplexes.
Figure 14 shows LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA:poly(IC); LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA:poly(IC); LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA:poly(IC); and LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA:poly(IC) multicomplexes.
Figure 15 shows cell survival in SKOV3 and MCF7 cells treated with various concentrations of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -folate:poly(IC) multicomplex.
Figure 16A shows LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly in MCF7 cells. Cell survival is shown as a function of treatment with (Glu). The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 16B shows LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly in SKBR3 cells. Cell survival is shown as a function of treatment with (Glu). The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 16C shows LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly in BT474 cells. Cell survival is shown as a function of treatment with (Glu). The X axis represents the log concentration of poly(IC) or poly(Glu) delivered.
Figure 17 shows IP-10 secretion as a function of treatment with 0.125, 0.25, 0.5 and 1 μg/mL LPEI- l -PEG ₂₄ -hEGF:poly(IC) multicomplex in A431 and MCF7 cells.
Figure 18 shows Western blot image analysis showing qualitative levels of EGFR phosphorylation as a function of complete serum, serum starvation, treatment with LPEI -1 -PEG ₂₄ -EGF, LPEI - 1-PEG ₂₄ -hEGF:poly(IC), and hEGF. indicates.
Figure 19A shows IP-10 secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells.
Figure 19B shows IP-10 secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells.
Figure 19B shows IP-10 secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and DU145 cells.
Figure 20A shows RANTES secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells.
Figure 20B shows RANTES secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells.
Figure 20C shows RANTES secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and DU145 cells.
Figure 21A shows INF-β secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells.
Figure 21B shows INF-β secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells.
Figure 21C shows INF-β secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and DU145 cells.
Figure 22 shows LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly at 0, 0.0625 and 0.625 μg/mL. (Glu) Western blot image analysis showing qualitative levels of caspase 3, cleaved caspase 3, PARP, cleaved PARP, RIG-1, MDA5, and ISG15 as a function of treatment with the multicomplex.
Figure 23 shows compounds 31 and 31b and poly(IC), LPEI- l -[N ₃ ) formed at an N/P ratio of 4 and a concentration of 0.1875 mg/mL in HEPES 20 mM buffer, 5% glucose (HBG), pH 7.2. :DBCO]-PEG ₃₆ -DUPA:SEM image of multicomposite particles containing poly(IC) is shown.
Figure 24A shows luminescence (AU) of Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to control delivery vehicle Messenger Max. Luminescence was observed after treatment with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and Lipofectamine Messenger Max at N/P ratios of 4, 6, 12 and concentrations of 0.125 to 1.0 mg/mL for 24 hours. Measurements were made over time.
Figure 24B shows luminescence (AU) of Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to the control delivery vehicle jetPEI. Luminescence was measured 24 hours after treatment with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and jetPEI at N/P ratios of 4, 6, and 12 and concentrations of 0.125 to 1.0 mg/mL. .
Figure 24C shows the luminescence (AU) ratio between Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to Messenger Max as a comparative delivery vehicle. . Luminescence was observed after treatment with LPEI- l- [ _N3 :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and Lipofectamine Messenger Max at N/P ratios of 4, 6, and 12 and concentrations of 0.125 to 1.0 mg/mL. Measurements were made over time, and the ratio was calculated by dividing the luminescence signal from Lenca EGFR M1H cells by the luminescence signal from Lenca blast cells.
Figure 24D shows the luminescence (AU) ratio between Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to jetPEI as a comparative delivery vehicle. Luminescence was measured 24 hours after treatment with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and jetPEI at N/P ratios of 4, 6, and 12 and concentrations of 0.125 to 1.0 mg/mL. , the ratio was calculated by dividing the luminescence signal from Lenca EGFR M1H cells by the luminescence signal from Lenca parent cells.
Figure 24E shows the percent survival of Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to the control delivery vehicle Messenger Max. Percent survival was determined at 24 hours after treatment with N/P ratios of 4, 6, and 12 and LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and Messenger Max at concentrations of 0.125 to 1.0 mg/mL. Measurements were made, and the ratio was calculated by dividing the luminescence signal from Lenca EGFR M1H cells by the luminescence signal from Lenca parent cells.
Figure 25A shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 4 at 24 hours post-treatment. indicates.
Figure 25B shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 6 at 24 hours post-treatment. indicates.
Figure 25C shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 4 at 48 hours post-treatment. indicates.
Figure 25D shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 6 at 48 hours post-treatment. indicates.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The embodiments, preferred embodiments and highly preferred embodiments described and disclosed herein should again apply to all aspects and other embodiments, preferred embodiments and highly preferred embodiments, whether or not specifically mentioned.

The present invention provides linear conjugates of LPEI and PEG that can form multicomplexes with nucleic acids such as polyanions and poly(IC), as outlined herein below. The conjugate preferably comprises an LPEI fragment, a PEG fragment and a targeting fragment. In a preferred embodiment, the LPEI fragment and the PEG fragment are linked in a clear end-to-end fashion. In some preferred embodiments, the LPEI fragment and the PEG fragment are linked via covalent attachment of an azide to an alkene or alkyne to form 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3 ]Forms triazole.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

The articles “a” and “an” are used in the present invention to refer to the grammatical object of one or more (i.e., at least one) articles. By way of example, “element” means one element or more than one element.

The term “and/or” is used herein to mean either “and” or “or” unless otherwise indicated.

As used herein, the term “about” shall have the meaning of +/- 10%. For example, about 50% would mean 45% to 55%. Preferably, the term “about” as used herein shall have the meaning of +/- 5%. For example, about 50% would mean 47.5% to 52.5%.

As used herein, the phrase “number X to number Y” shall be meant to include the number X and the number Y. For example, the phrase “0.01 μmol to 50 μmol” refers to 0.01 μmol and 50 μmol and values in between. The same applies to “about the number X to about the number Y”.

The term “optionally substituted” is understood to mean that a given chemical moiety (e.g. an alkyl group) may (but is not required to) be attached to another substituent (e.g. a heteroatom). For example, an optionally substituted alkyl group can be a fully saturated alkyl chain (i.e., a pure hydrocarbon). Alternatively, the same optionally substituted alkyl group may have a hydrogen and a different substituent. For example, it may be attached to a halogen atom, an alkoxy group, or any other substituent described herein at any point along the chain. Accordingly, the term “optionally substituted” means that a given chemical moiety may contain other functional groups, but does not necessarily have additional functional groups.

The term "optionally replaced" is understood to mean that the carbon atom of the methylene group (i.e. -CH ₂ -) may, but is not required to, be replaced by a heteroatom (e.g. -NH-, -O-). do. For example, a C ₃ alkylene (i.e., propylene) group in which one methylene group is “optionally substituted” may have the structure -CH ₂ -O-CH ₂ - or -O-CH ₂ -CH ₂ -. Those skilled in the art will understand that methylene groups cannot be substituted when such substitution would create an unstable chemical moiety. For example, those skilled in the art will understand that four methylene groups cannot be substituted with oxygen atoms at the same time. Accordingly, in some preferred embodiments when one methylene group of an alkylene segment is substituted with a heteroatom, one or both neighboring carbon atoms are not substituted with a heteroatom.

The term “aryl” refers to a cyclic hydrocarbon group having one to two aromatic rings, including mono- or bicyclic groups such as phenyl, biphenyl or naphthyl. C ₆ -C ₁₀ aryl groups contain 6 to 10 carbon atoms. When comprising two aromatic rings (such as on a dicyclic), the aromatic rings of the aryl group may be connected at a single point (e.g., biphenyl) or fused (e.g., naphthyl). An aryl group may be optionally substituted with one or more substituents, such as 1 to 5 substituents, at any point of attachment. A substituent may itself be optionally substituted. Additionally, an aryl group as defined herein when comprising two fused rings may have a fully saturated ring and an unsaturated or partially saturated ring fused. Exemplary ring systems for such aryl groups include indanyl, indenyl, tetrahydronaphthalenyl, and tetrahydrobenzoannulenyl. In some preferred embodiments, the aryl group is a phenyl group.

Unless specifically defined otherwise, “heteroaryl” refers to a monocyclic group of 5 to 24 ring atoms, containing one or more ring heteroatoms selected from N, S, P, or O, with the remaining ring atoms being C. It refers to an aromatic ring or a multi-ring aromatic ring. A 5- to 10-membered heteroaryl group contains 5 to 10 atoms. In addition, heteroaryl as defined herein refers to a bicyclic heteroaromatic group in which the heteroatoms are selected from N, S, P or O. Aromatic radicals are optionally independently substituted with one or more substituents described herein. Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl, pyrimidyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiopine-2 -yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazo[ 1,2-b]pyrazolyl, furo[2,3-c]pyridinyl, imidazo[1,2-a]pyridinyl, indazolyl, pyrrolo[2,3-c]pyridinyl, pyrrolo[ 3,2-c]pyridinyl, pyrazolo[3,4-c]pyridinyl, thieno[3,2-c]pyridinyl, thieno[2,3-c]pyridinyl, thieno[2, 3-b] pyridinyl, benzothiazolyl, indolyl, indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, di Hydrobenzothiazine, dihydrobenzooxanyl, quinolinyl, isoquinolinyl, 1,6-naphthyridinyl, benzo[de]isoquinolinyl, pyrido[4,3-b][1,6 ]Naphthyridinyl, thieno[2,3-b]pyrazinyl, quinazolinyl, tetrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl Dinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b] ]Pyridinyl, pyrrolo[1,2-a]pyrimidyl, tetrahydropyrrolo[1,2-a]pyrimidyl, 3,4-dihydro-2H-1λ2-pyrrolo[2,1-b] Pyrimidine, dibenzo[b,d]thiophene, pyridin-2-one, furo[3,2-c]pyridinyl, furo[2,3-c]pyridinyl, 1H-pyrido[3,4- b][1,4]thirazinyl, benzoxazolyl, benzoisoxazolyl, furo[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, furo[3,2-b] Pyridine, [1,2,4]thiazolo[1,5-a]pyridinyl, benzo[1,2,3]thiazolyl, imidazo[1,2-a]pyrimidinyl, [1,2, 4]triazolo[4,3-b]pyridazinyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-di Hydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetra Hydropyrazolo[1,5-a]pyridinyl, thiazolo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2, 3-b]pyrrolyl, 3H-indolyl and their derivatives. Additionally, heteroaryl groups as defined herein when comprising two fused rings may have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems for such aryl groups include indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3,4- Includes dihydro-1H-isoquinolinyl, 2,3-dihydrobenzofuran, indolinyl, indolyl and dihydrobenzooxanyl.

The term “alkyl” refers to a straight or branched chain saturated hydrocarbon. C ₁ -C ₆ alkyl groups contain 1 to 6 carbon atoms. Examples of C ₁ -C ₆ alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and neopentyl.

As used herein, the term “alkylene” refers to straight or branched chain saturated and divalent hydrocarbon fragments. C ₀ -C ₆ alkyl groups contain 0 to 6 carbon atoms. Examples of C ₀ -C ₆ alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, isopropylene, isobutylene, sec-butylene, tert-butylene, isopentylene and neo. Contains pentylene.

As used herein, the term “C ₁ -C ₆ alkoxy” refers to a substituted hydroxyl of the formula (-OR'), wherein R' is substituted with C ₁ -C ₆ alkyl as defined herein, and is substituted with an oxygen moiety. The tee is attached directly to the parent molecule, and therefore, as used herein, the term "C ₁ -C ₆ alkoxy" refers to straight or branched chain C ₁ -C ₆ alkoxy, such as methoxy, ethoxy, n-propoxy, It may be isopropoxy, n-butoxy, isobutoxy, sec-butoxy, ter-butoxy, straight or branched chain pentoxy, straight or branched chain hexyloxy. C ₁ -C ₄ alkoxy and C ₁ -C ₃ alkoxy are preferred.

The term “cycloalkyl” refers to a monocyclic or polycyclic saturated carbon ring containing from 3 to 18 carbon atoms. C ₃ -C ₈ cycloalkyl contains 3 to 8 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclocyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo. Contains rho[2.2.2]octenyl. C ₃ -C ₈ Cycloalkyl is a cycloalkyl group containing 3 to 8 carbon atoms.

The term “cycloalkenyl” refers to a monocyclic, non-aromatic carbon ring containing 5 to 18 carbon atoms. Examples of cycloalkenyl groups include, but are not limited to, cyclopeptenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and norborenyl. C ₅ -C ₈ Cycloalkenyl is a cycloalkenyl group containing 5 to 18 carbon atoms.

The term "heterocyclyl", "heterocycloalkyl" or "heterocycle" refers to a monocyclic or polycyclic 3- to 24-membered ring containing carbon and heteroatoms derived from oxygen, nitrogen or sulfur, wherein There are no mobile π electrons (directional) shared between ring carbons or heteroatoms. 3- to 10-membered heterocycloalkyl groups contain 3 to 10 atoms. Heterocyclyl rings include, but are not limited to, oxetanyl, azetadinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl. , tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl, S-oxide, thiomorpholinyl, S-dioxide, piperazinyl, azepinyl, jade Includes cefinil, diazefinil, tropanil and homotropanil.

The term "heterocycloalkenyl" refers to a monocyclic or polycyclic 3- to 24-membered ring containing carbon and heteroatoms derived from oxygen, nitrogen or sulfur, wherein the ring carbons or heteroatoms are shared between the ring carbons or heteroatoms. There are no mobile π electrons (directional), but there is at least one unsaturated element in the ring. A 3- to 10-membered heterocycloalkenyl group contains 3 to 10 atoms.

As used herein, the term “halo” or “halogen” means fluoro (F), chloro (Cl), bromo (Br) or iodo (I).

The term “carbonyl” refers to a functional group consisting of a carbon atom double bonded to an oxygen atom. This may be abbreviated herein as “oxo”, C(O) or C-O.

The term “overexpression” refers to increased gene or protein expression within a cell or on the cell surface compared to basal or normal expression. In a preferred embodiment, the targeting fragment is capable of binding to cells overexpressing a cell surface receptor. In one embodiment, the cells overexpressing the cell surface receptor are such that the level of the cell surface receptor expressed therein in a particular tissue is compared to the level of the cell surface receptor as measured in healthy normal cells of the same tissue type under similar conditions. This means that it has risen. In one embodiment, a cell overexpressing the cell surface receptor refers to an increase in the level of the cell surface receptor in the cell compared to the level in the same cell or a closely related non-malignant cell under normal physiological conditions.

As used herein, the term “polyanionic” refers to a polymer, preferably a biopolymer, having one or more moieties carrying a negative charge. Typically and preferably, the term "polyanionic" as used herein refers to a polymer, preferably a biopolymer, composed of repeating units comprising moieties capable of carrying a negative charge. In a further embodiment, a polyanion refers to a polymer, preferably a biopolymer, composed of repeating units comprising negatively charged residues. In another preferred embodiment, the polyanion is a nucleic acid, more preferably DNA, RNA, polyglutamic acid or hyaluronic acid.

As used herein, the term “nucleic acid” includes deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) or combinations thereof. In a preferred embodiment, the term “nucleic acid” refers to deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA), herein referred to as genomes, viruses, and recombinantly produced and chemically synthesized molecules. Nucleic acids may be single- or double-stranded and in the form of linear or covalently closed circular molecules, and may include chemical derivatization of nucleic acids onto nucleotide bases, sugars or phosphates, and may include non-natural nucleotides and nucleotide analogs. It can be included.

As used herein, the term "dispersity" (abbreviated as D) refers to the distribution of molar mass in a given polymeric sample, such as polymeric fragments in the case of the conjugates and multicomplexes of the invention. Herein it is defined as D = (M _w /M _n ), where D is the degree of dispersion, M _w is the weight, average molecular weight of the polymeric sample or polymeric fragment, and M _n is the polymeric sample or polymeric fragment. is the number and average molecular weight.

As used herein, the term “polydispersity index” (abbreviated as PDI) refers to the polydispersity in dynamic light scattering measurements of multicomplex nanoparticles, such as the multicomplex according to the present invention. These indices are numbers calculated from two simple parameters tailored to correlation data (accumulation rate analysis). The polydispersity index is dimensionless and is calculated so that values less than 0.05 are rarely observed other than the high monodispersity standard. Values above 0.7 indicate that the sample has a very broad size distribution and is probably unsuitable for dynamic light scattering (DLS) techniques. Various size distribution algorithms operate with data that falls between these two extremes. The zeta-average diameter (z-average diameter) and polydispersity of the multicomplexes of the invention are determined by dynamic light scattering (DLS) based on the assumption that the multicomplexes are isotropic and spherical. Calculations for these parameters are defined and determined according to the ISO standard document ISO 22412:2017.

As used herein, the term "amino acid residue" refers to a divalent residue derived from an organic compound comprising the functional groups amine (-NH ₂ ) and carboxylic acid (-COOH), typically and preferably together with side chains specific to each amino acid. says In a preferred embodiment of the invention, the amino acid residue is a divalent residue derived from an organic compound comprising the functional groups amine (-NH ₂ ) and carboxylic acid (-COOH), wherein the divalent valence is the amine and the carboxylic acid. The functional group is created by -NH- and -CO- moieties accordingly. In an alternative preferred embodiment of the invention, the amino acid residue is a divalent residue derived from an organic compound comprising the functional groups amine (-NH ₂ ) and carboxylic acid (-COOH), wherein the dual valence is It is created with a boxylic acid functional group and additional functional groups present on the amino acid residue. By preferred examples and embodiments, the amino acid residue derived from cysteine according to the invention comprises the divalent structure -S-(CH2)-CH(COOH)-NH-, wherein the divalence is an amino functional group and It is produced by a thiol functional group. As used herein, the term “amino acid residue” typically and preferably includes amino acid residues derived from naturally occurring or non-naturally occurring amino acids. Additionally, the term "amino acid residue" as used herein typically and preferably includes amino acids such as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-), as well as It also includes amino acid residues derived from chemically synthesized non-natural amino acids, including mixtures of these in any proportion. Additionally, as used herein, the term "amino acid residue" typically and preferably refers to any isomeric form, particularly its D-stereoisomer and L-stereoisomer (alternatively, denoted by the (R) and (S) nomenclature ), as well as amino acid residues derived from alpha amino acids, including mixtures of these in any ratio, preferably a racemic ratio of 1:1. Accordingly, in a preferred embodiment the amino acid residue is a divalent group of the structure -NH-CHR-C(O)-, where R is the amino acid side chain. Two or more adjacent amino acid residues preferably form peptide (i.e., amide) bonds to both the amine and carboxylic acid portions of the amino acid residues, respectively. When di, tri or polypeptides are described herein as amino acid residues, typically (AA) _a , the provided sequences are written in the NC direction from left to right. Thus, as an example, (AA) _a , which becomes Trp-Trp-Gly, corresponds to the N-terminus of the tripeptide where Trp has the -NH- valency and Gly corresponds to the C-terminus of the tripeptide with the -CO- valency. The corresponding amino acid residue must be stated.

As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to a substance comprising two or more adjacent amino acid residues linked to another residue through a peptide bond. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues of any length. In one embodiment, the term “protein” refers to a long peptide, specifically a peptide having at least about 151 amino acids, while in one embodiment the term “peptide” refers to a peptide having at least about 2, at least about 3, at least about 8. or about 20 or more substances, and up to about 50 substances, about 100 substances or more, or about 150 substances or more.

As used herein, the term “epitope” refers to an antigenic determinant on a molecule, such as an antigen. The epitope of the protein preferably comprises continuous or discontinuous portions of the protein, preferably 5 to 100 epitopes, preferably 5 to 50 epitopes, more preferably 8 to 30 epitopes, most preferably 10 to 25 epitopes. The epitope is preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length. It can be.

The term “antibody” refers to any immunoglobulin and derivatives thereof and their characteristic portions, whether natural or synthetically produced in whole or in part. Antibodies may be monoclonal or polyclonal. The antibody may be a member of any immunoglobulin, including any of the human classes IgG, IgM, IgA, IgD, and IgE. As used herein, antibody fragment (i.e., characteristic portion of an antibody) refers to any antibody derivative that is shorter than the full length. Typically, antibody fragments retain at least a significant portion of the specific binding capacity of the full-length antibody. Examples of antibody fragments include, but are not limited to, single or double chain fragments, Fab, Fab', F(ab') ₂ , scFv, Fv, dsFv diazites, and Fd fragments. Antibody fragments can be produced by any means. For example, antibody fragments can be produced by enzymatic or synthetic fragmentation of an intact antibody and/or they can be produced recombinantly from genes encoding partial antibody sequences. Alternatively or additionally, antibody fragments may be produced, in whole or in part, synthetically. Antibody fragments may optionally include single chain antibody fragments. Alternatively or additionally, antibody fragments may comprise multiple chains linked together, for example by disulfide bonds. Antibody fragments may optionally comprise multimolecular complexes. A functional antibody fragment will typically contain at least about 50 amino acids, more typically at least about 200 amino acids. In some embodiments, antibodies may include chimeric (e.g., “humanized”) and single chain (recombinant) antibodies. In some embodiments, the antibody may have reduced effector function and/or be a bispecific molecule. In some embodiments, an antibody may comprise a fragment produced by a Fab expression library. Single chain Fv (scFv) is a recombinant antibody fragment consisting solely of the variable light (VL) and variable heavy (VH) chains covalently linked to each other by a polypeptide linker. Either VL or VH may contain an NH2-terminal domain. Polypeptide linkers can be of various lengths and compositions as long as the two variable domains are bridged without significant steric hindrance. Typically, the linker contains primarily stretches of glycine and serine residues interspersed with some glutamic acid or lysine to impart solubility. The diabody is a dimeric scFv. Diamers typically have shorter peptide linkers than most scFvs, and they often show a preference for binding as dimers. An Fv fragment is an antibody fragment consisting of one VH and one VL domain held together by non-covalent interactions.

As used herein, the term “dsFv” refers to an Fv with engineered intermolecular disulfide bonds that stabilize the VH-VL pair. The F(ab') ₂ fragment is an antibody fragment essentially equivalent to that obtained from immunoglobulins by digestion with the enzyme pepsin at pH 4.0 to 4.5. Fragments can be produced recombinantly. Fab' fragments are antibody fragments that are essentially equivalent to those obtained by reduction of the disulfide bridge or bridges connecting the two heavy chain fragments in the F(ab') ₂ fragment. Fab' fragments can be produced recombinantly. Fab fragments are antibody fragments that are essentially equivalent to those obtained by digestion of immunoglobulins with enzymes (eg, papain). Fab fragments can be produced recombinantly. The heavy chain segment of the Fab fragment is the Fd fragment.

As used herein, the term "alpha terminus of a linear polyethyleneimine fragment" (α-terminus of an LPEI fragment) refers to the initiation of polymerization using an electrophilic initiation element, as further explained below for the term "initiation moiety". This refers to the terminal end of the LPEI fragment that occurs.

As used herein, the term "omega terminus of a linear polyethyleneimine fragment" (ω-terminus of an LPEI fragment) means that termination of polymerization, as described herein, utilizes nucleophilic species such as azides, thiols and other nucleophilic species. This refers to the terminal end of the LPEI fragment that occurs.

The term “organic moiety” refers to any suitable organic group capable of bonding to a nitrogen atom contained within the PEI fragment. In a preferred embodiment, the organic moiety is linked to the nitrogen atom through a carbonyl group to form an amide bond. Without wishing to be bound by theory, the organic moiety is introduced on the nitrogen atom of poly(2-oxazoline) during ring-opening polymerization of 2-oxazoline (e.g., Glassner et al. , (2018), Poly( 2-oxazoline)s: A comprehensive overview of polymer structures and their physical properties., 67: 32-45. Typically and preferably, the organic moiety is cleaved from the poly(2-oxazoline) (i.e. typically the amide is cleaved) to form the LPEI and LPEI fragments and thus -(NH-) contained within the conjugates of the invention. CH ₂ -CH ₂ )- moiety is obtained. However, if the cleavage reaction is not complete, a fraction of the organic residues are not cleaved. Accordingly, in a preferred embodiment of the invention, at least 80% of R ² in the R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety of the conjugates of the invention, including conjugates of formulas I ^* and I, %, preferably 90%, is H, preferably of R ² in the R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety of the conjugates of the invention, including conjugates of formulas I ^* and I. at least 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, most preferably 99%. % is H.

The term “initiation residue” refers to the residue present in the LPEI fragment and R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -moiety of the conjugate of the invention, which residue is selected from 2-oxazoline to poly(2- oxazoline), typically and preferably from any electrophilic initiator. Glassner et al ., (2018), Poly(2-oxazoline)s: A comprehensive overview of polymer structures and their physical properties. Polym. Int., 67: 32-45, https://doi.org /10.1002/pi.5457), "toluenesulfonic acids (MeOTs) or alkyl sulfonates, such as methyl p-toluenesulfonate (MeOT), which is the most commonly found in the literature, p-nitrobenzenesulfonates (nosylate ) and trifluoromethanesulfonate (triflate), alkyl, benzyl and acetyl halides, oxazolinium salts and Lewis acids." Accordingly, although in a preferred embodiment R ¹ is -H or -CH ₃ , those skilled in the art will understand that R ¹ may be other groups such as, but not limited to, a C _n alkyl group with n greater than 1, typically a C _1-6 alkyl group, a benzyl group or an acetyl group. It will be appreciated that suitable moieties may also be included.

Accordingly, in one aspect, the present invention provides a composition comprising a conjugate, wherein the conjugate includes a linear polyethyleneimine (LPEI) fragment comprising an alpha terminus and an omega terminus; and a polyethylene glycol (PEG) fragment, preferably a linear polyethylene glycol (PEG) fragment, comprising a first terminal end and a second terminal end; The omega terminus of the LPEI fragment is connected by a covalent moiety to the first terminal end of the PEG fragment; The covalent moiety is not an amide; Preferably the alpha terminus of the LPEI fragment is bonded to a methyl group or a hydrogen atom, more preferably the alpha terminus of the LPEI fragment is bonded to a hydrogen atom; Preferably the alpha terminus of the PEG fragment is linked to the targeting fragment.

In one aspect, the present invention provides a composition comprising a conjugate, wherein the conjugate includes a linear polyethyleneimine fragment comprising an alpha terminus and an omega terminus; and a polyethylene glycol segment comprising a first terminal end and a second terminal end; The alpha terminus of the polyethyleneimine fragment is the initiation residue; The omega terminus of the polyethyleneimine fragment is connected to the first end of the polyethylene glycol fragment by a covalent bond -ZX ¹ -, where -Z- is not a single bond, -Z- is not an amide, and -X ¹ - is a divalent bond. is a covalent moiety; The second terminal end of the polyethylene glycol fragment is capable of binding, preferably wherein the polyethylene glycol fragment binds the targeting fragment. In a preferred embodiment of this aspect, the composition consists of the conjugate. In a preferred embodiment, the linear polyethyleneimine fragment has the formula R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -, where n is any integer from 1 to 1,500. In a further preferred embodiment, the R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety has a repeating unit n of about 115 to about 1,150 and a dispersity of about 5 or less, preferably about 3 or less. A dispersed polymer having a dispersion degree of about 280 to about 700 repeat units n, more preferably a dispersion degree of about 2 or less, about 350 to about 630 repeat units n, and preferably R ¹ is -H or -CH ₃ It is a polymeric moiety.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety and Z is not -NHC(O)-; L is preferably a targeting fragment capable of binding to a cell, and preferably the composition consists of the conjugate.

In another aspect, the present invention provides a conjugate of Formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety and Z is not -NHC(O)-; L provides a conjugate, preferably a targeting fragment capable of binding to cells.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety, Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor,

Preferably, the composition provides a composition comprising the conjugate.

In another aspect, the present invention provides a conjugate of Formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety, Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here is a single bond or a double bond; n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety are H; Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H; ^{where ​} _​ ^{​ ​ ​} -O-, -S-, -NR ¹³ -, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, each divalent phenyl and heteroaryl being independently selected from one or more R ¹³ and each divalent heterocycle is optionally substituted with one or more R ¹⁴ ; ^R11 , R ¹² and R ¹³ are independently -H or C ₁ -C ₆ alkyl at each occurrence; R ¹⁴ is independently at each occurrence -H, C ₁ -C ₆ alkyl or oxo; ^{and ​} _​ ^{​ ​ ​ ​} , -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, and each divalent phenyl and divalent heteroaryl is independently selected from one or more R ²³ and each divalent heterocycle moiety is optionally substituted with one or more R ²⁴ ; R ²¹ , R ²² and R ²³ are each independently -H, -CO ₂ H or C ₁ -C ₆ alkyl at each occurrence, and each C ₁ -C ₆ alkyl is one or more -OH, oxo, C ₆ -C ₁₀ optionally substituted with aryl or 5- to 8-membered heteroaryl; R ²⁴ is independently at each occurrence -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo; L is preferably a targeting fragment capable of binding to a cell, and preferably the composition consists of the conjugate.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here is a single bond or a double bond; n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety are H; Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H; ^{where ​} _​ ^{​ ​ ​} -O-, -S-, -NR ¹³ -, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, each divalent phenyl and heteroaryl being independently selected from one or more R ¹³ and each divalent heterocycle is optionally substituted with one or more R ¹⁴ ; ^R11 , R ¹² and R ¹³ are independently -H or C ₁ -C ₆ alkyl at each occurrence; R ¹⁴ is independently at each occurrence -H, C ₁ -C ₆ alkyl or oxo; ^{and ​} _​ ^{​ ​ ​ ​} , -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, and each divalent phenyl and divalent heteroaryl is independently selected from one or more R ²³ and each divalent heterocycle moiety is optionally substituted with one or more R ²⁴ ; R ²¹ , R ²² and R ²³ are each independently -H, -CO ₂ H or C ₁ -C ₆ alkyl at each occurrence, and each C ₁ -C ₆ alkyl is one or more -OH, oxo, C ₆ -C ₁₀ optionally substituted with aryl or 5- to 8-membered heteroaryl; R ²⁴ is independently at each occurrence -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo; L provides a conjugate, preferably a targeting fragment capable of binding to cells. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In some embodiments, the covalent moiety Z comprises a triazole.

In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the LPEI in the composition is linked to the PEG fragment by a single covalent moiety, preferably a covalent moiety Ti produces a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the LPEI fragments comprised in the composition are preferably determined by UV spectrophotometry or mass spectrometry, Included in the conjugate. In some embodiments, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% of the LPEI fragments comprised in the composition are preferably determined by UV spectrophotometry or mass spectrometry, Included in the conjugate. In some embodiments, the composition consists essentially of the conjugate. In some embodiments, the composition consists of the conjugate.

In some embodiments, at least 60% of the LPEI in the composition is linked to a single PEG fragment by a single covalent moiety Z, preferably the covalent moiety Z forms a linear end-to-end linkage between the LPEI fragment and the PEG fragment. produce. In some embodiments, at least 60% of the LPEI fragments included in the composition are linked to the PEG fragment by a single triazole linker, preferably as determined by UV spectrophotometry or mass spectrometry. In some embodiments, at least 70% of the LPEI in the composition is linked to the PEG fragment by a single covalent moiety Z, preferably the covalent moiety Z produces a linear end-to-end linkage between the LPEI fragment and the PEG fragment. do. In some embodiments, at least 70% of the LPEI fragments comprised in the composition are comprised in the conjugate, preferably as determined by UV spectrophotometry or mass spectrometry. In some embodiments, at least 80% of the LPEI in the composition is linked to the PEG fragment by a single covalent moiety Z, preferably the covalent moiety Z produces a linear end-to-end linkage between the LPEI fragment and the PEG fragment. do. In some embodiments, at least 80% of the LPEI fragments comprised in the composition are comprised in the conjugate, preferably as determined by UV spectrophotometry or mass spectrometry. In some embodiments, at least 90% of the LPEI in the composition is linked to the PEG fragment by a single covalent moiety Z, preferably the covalent moiety Z produces a linear end-to-end linkage between the LPEI fragment and the PEG fragment. do. In some embodiments, at least 90% of the LPEI fragments comprised in the composition are comprised in the conjugate, preferably as determined by UV spectrophotometry or mass spectrometry. In some embodiments, at least 95% of the LPEI in the composition is linked to the PEG fragment by a single covalent moiety Z, preferably the covalent moiety Z produces a linear end-to-end linkage between the LPEI fragment and the PEG fragment. do. In some embodiments, at least 95% of the LPEI fragments comprised in the composition are comprised in the conjugate, preferably as determined by UV spectrophotometry or mass spectrometry. In some embodiments, at least 99% of the LPEI in the composition is linked to the PEG fragment by a single covalent moiety Z, preferably the covalent moiety Z produces a linear end-to-end linkage between the LPEI fragment and the PEG fragment. do. In some embodiments, at least 99% of the LPEI fragments comprised in the composition are comprised in the conjugate, preferably as determined by UV spectrophotometry or mass spectrometry. In some embodiments, the composition consists essentially of the conjugate. In some embodiments, the composition consists of the conjugate. In some embodiments, the LPEI fragment does not include substitutions beyond its first and second terminal ends.

In some embodiments, Formula I ^* does not include the structure R ¹ -(NH-CH ₂ -CH ₂ ) _n -NHC(O)-(CH ₂ -CH ₂ -O) _m -X ² -L. In some embodiments, Formula I ^* comprises the structure R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -NHC(O)-X ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L I never do that. In some embodiments, the composition comprises a conjugate of the structure R ¹ -(NH-CH ₂ -CH ₂ ) _n -NHC(O)-X ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L I never do that. In some embodiments, the composition does not include a conjugate of the structure R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -NHC(O)-(CH ₂ -CH ₂ -O) _m -X ² -L .

In some embodiments, R ¹ is -H.

In some embodiments, at least 80% of R ² in the composition is -H. In some embodiments, at least 85%, preferably 90%, preferably 95%, more preferably 99% of the R ² in the composition is -H. In a preferred embodiment, R ² is independently -H or an organic moiety and at least 85%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety are H. In another preferred embodiment, R ² is independently -H or an organic moiety, and at least 90% of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H. In another preferred embodiment, R ² is independently -H or an organic moiety, and at least 90% of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H. In another preferred embodiment, R ² is independently -H or an organic moiety and represents at least 91%, preferably at least 92%, of said R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety; More preferably 93% is H. In another preferred embodiment, R ² is independently -H or an organic moiety and represents at least 94%, preferably at least 95% of said R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety, More preferably 96% is H. In another preferred embodiment, R ² is independently -H or an organic moiety and represents at least 95%, preferably at least 97%, of said R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety; More preferably at least 98%, more preferably 99% is H.

In some embodiments, Ring A is 8-membered cycloalkenyl, 5-membered heterocycloalkyl, or 7- to 8-membered heterocycloalkenyl, and each cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl is optional. is optionally substituted with one or more R ^A1 at the position.

In some embodiments, Ring A is cyclooctene, succinimide, or 7- to 8-membered heterocycloalkenyl, and the heterocycloalkyl or heterocycloalkenyl does not contain heteroatoms other than N, O, and S, and Each cyclooctene, heterocycloalkyl or heterocycloalkenyl is optionally substituted at any position with one or more R ^A1 .

In some embodiments, Ring A is cyclooctene, succinimide, or 7- to 8-membered heterocycloalkenyl, and the heterocycloalkyl or heterocycloalkenyl is selected from one or more heteroatoms, preferably N, O, and S. It contains one or more heteroatoms, and each cyclooctene, heterocycloalkyl or heterocycloalkenyl is optionally substituted at any position with one or more R ^A1 .

In some embodiments, Ring A is cyclooctene, succinimide, or 8-membered heterocycloalkene, wherein the heterocycloalkene comprises exactly one heteroatom selected from N, O, and S, and each cyclooctene or heterocycloalkene The alkene is optionally substituted with one or more R ^A1 at any position.

In some embodiments, R ^A1 is -H, oxo or fluorine, or two R ^A1 combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each phenyl ring optionally substituted with one or more -SO ₃ H or -OSO ₃ H.

In some embodiments, Ring A is cyclooctene, succinimide, or 8-membered heterocycloalkene, wherein the heterocycloalkene comprises exactly one heteroatom selected from N, O, and S, and each cyclooctene or heterocycloalkene The alkene is optionally substituted with one or more R ^A1 and two R ^A1 combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings.

In some embodiments, Ring A is a cyclooctene, succinimide, or 8-membered heterocycloalkene, wherein the heterocycloalkene comprises exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one heterocycloalkene. or is optionally substituted with two R ^A1 .

In some embodiments, R ^A1 is -H, oxo or fluorine, or two R ^A1 combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each phenyl ring is optionally substituted with one or more R ^A2 .

In some embodiments, Ring A is a cyclooctene, succinimide, or 8-membered heterocycloalkene, wherein the heterocycloalkene comprises exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one heterocycloalkene. or optionally substituted with two R ^A1s , wherein R ^A1 is -H, oxo or fluorine, or two R ^A1s combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings; , each phenyl ring is optionally substituted with one or more -SO ₃ H or -OSO ₃ H.

In some embodiments, Ring A is a cyclooctene, succinimide, or 8-membered heterocycloalkene, wherein the heterocycloalkene comprises exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one heterocycloalkene. or optionally substituted with two R ^A1s , wherein R ^A1 is -H, or two R ^A1s combine to form one or more fused phenyl rings, preferably one or two fused phenyl rings, and each phenyl The ring is optionally substituted with one or more -SO ₃ H or -OSO ₃ H.

Preparation of linear conjugates

The conjugates of the present invention can be prepared in many ways well known to those skilled in the art of polymer synthesis. By way of example, the compounds of the present invention can be synthesized using the methods described below, along with synthetic methods known in the art of polymer chemistry or variations thereof as understood by those skilled in the art. The methods include, but are not limited to, those described below. Conjugates of the invention can be synthesized by following the steps outlined in general Schemes 1, 2, 3, 4, 5, 6, 7 and 8, or alternative sequences of assembling the intermediates can be used without departing from the invention. It can be manufactured. Conjugates of the present invention can also be synthesized by making minor modifications to the steps outlined below. For example, while Scheme 3 demonstrates the use of tetrafluorophenyl ester as an electrophilic functional group for binding to hEGF, those skilled in the art will recognize other suitable electrophilic functional groups that can be used for the same purpose.

In some preferred embodiments, the LPEI fragment and the PEG fragment are linked, preferably via [3 + 2] cycloaddition between the azide and the alkene or alkyne to form 1,2,3-triazole or 4,5-dihydro-1H. -Produces [1,2,3]triazole. In some preferred embodiments, the LPEI fragment comprises an azide functional group and the PEG fragment comprises an alkene or alkyne functional group.

LPEI fragment

Conjugates of the invention may include LPEI fragments and PEG fragments. Linear polyethyleneimine (LPEI) has the formula -[NH-CH ₂ -CH ₂ ]-. LPEI can be synthesized according to many methods known in the art, specifically involving polymerization of 2-oxazoline followed by hydrolysis of the pendant amide bond (e.g., Brissault et al ., Bioconjugate Chem., 2003, 14: 581-587). As mentioned above, the polymerization of poly(2-oxazoline) from 2-oxazoline (i.e., a suitable precursor of LPEI) can be initiated with any suitable initiator. In some embodiments, the initiator leaves an initiation residue at the alpha terminus of the poly(2-oxazoline). In a preferred embodiment, the initiating moiety (i.e. Formula I ^* or R ¹ of Formula I) is a hydrogen atom or C ₁ -C ₆ alkyl, preferably hydrogen or C ₁ -C ₄ alkyl, more preferably hydrogen or a methyl group, most Preferably it is a hydrogen atom. In a preferred embodiment, the starting moiety R ¹ of formula (I ^*) or formula (I) is a hydrogen atom or C ₁ -C ₆ alkyl, preferably hydrogen or C ₁ -C ₄ alkyl, more preferably hydrogen or a methyl group, most preferably a hydrogen atom. am. In a preferred embodiment, the initiating moiety (i.e. Formula I ^* or R ¹ of Formula I) is -H or -CH ₃ , most preferably -H. In a preferred embodiment, the starting residue R ¹ of formula I ^* is -H. In a preferred embodiment, the starting moiety R ¹ of formula (I) is -H. In a preferred embodiment, the starting moiety R ¹ of formula I ^* is -CH ₃ . In a preferred embodiment, the starting moiety R ¹ of formula I is -CH ₃ . However, those skilled in the art will understand that the initiator may be a residue remaining from any suitable initiator capable of initiating the polymerization of poly(2-oxazoline) from 2-oxazoline.

In some embodiments, the LPEI fragment is linked to the PEG fragment via [3 + 2] cycloaddition between the azide and the alkene or alkyne to form 1,2,3-triazole or 4,5-dihydro-1H-[1 ,2,3]triazoles, where the LPEI fragment contains an azide (-N ₃ ) functional group at the omega terminus of the chain. In some preferred embodiments, the LPEI fragment is not further substituted except for a single substitution at the alpha terminus. For example, in some preferred embodiments, the LPEI fragment comprises the repeating formula -[NH-CH ₂ -CH ₂ ]- and is substituted at the omega terminus with an azide group that can be bonded to an alkyne or alkene substituent on the PEG fragment. do. In some preferred embodiments, the alpha terminus of the LPEI fragment may be substituted with a hydrogen atom or C ₁ -C ₆ alkyl, preferably with hydrogen or C ₁ -C ₄ alkyl, more preferably with hydrogen or a methyl group, most preferably with a hydrogen atom.

For example, in some preferred embodiments the LPEI fragment has a hydrogen atom or C ₁ -C ₆ alkyl at the alpha terminus, preferably a hydrogen atom or C ₁ -C ₄ alkyl, more preferably a hydrogen atom or a methyl group and an azide at the omega terminus. groups, and in some preferred embodiments no additional substitutions are present on the LPEI fragment. For example, the conjugates of the invention can be prepared from an LPEI fragment of the formula:

where R ¹ may be any suitable starting moiety, preferably hydrogen or C ₁ -C ₆ alkyl, more preferably hydrogen or C ₁ -C ₄ alkyl, even more preferably hydrogen or a methyl group, most preferably hydrogen.

In some embodiments, the LPEI fragment may be terminated with a thiol group, such that in some embodiments the omega terminus of the LPEI fragment is preferably a thiol group, which may be attached to a reactive alkene group on the PEG fragment by a thiol-ene reaction. Accordingly, in some embodiments, conjugates of the invention may be prepared from LPEI fragments of the formula:

where R ¹ may be any suitable starting moiety, preferably hydrogen or a methyl group, preferably hydrogen.

In some embodiments, the LPEI fragment may be terminated with an alkene group, such that in some embodiments the omega terminus of the LPEI fragment comprises an alkene group, preferably an alkene group, which is converted to a reactive thiol group on the PEG fragment by a thiol-ene reaction. can be combined Accordingly, in some embodiments the conjugates of the invention may be prepared from an LPEI fragment of the formula:

where R ¹ may be any suitable starting moiety, preferably hydrogen or a methyl group, preferably hydrogen.

LPEI fragments can encompass a wide range of lengths (i.e., repeat units, indicated above by the variable “n”). For example, an LPEI fragment may contain from 1 to 1,000 repeat units (i.e., -NH-CH ₂ -CH ₂ -). In some embodiments, LPEI fragments may exist as dispersed polymeric moieties and do not contain a clear number of -NH-CH ₂ -CH ₂ - repeat units. For example, the LPEI fragment is preferably a dispersed fragment having a molecular weight of about 5 to 50 kDa with a dispersion of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. It may exist as a polymeric moiety. In some embodiments, the LPEI fragments may exist as dispersed polymeric moieties having a molecular weight of about 10 to 40 kDa with a degree of dispersion of about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. there is. In some embodiments, the LPEI fragments may exist as dispersed polymeric moieties having a molecular weight of about 12 to 30 kDa with a degree of dispersion of about 3 or less, preferably about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI fragments may exist as dispersed polymeric moieties having a molecular weight of about 15 to 27 kDa with a degree of dispersion of about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI fragments may exist as dispersed polymeric moieties having a molecular weight of about 17 to 25 kDa with a degree of dispersion of about 1.2 or less.

For example, the LPEI fragment preferably contains about 115 to 1,150 repeat units with a dispersion of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. It may exist as dispersed polymeric moieties. In some embodiments, the LPEI fragment is a dispersed polymeric moiety comprising about 230 to 930 repeat units with a dispersion of about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. It can exist. In some embodiments, the LPEI fragments may exist as dispersed polymeric moieties comprising about 280 to 700 repeat units with a degree of dispersion of about 3 or less, preferably about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI fragments may exist as dispersed polymeric moieties comprising about 350 to 630 repeat units with a degree of dispersion of about 2 or less, preferably about 1.5 or less. In some embodiments, the LPEI fragments may exist as dispersed polymeric moieties comprising about 400 to 580 repeat units with a dispersion degree of about 1.2 or less.

In some embodiments, the R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety is a dispersed polymeric moiety having about 115 to about 1,150 repeat units n and a degree of dispersion of about 5 or less. , preferably the R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety is a dispersed polymeric moiety having about 280 to about 700 repeat units n and a dispersity of about 3 or less, and further Preferably, the R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety is a dispersed polymeric moiety having a repeating unit n of about 350 to about 630 and a dispersity of about 2 or less, Even more preferably the R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety is a dispersed polymeric moiety having a repeating unit n of about 400 to about 580 and a dispersity of about 1.2 or less.

In a preferred embodiment, the polyethyleneimine fragment has about 230 to 930 repeat units, with about 115 to about 1,150 repeat units and a dispersion of about 5 or less, preferably about 4 or less, more preferably about 3 or less. About 280 to 700 repeat units with a dispersion degree of about 280 to 700 repeat units, more preferably about 350 to 630 repeat units with a dispersion degree of about 2 or less, even more preferably about 400 to 580 repeat units with a dispersion degree of about 1.2 or less. It is a dispersed polymeric moiety having.

In a preferred embodiment, the polyethyleneimine fragment has about 115 to about 1,150 repeat units and a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. It is a dispersed polymeric moiety having. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymeric moiety having from about 230 to about 930 repeat units with a dispersion of about 4 or less, preferably about 3 or less, preferably about 2 or less, preferably about 1.5 or less. It's tee. In a preferred embodiment, the polyethyleneimine segments are dispersed polymeric moieties having from about 280 to about 700 repeat units with a dispersion of about 3 or less, preferably about 2 or less, preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine segments are dispersed polymeric moieties having from about 350 to about 630 repeat units with a dispersion of about 2 or less, preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine segments are dispersed polymeric moieties having from about 400 to about 580 repeat units with a degree of dispersion of about 1.2 or less.

As noted above, those skilled in the art will appreciate that in some embodiments the LPEI fragment may comprise an organic moiety (i.e., a pendant amide group) linked to a nitrogen atom embedded within the LPEI chain. Those skilled in the art will understand that such organic moieties (i.e., amide groups) may be formed during ring-opening polymerization of 2-oxazoline to form poly(2-oxazoline). Without wishing to be bound by theory, LPEI can be formed by cleavage of the amide group from poly(2-oxazoline) (e.g., using an acid such as HCl). However, in some cases not all amide bonds are cleaved under these conditions. Accordingly, in some embodiments, up to about 5% of the nitrogen atoms of the LPEI fragment may be linked to an organic moiety to form an amide. In some embodiments, in the LPEI fragment, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%. Up to % of the nitrogen atoms may be linked to an organic moiety to form an amide. Those skilled in the art will understand that the molecular weight of an LPEI fragment includes the percentage of the LPEI fragment that is bound to an organic moiety as an amide. Moreover, although the chemical structures presented herein show repeating NH-CH ₂ -CH ₂ - fragments, those skilled in the art will recognize that trace amounts of residual organic moieties, such as pendant amide groups (e.g., as defined above), are present in the present invention. It will be appreciated that it may still exist in a triconjugate of multiple complexes. The term "triconjugate", as sometimes used herein, will refer to the conjugates of the invention. The prefix “triple” is derived from the three components included in the conjugates of the invention: the LPEI fragment, the PEG fragment and the targeting fragment.

PEG fragment

Polyethylene glycol (PEG) has the formula -[O-CH ₂ -CH ₂ ]-. In some preferred embodiments, the PEG fragment is linked to the LPEI fragment via [3 + 2] cycloaddition between the azide and the alkene or alkyne to form 1,2,3-triazole or 4,5-dihydro-1H-[ 1,2,3]triazoles, wherein the PEG fragment contains an alkene or alkyne functional group. For example, in some preferred embodiments the PEG fragment comprises the repeating formula -[O-CH ₂ -CH ₂ ]- and is bound at the first terminus (i.e., the end) to the azide group of the corresponding LPEI fragment. may be substituted with an alkene or alkyne group (e.g., via a linking moiety “X ¹ ” as discussed below).

In some preferred embodiments, the alkene or alkyne group is an activated alkene or alkyne group that can react spontaneously with an azide (eg, without the addition of a catalyst, such as a copper catalyst). For example, an activated alkyne group can be introduced into a 7- or 8-membered ring, creating a chained species that spontaneously reacts with the azide group of the LPEI fragment. Activated alkenes may contain a maleimide moiety and are activated by conjugation of the alkene to a neighboring carbonyl group. In some preferred embodiments, the second terminal end (i.e., terminus) of the PEG terminus is ^a targeting fragment (e.g., hEGF, HER2, folate or DUPA) (e.g., a binding moiety “ via), and in some preferred embodiments there are no additional substitutions present on the PEG fragment.

PEG fragments can cover a wide range of lengths (i.e., repeat units, denoted by the variable “m”). In other embodiments, PEG fragments may contain a definite number of repeating -O-CH ₂ -CH ₂ - units and are not defined in terms of average chain length. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety is a dispersed polymeric moiety. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, a definite number of repeat units m. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, a definite number of contiguous repeat units m.

In some preferred embodiments, the PEG fragment is a dispersed polymeric moiety comprising from about 1 to about 200 repeat units, preferably from about 1 to about 100 repeat units. In some preferred embodiments, the PEG fragment may comprise from about 1 to about 100 repeat units (i.e., -O-CH ₂ -CH ₂ -). Preferably, the PEG fragment of the invention has about 1 to about 100 repeat units, about 1 to about 100 repeat units, about 1 to about 90 repeat units, about 1 to about 80 repeat units, about 1 to about 70 repeat units, about 1 to about 60 repeat units, about 1 to about 50 repeat units, about 1 to about 50 repeat units, about 1 to about 40 repeat units, about It contains from 1 to about 30 repeat units, or from about 1 to about 20 repeat units. In some other preferred embodiments, the PEG fragment comprises a definite number m of repeat units, preferably 12 repeat units or 24 repeat units. In some embodiments, the polyethylene glycol fragments have from about 2 to about 80 repeat units and a dispersion of about 2.0 or less, preferably about 1.8 or less, more preferably about 1.5 or less; from about 2 to about 70 repeat units, preferably with a dispersion of about 1.8 or less, preferably about 1.5 or less; More preferably, it is a dispersed polymeric moiety having from about 2 to about 50 repeat units with a degree of dispersion of about 1.5 or less. In some embodiments, the -(O-CH ₂ -CH ₂ ) _m - moiety has from about 2 to about 80 repeat units and a dispersion of about 2.0 or less, preferably about 1.8 or less. It is a dispersed polymeric moiety having about 70 repeat units, more preferably about 2 to about 50 repeat units with a degree of dispersion of about 1.5 or less.

In a preferred embodiment, the polyethylene glycol fragments, PEG fragments, comprise and preferably consist of a definite number of repeat units m, preferably 12 or 24 repeat units. In a preferred embodiment, said m (of said -(O-CH ₂ -CH ₂ ) _m - moiety) comprises and preferably consists of a definite number of repeat units m, preferably 12 or 24 repeat units. do.

In a preferred embodiment, the PEG fragment comprises and preferably consists of repeat units m in a definite number of 2 to 100, preferably in a definite number m of 4 to 60. In a preferred embodiment, the PEG fragment comprises and preferably consists of repeat units m in a definite number of 4 to 60, preferably in a definite number m of 10 to 60. In a preferred embodiment, the PEG fragments have 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 It comprises, and preferably consists of, a definite number of repeat units m, 52, 53, 54, 55, 56, 57, 58, 59 or 60. In a preferred embodiment, the PEG fragments have 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 or It contains, and preferably consists of, a definite number of repeat units m, 60. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a definite number of repeat units m of 4. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, repeat units m with a definite number of 12. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a definite number of repeat units m of 24. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a distinct number of repeat units m of 36.

In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a definite number of m contiguous repeat units from 2 to 100, preferably from 4 to 60 contiguous repeat units m. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a definite number of m contiguous repeat units of from 4 to 60, preferably a definite number of m contiguous repeat units of from 10 to 60. In a preferred embodiment, the PEG fragments have 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 It comprises, and preferably consists of, a definite number of contiguous repeat units m, 52, 53, 54, 55, 56, 57, 58, 59 or 60. In a preferred embodiment, the PEG fragment has 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 or It comprises, and preferably consists of, a definite number of m adjacent repeat units of 60. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a definite number of m adjacent repeat units. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a distinct number of m contiguous repeat units of 12. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a definite number of m contiguous repeat units of 24. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, a definite number of m contiguous repeat units of 36.

In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety of formula (I*) or formula (I) has a repeating unit m in a definite number of 2 to 100, preferably in a definite number of 4 to 60. It comprises, and preferably consists of, a repeating unit m. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises a definite number of repeat units m, preferably between 4 and 60, preferably between 10 and 60, Preferably it consists of this. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moieties are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, contains a definite number of repeat units m of 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60; , preferably consists of this. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety is 4, 8, 12, 16, 20, 24, 28, 32, 36, 40. , and preferably consists of a definite number of repeat units m, 44, 48, 52, 56 or 60. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, four distinct numbers of repeat units m. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, a distinct number of repeat units m of 12. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, a repeating unit m with a definite number of 24. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, a repeating unit m with a definite number of 36.

In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety of formula (I*) or formula (I) has a distinct number of contiguous repeat units m, preferably from 2 to 100, preferably from 4 to 60. It contains, and preferably consists of, adjacent repeating units m. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises a distinct number of contiguous repeat units m of 4 to 60, preferably a distinct number of contiguous repeat units m of 10 to 60. And, preferably, it consists of this. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moieties are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, Contains a definite number of contiguous repeat units m of 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 And, preferably, it consists of this. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety is 4, 8, 12, 16, 20, 24, 28, 32, 36, 40. , and preferably consists of a definite number of contiguous repeat units m, 44, 48, 52, 56 or 60. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, four distinct numbers of contiguous repeat units m. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises, and preferably consists of, a distinct number of m adjacent repeat units. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises and preferably consists of 24 distinct numbers of contiguous repeat units m. In a preferred embodiment, the -(O-CH ₂ -CH ₂ ) _m - moiety comprises and preferably consists of 36 distinct numbers of contiguous repeat units m.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 2 to 200, preferably a definite number m of repeat units from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 2 to 200, preferably a definite number m of repeat units from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 2 to 200, preferably a definite number of m contiguous repeat units of 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 2 to 200, preferably a definite number of m contiguous repeat units of 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably a conjugate wherein the targeting fragment is capable of binding to a cell, more preferably wherein the targeting fragment is capable of binding to a cell surface receptor; to provide. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In some preferred embodiments, the conjugates of the invention comprise LPEI fragments present as dispersed polymeric moieties, where n is from about 280 to about 700 with a degree of dispersion of about 3 or less, preferably with a degree of dispersion of about 2 or less. a dispersity of about 350 to about 630, more preferably about 1.2 or less, about 400 to about 580, and the conjugate of the present invention further has (i) m a dispersity of about 2 to about 80 and about 2 or less, preferably preferably from about 2 to about 70 with a dispersion of up to about 1.8, more preferably from about 2 to about 50 with a dispersion of up to about 1.5; (ii) preferably the PEG fragment is present as a number of distinct repeat units, wherein the distinct number of repeat units m is from 12 to 24 repeat units.

In some embodiments, the conjugate of the invention comprises an LPEI fragment existing as dispersed polymeric moieties of about 17 to 25 kDa with a dispersity of about 1.2 or less, and PEG, preferably consisting of 12 repeat units. Includes fragments. In some preferred embodiments, the conjugate of the invention comprises an LPEI fragment present as a dispersed polymeric moiety having a molecular weight of about 17 to 25 kDa with a dispersion of about 1.2 or less, and 24 repeat units, preferably It may include PEG fragments composed of this.

targeting fragment

Conjugates of the invention include targeting fragments that allow directing the conjugates of the invention and multicomplexes of the invention to specific target cell types, cell populations, organs or tissues. Typically and preferably, the targeting fragment is capable of binding to a target cell, preferably a cell receptor or a cell surface receptor thereof.

As used herein, the term “cell surface receptor” refers to a protein, glycoprotein or lipoprotein that is present on the surface of a cell and is typically and preferably a unique marker for cell recognition. Typically and preferably, the cell surface receptors include hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules or nutrients in the form of peptides, small molecules, saccharides and oligosaccharides, lipids, amino acids, and antibodies, aptamers. , apibody, antibody fragments, etc. can bind to a ligand containing such other binding moieties.

Conjugates and multicomplexes containing targeting fragments of the invention aim to mimic this ligand-receptor interaction. Accordingly, in a preferred embodiment the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, the cell surface receptors include growth factor receptors, extracellular matrix proteins, cytokine receptors, hormone receptors, glycosylphosphatidylinositol (GPI) anchored membrane proteins, carbohydrate-binding membrane integral proteins, lectins, ion channels, G -selected from protein-coupled receptors and enzyme-coupled receptors, such as tyrosine kinase-coupled receptors.

In a preferred embodiment, the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, the cell surface receptors include growth factor receptors, extracellular matrix proteins, cytokine receptors, hormone receptors, glycosylphosphatidylinositol (GPI) anchored membrane proteins, carbohydrate-binding membrane integral proteins, lectins, ion channels, G -selected from protein-coupled receptors and enzyme-coupled receptors, such as tyrosine kinase-coupled receptors. In a preferred embodiment, the cell surface receptor is a growth factor receptor. In a preferred embodiment, the cell surface receptor is an extracellular matrix protein. In a preferred embodiment, the cell surface receptor is a cytokine receptor. In a preferred embodiment, the cell surface receptor is a hormone receptor. In a preferred embodiment, the cell surface receptor is a glycosylphosphatidylinositol (GPI) anchored membrane protein. In a preferred embodiment, the cell surface receptor is a carbohydrate-binding membrane-integrated protein. In a preferred embodiment, the cell surface receptor is a lectin. In a preferred embodiment, the cell surface receptor is an ion channel. In a preferred embodiment, the cell surface receptor is a G-protein coupled receptor. In a preferred embodiment, the cell surface receptor is an enzyme-linked receptor, preferably the enzyme-linked receptor is a tyrosine kinase-linked receptor.

In a preferred embodiment, the cell surface receptor is epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), prostate surface membrane antigen (PSMA), insulin-like growth factor 2 receptor (IGF1R), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR). In a preferred embodiment, the cell surface receptor is epidermal growth factor receptor (EGFR). In a preferred embodiment, the cell surface receptor is human epidermal growth factor receptor 2 (HER2). In a preferred embodiment, the cell surface receptor is prostate surface membrane antigen (PSMA). In a preferred embodiment, the cell surface receptor is insulin-like growth factor 2 receptor (IGF1R). In a preferred embodiment, the cell surface receptor is vascular endothelial growth factor receptor (VEGFR). In a preferred embodiment, the cell surface receptor is platelet-derived growth factor receptor (PDGFR). In a preferred embodiment, the cell surface receptor is fibroblast growth factor receptor (FGFR).

The targeting fragment according to the present invention aims to localize, deliver, and specifically selectively deliver the payload, such as the multicomplex and nucleic acid of the present invention, to a desired target, specifically a desired target cell. In addition, the conjugate containing the targeting fragment of the present invention not only allows selective delivery of the conjugate and polycomplex to a target, such as a target cell, but also allows internalization and polyanion transfer by the target cell specifically. Allows for easy selective cellular uptake of the rod. Therefore, targeting fragments according to the invention represent parts of the conjugates and multicomplexes of the invention capable of specific binding to a selected target, preferably to a selected target cell, more preferably to a selected cell receptor.

In a preferred embodiment, the targeting fragment is capable of binding to a target cell. In a preferred embodiment, the targeting fragment is capable of binding to a selected target cell type. In a preferred embodiment, the targeting fragment is capable of binding to a target cell receptor. In a preferred embodiment, the targeting fragment is capable of binding to a target cell surface receptor.

In a preferred embodiment, the targeting fragment functions to bind to a target cell. In a preferred embodiment, the targeting fragment functions to bind to a selected target cell type. In a preferred embodiment, the targeting fragment functions to bind to a target cell receptor. In a preferred embodiment, the targeting fragment acts to bind to a target cell surface receptor.

In a preferred embodiment, the targeting fragment is capable of specifically binding to target cells. In a preferred embodiment, the targeting fragment is capable of specifically binding to a selected target cell type. In a preferred embodiment, the targeting fragment is capable of specifically binding to a target cell receptor. In a preferred embodiment, the targeting fragment is capable of specifically binding to a target cell surface receptor.

In one embodiment, specific binding to said target cell, target cell receptor or target cell surface receptor is typically and preferably determined by the dissociation constant (KD) of the targeting fragment and conjugate of the invention and/ or the multicomplex of the present invention binds to the target cell, the target cell receptor or the target cell surface receptor, respectively, at least twice as much as the binding to another non-targeted target cell, target cell receptor or target cell surface receptor, preferably at least It means specific binding that is 3 times stronger, more preferably at least 4 times stronger, and even more preferably at least 5 times stronger. Preferably, the targeting fragment binds to the selected cell surface receptor with a KD of less than 10 ^-5 M, preferably less than 10 ^-6 M, more preferably less than 10 ^-7 M, even more preferably less than 10 ^-8 M.

In one embodiment, the targeting fragment and the conjugate of the present invention and/or the multicomplex of the present invention specifically bind to the target cell, the target cell receptor, or the target cell surface receptor, respectively. or, for said target cell surface receptor, similar to that illustrated in Example 23, consistent with the conjugates and/or multicomplexes of the invention, but instead of the targeting fragment, a hydroxyl group or -OMe moiety, preferably -at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, even more preferably at least 10-fold, even more so than corresponding conjugates and/or multicomplexes comprising non-specific fragments such as -OMe moieties. Preferably, this means specific binding that is at least 100 times stronger. Binding to a target cell, target cell receptor or target cell surface receptor is typically and preferably measured by the dissociation constant (KD). Preferably, the targeting fragment binds to the selected cell surface receptor with a KD of less than 10 ^-5 M, preferably less than 10 ^-6 M, more preferably less than 10 ^-7 M, even more preferably less than 10 ^-8 M. In a preferred embodiment, said binding or said specific binding, and therefore the level of binding, of the conjugates of the invention and the multicomplexes of the invention are determined by binding assays or displacement assays, respectively, or by FRET or targeting fragments and cellular receptors. It can be determined by other measures that demonstrate interactions between cell surface receptors.

As used herein, the term "binding" refers to the binding of a targeting fragment to a cell, cell receptor or cell surface receptor, preferably electrostatic interaction, van der Waals interaction, hydrogen bonding, hydrophobic interaction, ionic bonding, charge refers to interactions through non-covalent bonds, such as interactions, affinity interactions and/or dipole interactions.

In another embodiment, specific binding to the target cell, target cell receptor or target cell surface receptor is induced by binding of the targeting fragment and the conjugate of the invention and/or the multicomplex of the invention, respectively, and/ or produces a biological effect induced by the delivered conjugate and/or multicomplex and polyanion payload of the invention, wherein the biological effect is directed to a non-targeted cell, a non-targeted cell receptor, or a non-targeted cell surface receptor. Compared to the biological effect, it is at least 2 times, preferably at least 3 times, more preferably at least 5 times, even more preferably at least 10 times, even more preferably at least 25 times, at least 50 times, or at least 100 times.

In another embodiment, specific binding to the target cell, target cell receptor or target cell surface receptor is induced by binding of the targeting fragment and the conjugate of the invention and/or the multicomplex of the invention, respectively, and/ or produces a biological effect induced by the delivered conjugate and/or polycomplex and polyanionic payload, wherein the biological effect is similar to that exemplified in Example 23, by the conjugate and/or polyanionic payload of the present invention. or derived by a corresponding conjugate and/or multicomplex, which is consistent with the multicomplex of the invention but contains a non-specific fragment such as a hydroxyl group or an -OMe moiety, preferably an -OMe moiety, instead of the targeting fragment. Compared to the biological activity, it is at least 2 times, preferably at least 3 times, more preferably at least 5 times, even more preferably at least 10 times, more preferably at least 25 times, at least 50 times, or at least 100 times.

Binding and specific binding can likewise be measured by measures of activation of protein signaling and thus protein phosphorylation or protein expression, mRNA expression in cells or tissues (using Western blot analysis, PCR, RNAseq IHC, etc.). It can be. The level of delivery of the multicomplex of the present invention to a specific tissue can be determined by comparing the amount of protein produced in overexpressing cells compared to normal and low-expressing cells by Western blot analysis, luminescence/fluorescence assay, or flow cytometry assay, and by ELISA or ECLIA. It can be determined by measuring protein secretion using a scale such as . Downstream (from a delivered nucleic acid such as poly(IC)) in cells/tissues overexpressing the target receptor compared to normal cells/tissues or cells/tissues with low expression by Western blot analysis or luminescence/fluorescence assay, flow cytometry assay. Compare the amount of protein expression or secretion, or measure protein secretion using a scale such as ELISA or ECLIA. Transduction levels can also be measured by cytotoxicity using cell survival assays or cell death assays (including MTT, methylene blue assay, cell titerglow assay, propidium iodide assay). Comparing the amount of protein produced in the tissue relative to the weight of the tissue, comparing the amount of therapeutic and/or preventive agent in the tissue relative to the weight of the tissue, comparing the amount of protein produced in the tissue relative to the total amount of protein in the tissue, or , compare the amount of therapeutic/preventive agent in the tissue compared to the total amount of therapeutic/preventive agent in the tissue. It will be appreciated that delivery of the multicomplexes of the invention to target cells or target tissues need not be determined in the subject to be treated, but may be determined in an animal model or a surrogate such as a cell model.

Accordingly, in a preferred embodiment the biological effect includes (i) activation of protein signaling; (ii) protein expression; (iii) mRNA expression in cells or tissues; (iv) expression or secretion of downstream proteins from the delivered nucleic acid, such as the delivered poly(IC), in cells/tissues that overexpress the target cell surface receptor compared to normal cells/tissues or cells/tissues with low expression; and (v) cytotoxicity.

In one embodiment, the target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, nerve cells, cardiac cells, adipocytes, and vascular smooth muscle. cells, and in one embodiment, the target cells are cells in the liver. In one embodiment, the target cell is an epithelial cell. In one embodiment, the target cells are hepatocytes. In one embodiment, the target cells are hematopoietic cells. In one embodiment, the target cells are muscle cells. In one embodiment, the target cell is an endothelial cell. In one embodiment, the target cells are tumor cells or cells in the tumor environment. In one embodiment, the target cells are blood cells. In one embodiment, the target cells are cells in the lymph nodes. In one embodiment, the target cells are cells in the lung. In one embodiment, the target cells are cells in the skin. In one embodiment, the target cells are spleen cells. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In one embodiment, the target cells are dendritic cells in the spleen. In one embodiment, the target cell is a T cell. In one embodiment, the target cell is a B cell. In one embodiment, the target cells are NK cells. In one embodiment, the target cell is a mononuclear cell.

In some embodiments, the targeting fragment interacts selectively or preferentially with a particular cell type. Targeting fragments not only serve to selectively target the conjugates and multicomplexes of the invention to specific cells, but also typically facilitate selective uptake of the conjugates and corresponding multicomplexes of the invention within specific cell types. In some embodiments, the targeting fragment selectively or preferentially interacts with a specific cell surface receptor. When the targeting fragment of the conjugate and/or multicomplex selectively or preferentially interacts with a cell surface receptor, the conjugate and/or multicomplex may be selectively or preferentially absorbed into cells containing said cell surface receptor. .

In a preferred embodiment, the targeting fragment is a peptide, protein, small molecule, saccharide, oligosaccharide, lipid, amino acid, and the peptide, protein, small molecule, saccharide, oligosaccharide, lipid, amino acid is a cytokine, selected from growth factors, cell adhesion molecules or nutrients, and the targeting fragment is an antibody, antibody fragment, aptamer or epibody.

As used herein, the term "small molecule ligand" specifically refers to a targeting fragment of the invention having a molecular weight of at least 75 g/mol, preferably at least 100 g/mol, more preferably at least 200 g/mol, preferably refers to a chemical moiety having a molecular weight of less than about 2,000 g/mol. In some embodiments, the small molecule has a molecular weight of less than about 1,500 g/mol, preferably less than about 1,000 g/mol. In a further preferred embodiment, the small molecule has a molecular weight of less than about 800 g/mol, more preferably less than about 500 g/mol. As used herein, the term "small molecule ligand" specifically refers to a targeting fragment of the invention that is capable of binding, and preferably specifically binding, to a target cell, a target cell receptor, or preferably a target cell surface receptor. It is about these ligands that are present. In a preferred embodiment, the small molecule ligand has a molecular weight of at least 75 g/mol, preferably at least 100 g/mol, more preferably at least 200 g/mol, preferably at least less than 2,000 g/mol, preferably at least 1,500 g/mol. It has a molecular weight of less than g/mol. In a preferred embodiment, the small molecule ligand has a molecular weight of at least 75 g/mol, preferably at least 100 g/mol, more preferably at least 200 g/mol, preferably at least less than 2,000 g/mol, preferably at least 1,500 g/mol. Having a molecular weight of less than g/mol, the small molecule ligand is capable of binding, preferably specifically, to a target cell surface receptor.

In some embodiments, the targeting fragment is a native, natural or modified ligand or paralog thereof, or a non-natural ligand such as an antibody mimetic, such as an antibody, single chain variable fragment (scFv) or epibody. In a preferred embodiment, the targeting fragment is a native, native or modified cell surface antigen ligand or paralog thereof, or a non-natural cell surface antigen ligand such as an antibody parent such as an antibody, single chain variable fragment (scFv) or epibody. It's a corpse. In a preferred embodiment, the targeting fragment is a native, native or modified cell surface receptor ligand or paralog thereof, or a non-natural cell surface receptor ligand such as an antibody parent such as an antibody, single chain variable fragment (scFv) or epibody. It's a corpse. In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, native, natural or modified ligand and/or paralogs thereof. In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, native, natural or modified cell surface antigen ligand and/or paralogs thereof, wherein the small molecule ligand is at least 75 g/mol, preferably It has a molecular weight of at least 100 g/mol, more preferably at least 200 g/mol, preferably at least less than 2,000 g/mol, preferably less than at least 1,500 g/mol. In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, native, natural or modified cell surface receptor ligand and/or paralogs thereof, wherein the small molecule ligand is at least 75 g/mol, preferably It has a molecular weight of at least 100 g/mol, more preferably at least 200 g/mol, preferably at least less than 2,000 g/mol, preferably less than at least 1,500 g/mol. In a preferred embodiment, the targeting fragment is an antibody parent such as a small molecule ligand, peptide, protein, aptamer, native, natural or modified ligand and/or paralog thereof, antibody, single chain variable fragment (scFv) or epibody. It's a corpse.

In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, native, native or modified cell surface receptor ligand and/or paralogs thereof. In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, native, natural or modified ligand and/or paralogs thereof, wherein the small molecule ligand, peptide, protein, aptamer, or Native, natural or modified ligands and/or their paralogs are capable of binding, and preferably selectively, binding to a target cell surface receptor. In a preferred embodiment, the targeting fragment is a small molecule ligand. In a preferred embodiment, the targeting fragment is a small molecule ligand, which is capable of binding, preferably selectively, to a target cell surface receptor. In a preferred embodiment, the targeting fragment is a peptide. In a preferred embodiment, the targeting fragment is a peptide, said peptide being capable of binding, preferably selectively, to a target cell surface receptor. In a preferred embodiment, the targeting fragment is a protein. In a preferred embodiment, the targeting fragment is a protein, which protein is capable of binding, preferably selectively, to a target cell surface receptor. In a preferred embodiment, the targeting fragment is an aptamer. In a preferred embodiment, the targeting fragment is an aptamer, wherein the aptamer is capable of binding, preferably selectively, to a target cell surface receptor. In a preferred embodiment, the targeting fragment is a native, natural or modified ligand and/or paralogs thereof. In a preferred embodiment, the targeting fragment is a native, natural or modified ligand and/or their paralogs, wherein the native, natural or modified ligand and/or their paralogs are capable of binding to a target cell surface receptor. and can preferably be selectively combined. In a preferred embodiment, the targeting fragment is an antibody, a single chain variable fragment (scFv) or an antibody mimetic such as an epibody. In a preferred embodiment, the targeting fragment is an antibody mimetic, such as an antibody, a single chain variable fragment (scFv) or an apibody, and the antibody mimetic, such as an antibody, a single chain variable fragment (scFv) or an apibody, is a target cell surface receptor. Can bind to, and preferably can bind selectively.

In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, antibody, antibody fragment, preferably a single chain variable fragment (scFv), preferably an antibody mimetic selected from apibody, nanobody, diabody, designed Ankyrin repeat protein (DARPin), growth factor or functional fragment thereof, preferably hEGF, hormone or functional fragment thereof, preferably insulin, cytokine or functional fragment thereof, integrin, interleukin or functional fragment thereof, enzyme, nucleic acid, fatty acid, Carbohydrates, monosaccharides, oligosaccharides or polysaccharides, peptidoglycan, glycoprotein, asialoorosomucoid, mannose-6-phosphate, mannose, ^sialyl -Lewis Nuclear localization agents, preferably T antigen, tumor low-pH insertion peptide (PHLIP), p32 targeting peptide, preferably LyP-1 tumor homing peptide, insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor and /or fibroblast growth factor.

In some embodiments, the targeting fragment is an antibody or antibody fragment (e.g., a single chain variable fragment (scFv), an antibody mimetic selected from an apiform, a nanobody, a diabody, a designed ankyrin repeat protein (DARPin), or other antibody variant) It is a non-native ligand such as In a preferred embodiment, the targeting fragment is a growth factor or fragment thereof, preferably a functional fragment (e.g. hEGF), a hormone or a fragment thereof, preferably a functional fragment (e.g. insulin), asialoorosomucoid, mannose-6- Such as phosphate, mannose, sialyl- ^Lewis p32 targeting peptide, insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor and/or fibroblast growth factor. Additionally, non-limiting examples of targeting fragments include enzymes, nucleic acids, fatty acids, carbohydrates, monosaccharides, oligosaccharides or polysaccharides, peptidoglycan, and glycoproteins.

In a preferred embodiment, the targeting fragment is a small molecule ligand, peptide, protein, aptamer, antibody, antibody fragment, preferably a single chain variable fragment (scFv), preferably Fab, Fab', F(ab') ₂ or scFv fragment. , preferably an antibody mimetic selected from apibody, nanobody, diabody, designed ankyrin repeat protein (DARPin), growth factor or functional fragment thereof, preferably hEGF, hormone or functional fragment thereof, preferably insulin, cytokine or Functional fragments thereof, interleukins or functional fragments thereof, enzymes, nucleic acids, fatty acids, carbohydrates, monosaccharides, oligosaccharides or polysaccharides, peptidoglycan, glycoprotein, asialoorosomucoid, mannose-6-phosphate, mannose, sialyl ^-Lewis growth factor-like 1, vascular endothelial growth factor, platelet-derived growth factor, and/or fibroblast growth factor.

In some embodiments, the targeting fragment L is hEGF; anti-HER2 peptide, preferably anti-HER2 antibody or apibody; DUPA; Folate receptor targeting fragment, folic acid; A somatostatin receptor targeting fragment, preferably somatostatin and/or octreotide; an integrin targeting fragment, preferably a fragment comprising arginine-glycine-aspartic acid (RGD); low-pH insert peptide; an asialo glycoprotein receptor targeting fragment, preferably an asialoorosomucoid; Insulin receptor targeting fragment, preferably insulin; Mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; Mannose receptor targeting fragment, preferably mannose; Sialyl Lewis ^X antigen targeting fragment, preferably E-selectin; Sigma-2 receptor agonists, preferably N,N-dimethyltryptamine (DMT), sphingolipid-derived amines and/or steroids, more preferably progesterone; p32 targeting ligand, preferably anti-p32 antibody or p32 binding LyP-1 tumor homing peptide; Trop-2 targeting fragments, preferably anti-Trop-2 antibodies and/or antibody fragments; Insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and fibroblast growth factor.

In some embodiments, the targeting fragment L is hEGF; anti-HER2 peptide, preferably anti-HER2 antibody or apibody; DUPA; folic acid; A somatostatin receptor targeting fragment, preferably somatostatin and/or octreotide; an integrin targeting fragment, preferably a fragment comprising arginine-glycine-aspartic acid (RGD); low-pH insert peptide; an asialo glycoprotein receptor targeting fragment, preferably an asialoorosomucoid; Insulin receptor targeting fragment, preferably insulin; Mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; Mannose receptor targeting fragment, preferably mannose; Sialyl Lewis ^X antigen targeting fragment, preferably E-selectin; Sigma-2 receptor agonists, preferably N,N-dimethyltryptamine (DMT), sphingolipid-derived amines and/or steroids, more preferably progesterone; p32 targeting ligand, preferably anti-p32 antibody or p32 binding LyP-1 tumor homing peptide; Trop-2 targeting fragments, preferably anti-Trop-2 antibodies and/or antibody fragments; Insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and targeting fragments derived from fibroblast growth factors.

In a preferred embodiment, the targeting fragment is an hEGF targeting fragment; PSMA targeting fragment; anti-HER2 peptide, preferably anti-HER2 antibody or apibody; folic acid; A somatostatin receptor targeting fragment, preferably somatostatin and/or octreotide; an integrin targeting fragment, preferably a fragment comprising arginine-glycine-aspartic acid (RGD); low-pH insert peptide; an asialo glycoprotein receptor targeting fragment, preferably an asialoorosomucoid; Insulin receptor targeting fragment, preferably insulin; Mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; Mannose receptor targeting fragment, preferably mannose; Sialyl Lewis ^X antigen targeting fragment, preferably E-selectin; Sigma-2 receptor agonists, preferably N,N-dimethyltryptamine (DMT), sphingolipid-derived amines and/or steroids, more preferably progesterone; p32 targeting ligand, preferably anti-p32 antibody or p32-binding LyP-1 tumor homing peptide; Trop-2 targeting fragments, preferably anti-Trop-2 antibodies and/or antibody fragments; Insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and targeting fragments derived from fibroblast growth factors.

In a preferred embodiment, the targeting fragment is an epidermal growth factor, such as human epidermal growth factor (hEGF), and typically and preferably the linkage to the remainder of the conjugate is via the amino group of hEGF. hEGF can be selectively taken up by cells that have increased expression (e.g., overexpression) of human epidermal growth factor receptor (EGFR).

In a preferred embodiment, the targeting fragment is capable of binding to human epidermal growth factor receptor (EGFR), also referred to herein as an EGFR targeting fragment.

EGFR is a transmembrane glycoprotein that is a receptor for members of the protein kinase superfamily and members of the epidermal growth factor family. EGFR is a cell surface protein that binds to epidermal growth factor and induces receptor duplexing and tyrosine autophosphorylation, thereby promoting cell proliferation. In a preferred embodiment, the EGFR targeting fragment is capable of binding an epitope on the extracellular domain of EGFR.

In a preferred embodiment, the targeting fragment is capable of binding to cells expressing EGFR. In a preferred embodiment, the targeting fragment is capable of binding to cells overexpressing EGFR. In one embodiment, the cells overexpressing EGFR mean that the level of EGFR expressed in the cells of a specific tissue is elevated compared to the level of EGFR measured in healthy normal cells of the same type of tissue under similar conditions. In one embodiment, the cell overexpressing the EGFR refers to an increase in the level of EGFR in the cell compared to the level in the same cell or a closely related non-malignant cell under physiological normal conditions. In one embodiment, the cells overexpressing EGFR have EGFR expression at least 10-fold, preferably at least 20-fold compared to EGFR expression in normal cells or normal tissues.

In a preferred embodiment, the targeting fragment is capable of binding to cells expressing or overexpressing EGFR. For example, EGFR is found in neoplastic tissue, including head and neck, thyroid, breast, ovary, colon, stomach, colorectal, gastrointestinal, cervix, bladder, lung, nasopharyngeal, and esophageal tissues, glioblastoma, carcinoma, and squamous tissue. In cancer types such as cancer of epithelial origin (e.g., Gan et al ., J. Cell Mol. Med., September 2009, 13(9b): 3993-4001; Aratani et al ., Anticancer Research, June 2017, 37(6): 3129-3135), specifically glioblastoma, non-small cell lung carcinoma, breast cancer, glioblastoma, squamous cell carcinoma, such as head and neck squamous cell carcinoma, small intestine cancer, colorectal cancer, adenocarcinoma, ovarian It is overexpressed in cancer, bladder cancer, or prostate cancer and their metastatic cancer.

EGFR expression and overexpression is preferably measured using monoclonal antibodies targeting EGFR, for example, by immunohistochemical methods (e.g., Kriegs et al. , Nature, 2019, 9: 13564; Prenzel et al ., Endocr. Relat. Cancer, 8: 11-31, 2001). A cut-off of 5% or more of EGFR positive cells can be used to define EGFR expression in different tissues or cell types. Therefore, cells or tissues with <5% positive cells can be considered negative.

In a preferred embodiment, the targeting fragment is capable of specifically binding to EGFR. Typically, specific binding refers to the binding affinity or dissociation constant K _D of the targeting fragment in the range from about 1×10 ^-3 M to about 1×10 ^-12 M. In a preferred embodiment, said targeting fragment is capable of specifically binding to EGFR, typically and preferably said affinity or specific binding is measured by the dissociation constant (K _D ), and said affinity or specific binding is determined by the dissociation constant (K D). is less than 10 ^-3 M, preferably less than 10 ^-4 M, more preferably less than 10 ^-5 M, more preferably less than 10 ^-6 M, more preferably less than 10 ^-7 M, even more preferably 10 ^-8 M. It refers to a K _D of less than, more preferably less than 10 ^-9 M. In a preferred embodiment, said targeting fragment is capable of specifically binding to EGFR, typically and preferably said affinity or specific binding is measured by the dissociation constant (K _D ), and said affinity or specific binding is determined by the dissociation constant (K D). refers to K _D of less than 10 ^-3 M, less than 10 ^-4 M, less than 10 ^-5 M, less than 10 ^-6 M, less than 10 ^-7 M, less than 10 ^-8 M and less than 10 ^-9 M. To detect binding or complexes or measure affinity, molecules are subjected to competition binding assays, typically and preferably such as on a Biacore 3000 instrument (Biacore, Piscataway NJ; see, e.g., Wei-Ting Kuo et al. , PLoS One. 2015, 10(2): e0116610 or described in US patent application US 2017224620A1. Preferably, binding leads to the formation of a complex between the EGFR targeting fragment and EGFR, where binding or complexing can be detected.

In a preferred embodiment, the targeting fragment is an EGFR antibody, an EGFR epibody, an EGFR aptamer, an EGFR targeting peptide or an EGFR targeting tyrosine kinase inhibitor. In a preferred embodiment, the EGFR targeting fragment is an EGFR antibody, an EGFR epibody, an EGFR aptamer, an EGFR targeting peptide or an EGFR targeting tyrosine kinase inhibitor.

In a preferred embodiment, the targeting fragment is an EGFR targeting peptide. EGFR targeting peptide typically and preferably refers to a peptide ligand of EGFR. Such peptide ligands of EGFR are known to those skilled in the art and are described, for example, in US patent application US2017224620A1 and in Gent et al ., 2018, Pharmaceutics, 2018, 10:2; incorporated herein by reference in its entirety. It is described by. EGFR targeting peptides have low immunogenic potential and show good penetration into solid tumor tissues.

In a preferred embodiment, the EGFR targeting peptide is from about 1,000 g/mol to about 2,000 g/mol, preferably from about 1,100 g/mol to about 1,900 g/mol, more preferably from about 1,200 g/mol to about 1,800 g/mol. , and even more preferably have a molecular weight of about 1,300 g/mol to about 1,700 g/mol.

In a preferred embodiment, the EGFR targeting peptide comprises, or preferably consists of, the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 9). In a preferred embodiment, the targeting fragment comprises, or preferably consists of, the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 9). GE-11 has excellent affinity for EGFR and also exhibits binding affinity for EGFR (kd = 22 nM) (Ruoslahti et al ., Adv. Mater., 2012, 24: 3747-3756; Li et al. ., J. Res. Commun. 2005, 19: 1978-1985). GE11 migrates from EGFR following addition of the physiological ligand EGF, demonstrating both selective binding to EGFR and its receptor affinity. GE11 has been reported to have high potential to accelerate nanoparticle endocytosis due to an alternative EGFR-dependent actin-driven pathway (Mickeler et al ., Nano Lett. 2012, 12: 3417-3423; Song et al ., FASEB J. 2009, 23: 1396-1404). We observed that the level of EGFR on the surface of cancer cells remained constant following treatment with GE11 multicomplex, indicating an EGFR recycling process that extended the capacity of cells to cycle GE11 multicomplex.

In a preferred embodiment, the EGFR targeting peptide comprises, or preferably consists of, GE11 (SEQ ID NO: 9), which is specifically used to treat solid tumors characterized by EGFR overexpressing cells. Conjugates and multicomplexes of the invention comprising, or preferably consisting of, GE11 as a targeting fragment are stable multicomplexes that ensure that the polyanion and nucleic acid payload are not released before the multicomplex reaches its target cells. It is considered.

In a preferred embodiment, the targeting peptide is an EGFR antibody. EGFR antibody refers to an antibody that binds to EGFR. In a preferred embodiment, the EGFR antibody is a human antibody. In a preferred embodiment, the EGFR antibody is a humanized EGFR antibody. In a preferred embodiment, the EGFR antibody is a monoclonal human antibody. In a preferred embodiment, the EGFR antibody is a humanized EGFR antibody. In a preferred embodiment, the EGFR antibody is a monoclonal fully human EGFR antibody. In another preferred embodiment, the EGFR antibody is an scFv or Fab fragment.

EGFR antibodies are known to those skilled in the art and are described, for example, in international patent applications WO2008/105773 and WO2017/185662 (which are hereby incorporated by reference in their entirety), including bevacizumab, panitumumab, and cetuximab. , tomozotuximab, futuximab, zatuximab, modotuximab, imgatuzumab, zalutumumab, matuzumab, necitumumab, nimotuzumab, CEVIAvax EGF, clone EGFR, L8A4, E6.2, TH190DS, Pep2, Pep3, LR-DM1, P1X, YC088, ratML66, FM329, TGM10-1, F4, 2F8, 15H8, TAB-301MZ-S(P), mAb528, 2224, E7.6.3, C225, CBL155, MR1 , MR1, L211C, N5-4, TH83DS, L2-12B, 15H8, 12Do3, 7A7, 42C11 (MOB-1078z), PABL-080, HPAB-2204LY-S(P), VHH205, ABT-806, , Tab- 271MZ, Hu225, LA22, Fab fragment DL11, Fab fragment DX 1-6, VHH104, OA-cb6, 07D06, Fab fragment HPAB-0419-FY-F(E), Fab fragment TAB-285MZ-F(E), Fab Fragment TAB-293MZ-F(E), Fab fragment HPAB-0136-YJ-F(E), FGF-R2, EG-19-11, Fab fragment pSEX81-63, DX 1-4, scFv fragment DX 1-6 , EG-26-11, EG-26-11, DX1-4, TAB-326MZ, scFv fragment 528, scFv fragment LA1, scFv fragment 07D06, single domain antibody VHH139, scFv fragment EG-19-11, single domain antibody VHH134 , including single domain antibodies 9G8, ABT-414, AMG-595, and IMGN-289. Those skilled in the art will understand that any antibody that recognizes and/or specifically binds to EGFR may be used in accordance with the present invention.

In a preferred embodiment, the targeting peptide is an EGFR inhibitor. EGFR inhibitors include targeting fragments that block cell surface localization and signaling of EGFR, such as oligosaccharide transferase inhibitors such as nerve growth inhibitor 1 or EGFR kinase inhibitors such as afatinib, erlotinib, osimertinib, and gefitinib. says EGFR inhibitors are known to those skilled in the art and are described, for example, in international patent application WO2018078076 and US patent application US2017224620A1, which are hereby incorporated by reference in their entirety.

In a preferred embodiment, the targeting peptide is an EGFR aptamer. Preferred EGFR targeting aptamers include, but are not limited to, Na Li et al ., PLoS One, which are hereby incorporated by reference in their entirety. 2011; 6(6): e20299;, Deng-LiangWang et al ., Biochemical and Biophysical Res. Com., 453(4), 2014, pp 681-685), Min Woo Kim et al. , Theranostics 2019, 9(3): 837-852); Akihiro Eguchi et al ., JACS, August 2021, 1, 5: 578-585) or Yingpan Song et al ., RSC Adv., 2020, 10: 28355-28364).

The term EGFR aptamer also includes EGFR aptamer derivatives and/or functional fragments of an EGFR aptamer. In some embodiments, no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 nucleic acids in an EGFR aptamer derivative are associated with the corresponding EGFR aptamer. Substituted in contrast. In some embodiments, the sequence of the EGFR aptamer derivative is at least 80% identical, preferably 85% identical, more preferably 90% identical, even more preferably 95% identical, and most preferably 99% identical to the corresponding EGFR aptamer.

In a preferred embodiment, the targeting fragment is an EGFR epibody. Preferred EGFR epibodies include, but are not limited to, ZEGFR:1907, ZEGFR:2377, ZEGFR:03115 (available from Affibody Medical) or dimeric forms of these epibodies. In a preferred embodiment, the EGFR epibody has the sequence SEQ ID NO: 8.

In a preferred embodiment, the targeting fragment is the EGFR ligand epidermal growth factor (EGF). Accordingly, in a preferred embodiment the targeting fragment is epidermal growth factor (EGF). In a preferred embodiment, the targeting fragment is human EGF (hEGF), mouse EGF (mEGF), rat EGF or guinea pig EGF. In a very preferred embodiment, said targeting fragment is human EGF (hEGF). In a preferred embodiment, the targeting fragment comprises and preferably consists of the sequence of SEQ ID NO:7.

In some embodiments, EGF is modified, such as by deletion or replacement of one or more amino acids or by truncation of EGF. Modified and/or truncated EGF molecules are disclosed, for example, in international patent application WO2019023295A1. EGF has many residues that are conserved across rat, mouse, guinea pig, and human species (Savage et al ., J. Biol. Chem., 247: 7612-7621, 1973; Carpenter and Cohen, Ann. Rev. Biochem. , 48: 193-316, 1979; Simpson et al ., J. Biochem., 153: 629-37, 1985). Specifically, six cysteine residues at positions 6, 14, 20, 31, 33, and 42 are conserved because they form three disulfide bridges, providing a conserved tertiary protein structure. Locations 7, 9, 11, 12, 13, 15, 18, 21, 24, 29, 32, 34, 36, 37, 39, 41, 46 Residues 4 and 47 are also conserved across all four species. These various residues may be expected to facilitate or provide key binding interactions with the corresponding EGFR. It has been described that both full-length human EGF (53 residues) and the truncated form resulting from trypsin cleavage (48 residues) retain strong binding affinity and activation of EGFR (Calnan et al. , 47(5)): 622-7, 2000; Gregory, Regul., 22: 217-26, 1988). Mutagenesis studies have reported that various residues are correlated with the effect of replacement of specific residues or activation of EGFR upon EGF binding to EGFR (Campion et al ., Biochemistry, 29: 9988-9993, 1990; Engler et al. ., J. Biol. Chem., 267: 2274-2281, 1992; Tadaki and Niyogi., J. Biol., 268: 10114-10119, 1993). Analysis of the x-ray crystal structure of EGF bound to EGFR revealed key binding interactions and also identified residues not directly involved in binding (Ogiso et al ., Cell, 110: 775-787, 2002).

In another aspect, the present invention provides a composition comprising a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

here

n is any integer from 1 to 1,500;

m is any integer from 1 to 200, preferably m is any integer from 1 to 100;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

X ¹ and X ² are independently divalent covalent moieties;

Z is a divalent covalent moiety, Z is not a single bond and Z is not -NHC(O)-;

L is a targeting fragment, said targeting fragment comprising or preferably consisting of the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 9);

Preferably, the composition provides a composition comprising the conjugate.

In another aspect, the present invention provides a conjugate of Formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

here

n is any integer from 1 to 1,500;

m is any integer from 1 to 200, preferably m is any integer from 1 to 100;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

X ¹ and X ² are independently divalent covalent moieties;

Z is a divalent covalent moiety, Z is not a single bond and Z is not -NHC(O)-;

L is a targeting fragment, and the targeting fragment comprises or preferably consists of the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 9).

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety,

X ² is a divalent covalent moiety,

L is a targeting fragment, and the targeting fragment comprises or preferably consists of the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 9).

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety,

X ² is a divalent covalent moiety,

L is a targeting fragment, and the targeting fragment comprises or preferably consists of the sequence YHWYGYTPQNVI (GE11) (SEQ ID NO: 9).

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 2 to 200, preferably a definite number m of repeat units from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, and the targeting fragment is an EGFR targeting fragment, preferably the EGFR targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, EGFR.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 2 to 200, preferably a definite number of m contiguous repeat units of 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, and the targeting fragment is an EGFR targeting fragment, preferably the EGFR targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, EGFR.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 2 to 200, preferably a definite number m of repeat units from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 2 to 200, preferably a definite number of m contiguous repeat units of 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor.

In a preferred embodiment, the targeting fragment is capable of binding to prostate surface membrane antigen (PSMA), also referred to herein as the PSMA targeting fragment.

PSMA is a multifunctional transmembrane protein that functions as a glutamate carboxypeptidase and also exhibits rapid ligand-induced internalization and recycling (Liu H., et al ., 1998, Cancer Res., 58: 4055-4060). PSMA is expressed primarily in four body tissues, including the prostatic epithelium, the proximal tubules of the kidney, the jejunal border of the small intestine, and the ganglia of the nervous system (Mhawech-Fauceglia et al ., Histopathology 2007, 50: 472-483). In a preferred embodiment, the targeting fragment is capable of binding an epitope on the extracellular domain of PSMA.

In a preferred embodiment, said targeting fragment, preferably said PSMA targeting fragment, is capable of binding to cells expressing PSMA. In a preferred embodiment, said targeting fragment, preferably said PSMA targeting fragment, is capable of binding to cells overexpressing PSMA. For example, PSMA is overexpressed in neoplastic tissue and malignant prostate, especially prostate adenocarcinoma, compared to normal tissue, and PSMA expression levels are further upregulated as the disease progresses to the metastatic phase (Silver et al ., 1997, Clin Cancer Res., 3: 81). PSMA is also expressed and overexpressed in other tumor types (Mhawech-Fauceglia et al ., Histopathology, 2007, 50: 472-483; Israeli RS et al ., Cancer Res., 1994, 54: 1807-1811; Chang SS et al. , Cancer Res., 1999, 59: 3192-198).

In one embodiment, overexpressing PSMA means that the level of PSMA expressed in the cells of a specific tissue is elevated compared to the level of PSMA measured in healthy normal cells of the same type of tissue under similar conditions. In one embodiment, overexpressing PSMA refers to an increase in the level of PSMA in the cell compared to the level in the same cell or a closely related non-malignant cell under physiological normal conditions. In one embodiment, the cells overexpressing PSMA have PSMA expression at least 10-fold compared to normal cells or normal tissues. In one embodiment, the cells overexpressing PSMA are related to PSMA expression with a cut-off of 5% or more of PSMA positive cells as described for example in Mhawech-Fauceglia et al ., 2007, which differs It can be used to define PSMA expression in a tissue or cell type. Therefore, cells or tissues with <5% positive cells were considered negative, or PSMA expression was classified according to its intensity, 0 (no expression), 1 (low expression), 2 (moderate expression) and Scored as 3 (high expression) (Hupe MC et al ., Frontiers in Oncology, 2018, 8(623): 1-7).

In a preferred embodiment, the targeting fragment is capable of binding to cells expressing or overexpressing PSMA. Cells expressing PSMA typically include tumor cells such as prostate, bladder, pancreas, lung, kidney, colon tumor cells, melanoma, and sarcoma. In a preferred embodiment, the targeting fragment is capable of binding to a cell expressing or overexpressing PSMA, wherein the cell is a tumor cell preferably selected from prostate, bladder, pancreas, lung, kidney, colon tumor cells, melanoma, and sarcoma. am. In a preferred embodiment, the targeting fragment is capable of binding to a cell that expresses or overexpresses PSMA, wherein the cell is a tumor cell and the tumor cell is a prostate tumor cell.

In a preferred embodiment, the targeting fragment is capable of specifically binding PSMA, typically and preferably said affinity or specific binding is measured by the dissociation constant (K _D ), and said affinity or specific binding is less than 10 ^-3 M, preferably less than 10 ^-4 M, more preferably less than 10 ^-5 M, more preferably less than 10 ^-6 M, more preferably less than 10 ^-7 M, even more preferably 10 ^-8 M. It refers to a K _D of less than, more preferably less than 10 ^-9 M. In a preferred embodiment, the targeting fragment is capable of specifically binding PSMA, typically and preferably said affinity or specific binding is measured by the dissociation constant (K _D ), and said affinity or specific binding refers to K _D of less than 10 ^-3 M, less than 10 ^-4 M, less than 10 ^-5 M, less than 10 ^-6 M, less than 10 ^-7 M, less than 10 ^-8 M and less than 10 ^-9 M. Preferably, the binding leads to the formation of a complex between the targeting fragment and PSMA, and the binding or complex is typically and preferably Kularatne et al ., Mol. Pharm., 2009, 6(3): 790. -800), typically and preferably detected using a BIACOA 3000 instrument (BIACOA, Piscataway NJ) or a cell-based binding assay or flow induced dispersion analysis (FIDA).

In a preferred embodiment, the targeting fragment is a PSMA antibody, PSMA aptamer or small molecule PSMA targeting fragment. In a preferred embodiment, the PSMA targeting fragment is a PSMA antibody, PSMA aptamer or small molecule PSMA targeting fragment. As used herein, the term “small molecule PSMA targeting fragment” typically and preferably has a molecular weight of less than about 2,000 g/mol and refers to a chemical moiety that is capable of binding PSMA. In some embodiments, the small molecule PSMA targeting fragment has a molecular weight of less than about 1,800 g/mol. In some embodiments, the small molecule PSMA targeting fragment has a molecular weight of less than about 1,500 g/mol, more preferably less than about 1,000 g/mol. In a further preferred embodiment, the small molecule has a molecular weight of less than about 800 g/mol, even more preferably less than about 500 g/mol.

In some embodiments, the PSMA targeting fragment is a PSMA antibody, which is an antibody capable of specifically binding to PSMA. In some embodiments, the antibodies are monoclonal antibodies, polyclonal antibodies and/or antibody fragments, preferably functional fragments thereof, chimeric antibodies, recombinant antibodies, and/or bispecific or multispecific antibodies. These PSMA antibodies include, but are not limited to, scFv antibodies A5, G0, G1, G2 and G4, and mAbs 3/E7, 3/F11, 3/A12, K7, K12 and D20 (Elsasser-Beile et al ., 2006 , Prostate, 66: 1359); mAbs E99, J591, J533 and J415 (Liu et al ., 1997, Cancer Res., 57: 3629; Liu et al ., 1998, Cancer Res., 58: 4055; Fracasso et al ., 2002, Prostate, 53: 9; McDevitt et al ., 2000, Cancer Res ., 60: 6095; McDevitt et al . , 2001, Science, 294: 1537; ., 2004, Prostate, 58: 145; Bander et al ., 2003, J. Urol., 170: 1717; Patri et al ., 2004, Bioconj., 15: 1174; and US Pat. No. 7,163,680; mAb 7E11-C5.3 (Horoszewicz et al ., 1987, Anticancer Res., 7: 927); antibody 7E11 (Horoszewicz et al ., 1987, Anticancer Res., 7: 927; and US Pat. No. 5,162,504); and antibodies described below (Chang et al ., 1999, Cancer Res., 59: 3192; Murphy et al ., 1998, J. Urol., 160: 2396; Grauer et al ., 1998, Cancer Res., 58: 4787; and Wang et al ., 2001, J. Cancer, 92: 871). Those skilled in the art will understand that any antibody that recognizes and/or specifically binds PSMA may be used in accordance with the present invention. All of the foregoing documents and disclosures are hereby incorporated by reference in their entirety.

In some embodiments, the targeting fragment capable of binding PSMA is an aptamer. PSMA targeting aptamers include, but are not limited to, the A10 aptamer or the A9 aptamer (Lupold et al ., 2002, Cancer Res., 62: 4029; and Chu et al ., 2006, Nuc. Acid Res., 34: e73 ), derivatives thereof, and/or functional fragments thereof. In some embodiments, no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 nucleic acids in the PSMA aptamer derivative are substituted relative to the aptamer. do. In some embodiments, the sequences of the aptamer derivatives are at least 80% identical, preferably 85% identical, more preferably 90% identical, even more preferably 95% identical, and most preferably 99% identical.

In a preferred embodiment, the targeting fragment is a small molecule PSMA targeting fragment. In a preferred embodiment, the PSMA targeting fragment is a small molecule PSMA targeting fragment, preferably a small molecule PSMA targeting peptidase inhibitor. In a preferred embodiment, the small molecule PSMA targeting peptidase inhibitor is 2-PMPA, GPI5232, VA-033, phenylalkylphosphoramidate (Jackson et al ., 2001, Curr. Med. Chem., 8: 949; Bennett et al ., 1998, J. Am. Soc., 120: 12139 , J. Med. Chem., 2002, Bioorg Med . Lett., 12 : 2189; Biochem. Biophys. Commun . , 307 ; Bioorg. Chem., 11: 4455; , 2004, Bioorg. Med. Chem., 12: 4969), and/or analogs and derivatives thereof. All of the foregoing publications (scientific and other publications, patents and patent applications) are hereby incorporated by reference in their entirety. In some embodiments, the small molecule PSMA targeting fragment is a protein, peptide, amino acid, or derivative thereof. In a preferred embodiment, the small molecule PSMA targeting fragment is thiol and indole thiol derivatives such as 2-MPPA and 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid derivatives (Majer et al ., 2003, J. Med. Chem., 46: 11989; and US Patent Application US 2005/0080128. In some embodiments, the small molecule PSMA targeting fragment comprises a hydroxamate derivative (Stoermer et al ., 2003, Bioorg. Med. Chem. Lett., 13: 12097). In a preferred embodiment, the small molecule PSMA peptidase inhibitor is an androgen receptor targeting agent (ARTA), such as those described in US Pat. No. 7,026,500; US 7,022,870; US 6,998,500; US 6,995,284; US 6,838,484; US 6,569,896; US 6,492,554; US patent application US 2006/0287547; US 2006/0276540; US 2006/0258628; US 2006/0241180; US 2006/0183931; US 2006/0035966; US 2006/0009529; US 2006/0004042; US 2005/0033074; US 2004/0260108; US 2004/0260092; US 2004/0167103; US 2004/0147550; US 2004/0147489; US 2004/0087810; US 2004/0067979; US 2004/0052727; US 2004/0029913; US 2004/0014975; US 2003/0232792; US 2003/0232013; US 2003/0225040; US 2003/0162761; US 2004/0087810; US 2003/0022868; US 2002/0173495; US 2002/0099096; Including those described in US 2002/0099036. In some embodiments, the small molecule PSMA targeting fragment comprises polyamines such as putrescine, spermine, and spermidine (US patent applications US 2005/0233948 and US 2003/0035804). All of the foregoing documents and disclosures are hereby incorporated by reference in their entirety.

In a preferred embodiment, the small molecule PSMA peptidase inhibitor is PBDA- and urea-based inhibitors, such as ZJ 43, ZJ, ZJ 17, ZJ 38 (Nan et al ., 2000, J. Med. Chem., 43: 772 ; and Kozikowski et al ., 2004, J. Med. Chem., 47: 7, 1729-1738) and/or analogs and derivatives thereof. Other agents that bind PSMA are also, for example, targeting fragments identified in the literature (Clin. Cancer Res., 2008 14: 3036-43), prepared by sequential addition of components to preformed urea, such as Banerjee et al. et al ., J. Med. Chem., 51: 4504-4517, 2008). In a preferred embodiment, the at least one targeting fragment capable of binding to prostate-specific membrane antigen (PSMA) is a small molecule PSMA targeting fragment, more preferably a small urea-based inhibitor.

In a preferred embodiment, the small molecule PSMA targeting fragment is a urea-based inhibitor (also referred to herein as a urea-based peptidase inhibitor), more preferably Kularatne et al ., Mol. Pharmaceutics, 2009, 6: 780; Kularatne et al ., Mol. Pharmaceutics, 2009, 6: 790; Kopka et al ., J. Nucl. Med., 2017, 58: 17S-26S; Kozikowski et al ., J. Med. Chem., 2001, 44: 298-301; Kozikowski et al ., J. Med. Chem., 2004, 47: 1729-1738; International patent applications WO2017/044936, WO2011/084518, WO2011/084521, WO2011/084513, WO2012/166923, WO2008/105773, WO2008/121949, WO2012/135592, /005740, WO2015/168379, WO03/045436, WO03/045436 , WO2016/183447, US patent application US 2015/258102, international patent application WO2011/084513, WO 2017/089942, US patent application US 2010/278927, international patent application WO2012/016188, WO2008/124634, WO20 09/131435, US Patent Application US 2007/225213, international patent applications WO2017/086467, WO2009/026177, WO2012005572, WO2014/072357 and WO2011/108930 are small urea-based inhibitors. The foregoing documents and disclosures are hereby incorporated by reference in their entirety.

In a preferred embodiment, the targeting fragment is a dipeptide urea-based PSMA peptidase inhibitor, preferably a small molecule dipeptide urea-based PSMA peptidase inhibitor. In a preferred embodiment, the PSMA targeting fragment is a dipeptide urea-based PSMA peptidase inhibitor, preferably a small molecule dipeptide urea-based PSMA peptidase inhibitor.

The term “urea-based PSMA peptidase inhibitor” refers to a PSMA peptidase inhibitor comprising a urea group. The term "dipeptide urea-based PSMA peptidase inhibitor" refers to a PSMA peptidase inhibitor comprising two peptides or amino acids independently attached to a urea group and a urea-NH2 group, while the term "small molecule dipeptide “Urea-based PSMA peptidase inhibitor” also refers to a dipeptide urea-based PSMA peptidase inhibitor that has a molecular weight of less than about 2,000 g/mol and typically and preferably is capable of binding PSMA. In some embodiments, the small molecule dipeptide urea based PSMA peptidase inhibitor has a molecular weight of less than about 1,800 g/mol, less than about 1,500 g/mol, preferably less than about 1,000 g/mol. In a further preferred embodiment, the small molecule dipeptide urea based PSMA peptidase inhibitor has a molecular weight of less than about 800 g/mol, even more preferably less than about 500 g/mol. PSMA peptidase inhibitors can reduce the activity of PSMA transmembrane zinc(II) metalloenzyme, which catalyzes the cleavage of terminal glutamate. More preferably, the small molecule dipeptide urea-based PSMA peptidase inhibitor has a molecular weight of less than about 500 g/mol. Even more preferably, the PSMA peptidase inhibitor based on the small molecule dipeptide urea is preferably one of the compounds described (Kopka et al ., J. Nuc. Med., 58(9): suppl. 2, 2017; Wirtz et al ., EJNMMI Research (2018), 8: 84) and the references cited therein, which are incorporated herein by reference in their entirety.

In a preferred embodiment, the targeting fragment, preferably a urea-based PSMA peptidase inhibitor, is a glutamate-urea moiety of formula 1, preferably formula 1 ^* and their optical, stereoisomers, rotational isomers, tautomers, is a diastereomer or racemate,

where R is preferably substituted or unsubstituted alkyl, substituted or unsubstituted aryl and any combinations thereof; More preferably, R is C _1-6 alkyl, preferably C ₂ -C ₄ alkyl, substituted at least once, preferably once, by OH, SH, NH ₂ or COOH, wherein NH ₂ , OH or SH, or COOH One of the groups serves as the X ² binding moiety and the point of covalent attachment to the PEG fragment, respectively, and the alkyl group is optionally blocked by N(H), S or O. In another preferred embodiment, R is C _1-6 alkyl, preferably C ₂ -C ₄ alkyl, substituted once by OH, SH, NH ₂ or COOH, wherein the NH ₂ , OH or SH or COOH group is X acts as a ² binding moiety and a covalent attachment point for the PEG fragment, respectively. In a very preferred embodiment, R is C ₂ alkyl substituted once with COOH, wherein the COOH group serves as the X ² binding moiety and the covalent point of attachment to the PEG fragment, respectively.

In a preferred embodiment, the targeting fragment is a glutamate-urea moiety of formula (1),

where R is C _1-6 alkyl, preferably C ₂ -C ₄ alkyl, substituted one or more times, preferably once, by OH, SH, NH ₂ or COOH, among the NH ₂ , OH or SH, or COOH groups One serves as ^the In another preferred embodiment, R is C _1-6 alkyl, preferably C ₂ -C ₄ alkyl, substituted once by OH, SH, NH ₂ or COOH, wherein the NH ₂ , OH or SH or COOH group is X acts as a ² binding moiety and a covalent attachment point for the PEG fragment, respectively. In a very preferred embodiment, R is C ₂ alkyl substituted once with COOH, wherein the COOH group serves as the X ² binding moiety and the covalent point of attachment to the PEG fragment, respectively.

In a preferred embodiment, the targeting fragment is a glutamate-urea moiety of formula 1 ^* ,

where R is C _1-6 alkyl, preferably C ₂ -C ₄ alkyl, substituted one or more times, preferably once, by OH, SH, NH ₂ or COOH, among the NH ₂ , OH or SH, or COOH groups One serves as ^the In another preferred embodiment, R is C _1-6 alkyl, preferably C ₂ -C ₄ alkyl, substituted once by OH, SH, NH ₂ or COOH, wherein the NH ₂ , OH or SH or COOH group is X acts as a ² binding moiety and a covalent attachment point for the PEG fragment, respectively. In a very preferred embodiment, R is C ₂ alkyl substituted once with COOH, wherein the COOH group serves as the X ² binding moiety and the covalent point of attachment to the PEG fragment, respectively.

In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In a further aspect, the invention provides a composition comprising a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to cells overexpressing prostate surface membrane antigen (PSMA), preferably said L is a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH (COOH)-(CH ₂ ) ₂ -CO-), and preferably the composition consists of the conjugate.

In a further aspect, the invention provides a composition comprising a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to prostate surface membrane antigen (PSMA), preferably said L is a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)- (CH ₂ ) ₂ -CO-), and preferably the composition consists of the conjugate.

In a further aspect, the invention provides a composition comprising a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, preferably said targeting fragment L is a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably wherein the composition consists of the conjugate.

In a further aspect, the invention provides a conjugate of formula (I ^*) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof, comprising:

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to cells overexpressing prostate surface membrane antigen (PSMA), preferably said L is a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH (COOH)-(CH ₂ ) ₂ -CO-).

In another aspect, the present invention provides a conjugate of Formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to prostate surface membrane antigen (PSMA), preferably L is a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-( CH ₂ ) ₂ -CO-).

In another aspect, the present invention provides a conjugate of Formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )

where n is any integer from 1 to 1,500; m is any integer from 1 to 200, preferably m is any integer from 1 to 100; R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ; R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety is H; X ¹ and X ² are independently divalent covalent moieties; Z is a divalent covalent moiety where Z is not -NHC(O)-, preferably Z is a divalent covalent moiety where Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, preferably said targeting fragment L is a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , provides conjugates.

In some embodiments, the conjugate is Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or optical isomer thereof,

here is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is any integer from 1 to 200;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in the -(NR ² -CH ₂ -CH ₂ ) _n - moiety are H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ;

R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ;

R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

^{where ​} _​ ^{​ ​ ​} -O-, -S-, -NR ¹³ -, an amino acid residue, independently selected from a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocycle moiety, and a divalent heteroaryl moiety, each divalent phenyl or heteroaryl moiety aryl is optionally substituted with one or more R ¹³ and each divalent heterocycle is optionally substituted with one or more R ¹⁴ ; ^R11 , R ¹² and R ¹³ are independently H, -SO ₃ H, -NH ₂ , -CO ₂ H or C ₁ -C ₆ alkyl in each case, and each alkyl is optionally -CO ₂ H or -NH ₂ is replaced with; R ¹⁴ at each occurrence is independently H, C ₁ -C ₆ alkyl, oxo, C ₆ -C ₁₀ aryl or 5- to 8-membered heteroaryl;

^{and ​} _​ ^{​ ​ ​ ​} , -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent carbocyclic moieties, divalent heterocycle moieties and divalent heteroaryl moieties, each of the divalent phenyl and divalent heteroaryl moieties aryl is optionally substituted with one or more R ²³ and each divalent heterocycle moiety is optionally substituted with one or more R ²⁴ ; R ²¹ , R ²² and R ²³ are each independently H, -SO ₃ H, -NH ₂ , -CO ₂ H or C ₁ -C ₆ alkyl in each case, and each C ₁ -C ₆ alkyl is one or more -OH , oxo, -CO ₂ H, -NH ₂ , C ₆ -C ₁₀ aryl or 5- to 8-membered heteroaryl; R ²⁴ is independently at each occurrence -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo;

L is a targeting fragment, preferably the targeting fragment is a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-).

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 2 to 200, preferably a definite number m of repeat units from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, wherein the targeting fragment is a PSMA targeting fragment, preferably wherein the PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 2 to 200, preferably a definite number m of repeat units from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, wherein the targeting fragment is a PSMA targeting fragment, preferably wherein the PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, said targeting fragment is a PSMA targeting fragment, and preferably said PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a repeat unit m with a definite number of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, wherein the targeting fragment is a PSMA targeting fragment, preferably wherein the PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-steric structure as shown in Formula 1*.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 2 to 200, preferably a definite number of m contiguous repeat units of 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, said targeting fragment is a PSMA targeting fragment, and preferably said PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 2 to 200, preferably a definite number of m contiguous repeat units of 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, wherein the targeting fragment is a PSMA targeting fragment, preferably wherein the PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the present invention provides a composition comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, said targeting fragment is a PSMA targeting fragment, and preferably said PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the present invention provides a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, said targeting fragment is a PSMA targeting fragment, and preferably said PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the invention relates to a composition comprising, preferably consisting of, a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 2 to 200, preferably a definite number of m contiguous repeat units of 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

^{where ​} _​ ^{​ ​ ​} -O-, -S-, -NR ¹³ -, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, wherein each divalent phenyl or heteroaryl is independently selected from one or more R ¹³ and each divalent heterocycle is optionally substituted with one or more R ¹⁴ ; ^R11 , R ¹² and R ¹³ are independently -H or C ₁ -C ₆ alkyl at each occurrence; R ¹⁴ is independently at each occurrence H, C ₁ -C ₆ alkyl or oxo;

^{and ​} _​ ^{​ ​ ​ ​} , -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent heterocycle moieties and divalent heteroaryl moieties, and each divalent phenyl and divalent heteroaryl is independently selected from one or more R ²³ and each divalent heterocycle moiety is optionally substituted with one or more R ²⁴ ; R ²¹ , R ²² and R ²³ are each independently H, -CO ₂ H or C ₁ -C ₆ alkyl in each case, and each C ₁ -C ₆ alkyl is one or more -OH, oxo, C ₆ -C ₁₀ aryl. or optionally substituted with 5- to 8-membered heteroaryl; R ²⁴ is independently at each occurrence -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo;

L is a targeting fragment, said targeting fragment is a PSMA targeting fragment, and preferably said PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the invention relates to a composition comprising, preferably consisting of, a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is a definite number of m contiguous repeat units of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, said targeting fragment is a PSMA targeting fragment, and preferably said PSMA targeting fragment is capable of specifically binding to a cell expressing, preferably overexpressing, PSMA. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a further preferred embodiment, the targeting fragment comprises a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) , preferably consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In a preferred embodiment, the DUPA moiety is linked to the PEG targeting fragment by a binding ^moiety

Such binding moieties are known to those skilled in the art and are disclosed in US patent applications US 2020/0188523A1, US 2011/0288152A1, and US 2010/324008A1, the contents of which are hereby incorporated by reference in their entirety.

In a preferred embodiment, the binding moiety X ² is a peptide linker or a C ₁ -C ₁₀ alkylene linker or a combination of both. In a preferred embodiment, the binding moiety X ² is a peptide linker.

_{In a} preferred embodiment _, the binding ^moiety ₂ )-CO)-Asp-Cys-) or SEQ ID NO: 1 (-(NH-(CH ₂ ) ₇ -CO)-Phe-Gly-Trp-Trp-Gly-Cys-), preferably It consists of this. In a preferred _{embodiment ,} the binding ^moiety It contains, and preferably consists of, the sequence of. _{In a} further preferred embodiment, ^said binding moiety NH-CH(COOH)-(CH ₂ ) ₂ -CO- (DUPA residue). In _a very preferred embodiment _, ^said binding moiety COOH)-(CH ₂ ) ₂ -CO- (DUPA residue). In a preferred embodiment, said targeting fragment L is HOOC(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO, capable of binding to cells overexpressing PSMA. -, and the binding ^moiety

In another preferred embodiment, the targeting fragment is 2-[3-(1,3-dicarboxypropyl) ureido]pentanedioic acid (DUPA), and typically and preferably the linkage to the remainder of the conjugate is as described above. It is produced through the terminal carboxyl group of DUPA. Accordingly, in a further preferred embodiment the targeting fragment L is a DUPA moiety (HOOC(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-). DUPA can be selectively taken up by cells that have increased expression (e.g., overexpression) of prostate-specific membrane antigen (PSMA).

In a preferred embodiment, the targeting fragment is capable of binding to the asialo glycoprotein receptor (ASGPr), also referred to herein as the ASGPr targeting fragment. Accordingly, in some embodiments the targeting fragment is an ASGPr targeting fragment. The asialo glycoprotein receptor (ASGPr) is a carbohydrate binding protein (i.e., lectin) that binds asialo glycoproteins and glycoproteins, preferably galactose-terminal glycoproteins, preferably branched galactose-terminal glycoproteins. Preferably, the ASGPr targeting fragment is capable of binding to an epitope on the extracellular domain of ASGPr.

Preferably, the ASGPr targeting fragment is capable of binding to cells expressing ASGPr. In a preferred embodiment, the targeting fragment is capable of binding to cells overexpressing ASGPr, preferably hepatocytes. In a preferred embodiment, the targeting fragment is capable of binding to cells expressing ASGPr. In a preferred embodiment, the targeting fragment is capable of binding to cells overexpressing ASGPr. In one embodiment, the cells overexpressing ASGPr mean that the level of ASGPr expressed above in a specific tissue is elevated compared to the level of ASGPr measured in healthy normal cells of the same type of tissue under similar conditions. In one embodiment, the cell overexpressing ASGPr refers to an increase in the level of ASGPr in the cell compared to the level in the same cell or a closely related non-malignant cell under normal physiological conditions. In one embodiment, the cells overexpressing ASGPr have ASGPr expression at least 5-fold, preferably at least 10-fold, more preferably at least 20-fold compared to ASGPr expression in normal cells or normal tissues. For example, ASGPr is overexpressed in liver cells, preferably hepatocytes and liver cancer cells. In a preferred embodiment, the ASGPr targeting fragment is capable of binding to liver cells, preferably hepatocytes or cancerous liver cells and their metastases.

Preferably, the ASGPr targeting fragment can specifically bind to ASGPr. Typically, specific binding refers to the binding affinity or dissociation constant (K _D ) of the targeting fragment between about 1×10 ⁻³ M and about 1×10 ⁻¹² M. To detect binding of the complex or measure affinity, the molecule can be analyzed using a competition binding assay, such as a Biacore 3000 instrument (see, e.g., Kuo et al ., PLoS One, 2015, 10(2): e01166610). You can. Preferably, the ASGPr targeting fragment can specifically bind to ASGPr with a binding affinity greater than that of galactose.

In a preferred embodiment, the ASGPr targeting fragment is a small molecule or small molecule ligand, peptide, protein, more preferably an ASGPr antibody, ASGPr epibody, ASGPr aptamer, ASGPr targeting peptide, lactose, galactose, N-acetylgalactosamine (GalNAc) , galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-isobutanoylgalactosamine, and combinations thereof. (Iobst, S. T. and Drickamer, K., J. B. C., 1996, 271: 6686). In some embodiments, the ASGPr targeting fragment is monomeric (i.e., has a single galactosamine). In some embodiments, the ASGPr targeting fragment is multimeric (i.e., has multiple galactosamines).

In a preferred embodiment, the ASGPr targeting fragment is a galactose cluster. The galactose cluster is understood as molecules with two to four terminal galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose that have an affinity greater than that of galactose for the asialo glycoprotein receptor. Preferably, the galactose derivatives are galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butanoylgalactosamine and N-isobutanoylgalactosamine. are chosen from the common people. Preferably, the galactose derivative is N-acetylgalactosamine (GalNAc).

In a preferred embodiment, the galactose cluster comprises three galactose derivatives each attached to a central branch point, preferably with each terminal galactose derivative attached to the remainder of the galactose cluster via its C-1 carbon. In a preferred embodiment, the galactose derivative is linked to the branch point via a linker or spacer, preferably a flexible hydrophilic spacer, more preferably a PEG spacer and even more preferably a PEG3 spacer.

In a preferred embodiment, the galactose population has three terminal galactosamines or galactosamine derivatives, each of which has an affinity for ASGPr (i.e., is a tri-antennary galactose derivative population). In some embodiments, the galactose population includes tri-antennary galactose, trivalent galactose, and galactose trimer. Preferably, the galactose cluster has three terminal N-acetylgalactosamines.

In another preferred embodiment, the targeting fragment is folic acid, and typically and preferably the linkage to the remainder of the conjugate is via the terminal carboxyl group of said folic acid. In some preferred embodiments, the targeting fragment may be folate. Without wishing to be bound by theory, folate may be selectively taken up by cells that have increased expression (e.g., overexpression) of the folate receptor.

In a further preferred embodiment, the targeting fragment is HER2, and in some embodiments can be selectively taken up in cells that have increased expression (e.g., overexpression) of HER2.

In some embodiments, the targeting fragment can be a somatostatin receptor targeting fragment. Without wishing to be bound by theory, somatostatin receptor targeting fragments may be selectively taken up by cells that have increased expression (e.g., overexpression) of somatostatin receptor 2 (SSTR2).

In some embodiments, the targeting fragment may be an integrin targeting fragment, such as a ligand comprising arginine-glycine-aspartic acid (RGD) (e.g., a cyclic RGD ligand). Without wishing to be bound by theory, integrin targeting fragments may be selectively taken up by cells that have increased expression (e.g., overexpression) of integrins (e.g., RGD integrins such as α _γ β ₆ integrins or α _γ β ₈ integrins). .

In some embodiments, the targeting fragment can be a low-pH insertion peptide (pHLIP). Without wishing to be bound by theory, it is possible that low-pH insert peptides can be selectively taken up by cells residing in low pH microenvironments. In some embodiments, the targeting fragment may be an asialo glycoprotein receptor targeting fragment, such as asialoorosomucoid. Without wishing to be bound by theory, it is possible that the asialo glycoprotein receptor targeting fragment may be selectively taken up by cells that have increased expression (or overexpression) of the asialo glycoprotein receptor. In some embodiments, the targeting fragment may be an insulin receptor targeting fragment, such as insulin. Without wishing to be bound by theory, it is possible that insulin receptor targeting fragments may be selectively taken up by cells that have increased expression (or overexpression) of the insulin receptor. In some embodiments, the targeting fragment may be a mannose-6-phosphate receptor targeting fragment, such as mannose-6-phosphate. Without wishing to be bound by theory, the mannose-6-phosphate receptor targeting fragment may be selectively taken up by cells that have increased expression (or overexpression) of the mannose-6-phosphate receptor (e.g., monocytes). In some embodiments, the targeting fragment can be a mannose receptor targeting fragment, such as mannose. Without wishing to be bound by theory, it is possible that mannose receptor targeting fragments may be selectively taken up by cells that have increased expression (or overexpression) of the mannose receptor. In some embodiments, the targeting fragment may be a sialyl Lewis ^X antigen targeting fragment, such as E-selectin. Without wishing to be bound by theory, it is possible ^that the sialyl ^Lewis In some embodiments, the targeting fragment can be an N-acetyllactosamine targeting fragment. Without wishing to be bound by theory, it is possible that N-acetyllactosamine targeting fragments may be selectively taken up by cells that have increased expression (or overexpression) of N-acetyllactosamine. In some embodiments, the targeting fragment can be a galactose targeting fragment. Without wishing to be bound by theory, galactose targeting fragments may be selectively taken up by cells that have increased expression (or overexpression) of galactose. In some embodiments, the targeting fragment may be a sigma-2 receptor agonist, such as N,N-dimethyltryptamine (DMT), sphingolipid-derived amines, and/or steroids (e.g., progesterone). Without wishing to be bound by theory, sigma-2 receptor agonists may be selectively taken up by cells that have increased expression (or overexpression) of the sigma-2 receptor. In some embodiments, the targeting fragment may be an anti-p32 antibody or a p32 targeting ligand, such as a p32 binding LyP-1 tumor homing peptide. Without wishing to be bound by theory, p32 targeting ligands may be selectively taken up by cells that have increased expression (or overexpression) of the mitochondrial protein p32. In some embodiments, the targeting fragment may be a Trop-2 targeting fragment, such as an anti-Trp-2 antibody and/or antibody fragment. Without wishing to be bound by theory, it is possible that Trop-2 targeting fragments may be selectively taken up by cells that have increased expression (or overexpression) of Trop-2. In some embodiments, the targeting fragment can be an insulin-like growth factor 1 receptor targeting fragment. Without wishing to be bound by theory, the insulin-like growth factor 1 receptor targeting fragment may be selectively taken up by cells that have increased expression (or overexpression) of the insulin-like growth factor 1 receptor. In some embodiments, the targeting fragment may be a VEGF receptor targeting fragment, such as VEGF. Without wishing to be bound by theory, it is possible that VEGF receptor targeting fragments may be selectively taken up by cells that have increased expression (or overexpression) of the VEGF receptor. In some embodiments, the targeting fragment may be a platelet-derived growth factor receptor targeting fragment, such as platelet-derived growth factor. Without wishing to be bound by theory, platelet-derived growth factor receptor targeting fragments may be selectively taken up by cells that have increased expression (or overexpression) of platelet-derived growth factor receptor. In some embodiments, the targeting fragment may be a fibroblast growth factor receptor targeting fragment, such as fibroblast growth factor. Without wishing to be bound by theory, it is possible that fibroblast growth factor receptor targeting fragments may be selectively taken up by cells that have increased expression (e.g., overexpression) of fibroblast growth factor receptor.

Binding of PEG Fragments to Targeting Fragments

In some embodiments, the second terminal end of the PEG terminus is functionalized with a linking group (i.e., X ² ) that connects the PEG fragment to the targeting fragment. Typically, the linking ^group Those skilled in the art will understand the various complementary reactive groups of the coupling reaction between the X ² reactive group and the reactive group of the targeting fragment. In some embodiments, targeting fragment L is unmodified and can be used directly as a reaction partner for the respective covalent linkage to the PEG fragment and binding moiety X ² . For example, Scheme 3 shows the nucleophilic addition of hEGF to an electrophilic tetrafluorophenyl ester linked to a PEG fragment. As shown in Scheme 3, the nucleophilic amine of hEGF displaces the tetrafluorophenol of the tetrafluorophenyl ester to form covalent bonds with the PEG fragment and binding moiety X ² , respectively. In some embodiments, the targeting fragment L can be linked to the PEG fragment by the binding moiety X ² using a suitable chemical bond such as an amide or ester bond. For example, Scheme 4 and Scheme 5 show DUPA and folate groups, respectively, being linked to a PEG fragment by an X ² linker containing an amide bond. The amide group is formed by a dehydration synthesis reaction between the appropriate carboxylic acid group on DUPA and folate and the appropriate amine on the PEG-X ² fragment.

In some preferred embodiments, the first terminal end (i.e., terminal) of the PEG terminus is functionalized with an alkene or alkyne group, which in some embodiments can be used to react with azide functionalized LPEI; The second terminus (i.e., terminus) of the PEG terminus is functionalized with a targeting fragment, which in some embodiments can be used to facilitate uptake of the conjugates and corresponding multicomplexes in specific cell types. Accordingly, in some preferred embodiments the resulting conjugates of the invention may have the general structure LPEI-PEG-targeting fragments arranged in a linear end-to-end configuration.

Conjugates of the invention can be prepared using many different methods and steps. Scheme 1 and Scheme 2 below represent different strategies for arranging the conjugates of the invention. As shown in Scheme 1 below, the conjugate of the present invention can be prepared by first linking the PEG fragment to the targeting fragment and then linking the targeting fragment-modified PEG fragment to the LPEI fragment. As shown in Scheme 2 below, the conjugates of the present invention can be prepared by first linking the PEG fragment to the LPEI fragment and then linking the LPEI-modified PEG fragment to the targeting fragment.

Scheme 1: Binding of bifunctional PEG to exemplary targeting fragments followed by LPEI

As shown in Scheme 1, a bifunctional PEG (e.g., a PEG containing an alkene or alkyne and an electrophilic species) is first reacted with a targeting fragment (e.g., hEGF, DUPA or folate) to form a PEG fragment covalently linked to the targeting fragment. can produce. The alkene or alkyne group of the targeting fragment-modified PEG can then be reacted via [3 + 2] cycloaddition with the azide group of the LPEI fragment to produce a linear conjugate of the general structure LPEI-PEG-targeting fragment.

Scheme 2: Binding of Bifunctional PEG to Exemplary LPEI and Subsequent Targeting Fragments

As shown in Scheme 2, bifunctional PEG (e.g., PEG containing an alkene or alkyne and an electrophilic species) first reacts with the azide group of the LPEI fragment via [3 + 2] cycloaddition to form 1,2,3- Linear conjugates of LPEI and PEG covalently attached by triazole or 4,5-dihydro-1H-[1,2,3]triazole can be produced. The linear LPEI-PEG fragment can then be reacted with a targeting fragment (e.g., hEGF, DUPA or folate) to produce a linear conjugate of the general structure LPEI-PEG-targeting fragment.

Schemes 3 to 5 below represent general methods for linking PEG fragments to various targeting fragments. Those skilled in the art will understand that PEG fragments can be coupled to a variety of targeting fragments using any suitable chemistry (e.g., nucleophilic substitution, peptide bonds, etc.). For example, those skilled in the art will recognize that it is not necessary to use a tetrafluorophenyl ester as the electrophilic species for the PEG fragment to bind to hEGF as shown in Scheme 3, but rather other electrophilic groups such as maleate (Scheme 1 It will be understood that (as shown in) may also be used. Moreover, those skilled in the art will appreciate that the reactive group of the bifunctionalized PEG fragment need not necessarily be an electrophilic group, but may instead be, for example, a nucleophilic group that reacts with the electrophilic portion of the targeting fragment.

Scheme 3: Binding of Bifunctional PEG to Exemplary hEGF

As shown in Scheme 3 above, in some embodiments, PEG may be modified to include electrophilic groups such as tetrafluorotetrafluorophenyl ester and/or activated alkyne groups such as DBCO. Treatment of tetrafluorophenyl ester-modified PEG with hEGF in solution induces nucleophilic displacement of hEGF with a nucleophilic amine to produce hEGF-modified PEG. The DBCO group can be used in subsequent reactions to bind LPEI fragments. The variable m is the number of repeating PEG units described herein.

Scheme 4: Exemplary coupling of bifunctional PEG to DUPA

As shown in Scheme 4 above, PEG can be modified to contain an electrophilic maleimide (MAL) group and/or an activated alkyne group such as DBCO. Maleimide-substituted PEG is N-terminal with 2-[3-(1,3-dicarboxypropyl)ureido]pentanoic acid (DUPA), which contains a nucleophilic group, namely a thiol, due to an amino acid residue derived from cysteine. This derivatized DUPA derived moiety (comprising the peptidic spacer Aoc-Phe-Gly-Trp-Trp-Gly-Cys (SEQ ID NO: 1) as shown in the Scheme above) binds to a nucleophilic partner. It can be. Treatment of MAL-modified PEG in solution with a thiol-modified DUPA-derived moiety in solution induces nucleophilic 1,4-addition of the DUPA-derived moiety via a nucleophilic thiol to produce DUPA-modified PEG. The variable m is the number of repeating PEG units described herein.

Scheme 5. Binding of Bifunctional PEG to Exemplary Folate

As shown in Scheme 5 above, PEG can be modified to include an electrophilic maleimide (MAL) group. Maleimide substituted PEG can be linked to a nucleophilic partner, such as a folate moiety that is itself modified to contain a nucleophilic group (e.g., a thiol). Treatment of MAL-modified PEG in solution with folate thiol induces nucleophilic 1,4-addition via the nucleophilic thiol of folate to produce folate modified PEG. The variable m is the number of repeating PEG units described herein.

Binding of PEG fragments to LPEI fragments

Before or after linking the bifunctionalized PEG fragment to the targeting fragment, the bifunctionalized PEG fragment can be linked to the LPEI fragment. In a preferred embodiment, the bifunctionalized PEG fragment is linked to LPEI using cycloaddition chemistry, such as 1,3-dipolar cycloaddition between an azide and an alkene or alkyne, or [3 + 2] cycloaddition to form 1,2, It can form 3-triazole or 4,5-dihydro-1H-[1,2,3]triazole. In another preferred embodiment, the bifunctionalized PEG fragment is coupled to LPEI using thiol-ene chemistry between the thiol and the alkene to form a thioester.

Those skilled in the art will understand that any suitable alkene or alkyne group can be used to link the LPEI fragment to the PEG fragment by reacting with the azide group. In some preferred embodiments, introduction of an alkene or alkyne group into the ring system introduces a chain within the ring system. The chain of the ring system is released upon reaction of an alkene or alkyne group, preferably forming 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3]tria without the addition of a catalyst such as copper. Sol can be produced. Accordingly, in some preferred embodiments suitable ring systems include 7-, 8- or 9-membered rings containing alkyne groups, or 8-membered rings containing trans alkyne groups. For example, cyclooctyne (OCT), monofluorinated cyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctinol (DIBO), dibenzoazacyclooctyne (DIBAC), bicyclooctyne ( Suitable alkyne groups such as BCN), biarylazacyclooctyne (BARAC) and tetramethylthiepinium (TMTI) may be used. Additionally, suitable alkene groups such as trans cyclooctene, trans cycloheptene and maleimide may be used. For example, the conjugates of the invention can be prepared from a moiety comprising a PEG fragment and an alkene or alkyne group according to one of the following formulas:

where X ¹ , X ² , R ^1A , L and m are defined above.

Without wishing to be bound by theory, azide and alkene or alkyne groups react spontaneously (i.e. without the addition of a catalyst) to form 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3 ]Can form triazole. In some embodiments, the azide group reacts with an alkyne to form 1,2,3-triazole. In some embodiments, the azide group reacts with an alkene to form 4,5-dihydro-1H-[1,2,3]triazole.

Those skilled in the art will appreciate that both the LPEI fragment and the PEG fragment can be functionalized to contain an azide group, and both the LPEI fragment and the PEG fragment can be functionalized to contain an alkene or alkyne fragment (e.g., a chained alkene or alkyne) . Accordingly, in some embodiments the LPEI fragment comprises an alkene or alkyne group (e.g., a chained alkene or alkyne) and the bifunctionalized PEG fragment comprises an azide group. In some preferred embodiments, the bifunctionalized PEG fragment comprises an alkene or alkyne group (e.g., a chained alkene or alkyne). The LPEI fragment contains an azide group.

Those skilled in the art will also understand that [3 + 2] cycloaddition between an azide and an alkene or alkyne group can give adducts with different site chemistries as shown in Schemes 6 to 8 below. Those skilled in the art will understand that all possible site chemistries of [3 + 2] cycloaddition are contemplated by the present invention.

In some preferred embodiments, the [3 + 2] azide-alkyne cycloaddition reaction occurs at pH 5 or lower, preferably pH 4 or lower. As demonstrated in the comparative examples below, no reaction occurs when activated alkyne-modified PEG fragments are treated with azide-free LPEI fragments at pH 4. Without wishing to be bound by theory, these results suggest that the azide group of the LPEI fragment reacts chemoselectively with the alkyne or alkene (preferably chained alkyne or alkene) group of the PEG fragment. However, comparative examples at higher pH teach that by-products are formed between the nitrogen atom of the LPEI fragment and the alkene or alkyne, characterized by a hydroamination reaction. Without wishing to be bound by theory, the present invention provides that the LPEI fragment (e.g., comprising a terminal azide) may be combined with a PEG fragment (e.g., an activated, preferably chained alkene or It teaches that it can chemically selectively bind to (including alkynes).

Accordingly, in another aspect the invention provides a method of synthesizing a conjugate of Formula (I) comprising reacting an LPEI fragment comprising a thiol with a PEG fragment comprising an alkene.

In another aspect, the invention relates to a method of synthesizing a conjugate as described and defined herein, preferably a conjugate of formula (I), wherein the omega terminus of a linear polyethyleneimine fragment is coupled to the first terminus of a polyethylene glycol fragment. reacting, wherein the reaction occurs below about pH 5, preferably below about pH 4, and preferably the omega terminus of the linear polyethyleneimine fragment comprises an azide, and the first end of the polyethylene glycol fragment and an alkene or alkyne, wherein the reaction is between the azide and the alkene or alkyne.

Scheme 6: Binding of LPEI to dibenzocyclooctyne (DBCO) modified PEG

As shown in Scheme 6 above, in some embodiments, PEG may be modified to include a chain alkyne group such as DBCO. Treatment of DBCO-modified PEG with azide-modified LPEI in solution induces [3 + 2] cycloaddition of the azide to the alkyne of DBCO to produce 1,2,3-triazole. Those skilled in the art will appreciate that the reaction depicted in Scheme 6 below can produce triazole adducts with the different site chemistries shown above. The variables m and n are the number of repeating PEG and LPEI units described herein.

Scheme 7: Binding of LPEI to bicyclooctyne (BCN) modified PEG

As shown in Scheme 7 above, in some embodiments, PEG can be modified to include a chain alkyne group such as bicyclooctyne (BCN). Treatment of BCN-modified PEG with azide-modified LPEI in solution induces [3 + 2] cycloaddition of the azide to the alkyne of BCN to produce 1,2,3-triazole. Those skilled in the art will understand that the reaction depicted in Scheme 7 below can produce triazole adducts with the different site chemistries shown above. The variables m and n are the number of repeating PEG and LPEI units described herein.

Scheme 8: Binding of LPEI to maleimide (MAL) modified PEG

As shown in Scheme 8 above, in some embodiments, PEG may be modified to include an alkene group such as maleimide (MAL). Treatment of MAL-modified PEG with azide-modified LPEI in solution induces [3 + 2] cycloaddition of the azide to the alkene of MAL to form 4,5-dihydro-1H-[1,2,3]tria. It will produce pawns. The variables m and n are the number of repeating PEG and LPEI units described herein.

Scheme 9: Binding of LPEI to alkene modified PEG

As shown in Scheme 9 above, in some embodiments, PEG may be modified to include a terminal alkene group, and LPEI may be modified to include a terminal thiol group. Treatment of thiol-modified LPEI with alkene-modified PEG in solution can induce a thiol-ene reaction to produce the thioester. The variables m and n are the number of repeating PEG and LPEI units described herein.

X ^One and X ² binding moiety

In some embodiments, the PEG fragment of the conjugates of the invention can be linked to an alkene or alkyne group and/or targeting fragment by a covalent moiety.

X ^One binding moiety

In some embodiments, the PEG fragment of the conjugates of the invention can be linked to an activated (e.g., cyclic) alkene or alkyne group on the terminal end by a binding moiety. For example, ^the For example, the PEG fragment can be linked to an activated (e.g., cyclic) alkene or alkyne group by standard means such as a peptide bond (e.g., to form an amide), nucleophilic addition, or other means known to those skilled in the art.

_In ^{one embodiment , ​ ​} (O)-, -O-, -S-, -NR ¹³ -, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocycle moiety, and a divalent heteroaryl moiety, each the divalent phenyl or heteroaryl of is optionally substituted with one or more R ¹¹ and each divalent heterocycle is optionally substituted with one or more R ¹⁴ ; ^R11 , R ¹² and R ¹³ are independently at each occurrence H, -SO ₃ H, -NH ₂ or C ₁ -C ₆ alkyl; R ¹⁴ at each occurrence is independently H, C ₁ -C ₆ alkyl, oxo, C ₆ -C ₁₀ aryl or 5- to 8-membered heteroaryl.

In some embodiments, when Y ¹ is an amino acid residue it can be oriented in any direction, i.e., -C(O)-CHR-NH- or -NH-CHR-C(O)-, where "R" is It is the side chain of a naturally occurring amino acid.

In some embodiments, the divalent heteroaryl moiety is a divalent heteroaryl group comprising one or more heteroatoms selected from O, N, S and P, preferably 1 or 2 atoms selected from O and N. In some embodiments, the divalent heteroaryl moiety is a divalent furan, pyrrole, imidazole, pyrazole, triazole, pyridine, pyrimidine, pyridazine, pyrazine, thiophene, oxazole, or isoxazole, and the divalent heteroaryl is one Or, preferably, it is optionally substituted with 1 or 0 R ¹⁴ .

In the embodiments ^for

In some embodiments, the divalent heterocycle moiety is a divalent heterocycle group comprising one or more heteroatoms selected from O, N, S and P, preferably 1 or 2 atoms selected from O and N. In some embodiments, the divalent heterocycle moiety is a divalent tetrahydrofuran, pyrrolidine, piperidine, or 4,5-dihydroisoxazole, each optionally substituted with one or more R ¹⁴ . In some preferred embodiments, the divalent heterocycle moiety is succinimide. In some preferred embodiments, two Y ^1s are combined to form

Can form a binding moiety or a partial binding moiety.

In a further preferred embodiment, two Y ¹ are combined to form

may form a binding moiety or a partial binding moiety, where the wavy line next to the sulfur is the direction of connection to the targeting fragment.

In a further preferred embodiment, Y ¹ has the formula

It may contain a binding moiety or a partial binding moiety.

In a further preferred embodiment, Y ¹ has the formula

may comprise a binding moiety or a partial binding moiety, where the wavy line next to the sulfur is the direction of connection to the targeting fragment.

_In ^{some embodiments , ​} O)-, -O-, -S-, -NH-, -C ₆ H ₄ -,

is selected independently from

_In ^{some embodiments ,} _​ O)-, -O-, -S-, -NH-, -C ₆ H ₄ -,

is selected independently from

_In ^{some embodiments ,} _​ O)-, -O-, -S-, -NH-,

is selected independently from

_In ^{some embodiments ,} _​ O)-, -O-, -NH-, -C ₆ H ₄ -,

and is uniquely -NH- when Y ¹ is adjacent to a -C(O)- group to form a carbamate or amide.

In some embodiments, X ¹ is

, where r is an integer of 0 to 8, preferably 1 to 4, more preferably 1 to 2, and R ¹¹ and R ¹² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H.

In some embodiments, X ¹ is

, where r and s are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 7 or less, and R ¹¹ and R ¹² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "s" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where s and t are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 7 or less, and R ¹¹ , R ¹² and R ¹³ are independently It is -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "s" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where r is an integer of 0 to 3, preferably 1 to 3, more preferably 1 to 2, and s and t are each independently an integer of 0 to 2, preferably 0 to 1, r, s and t The sum of is 6 or less, and R ¹¹ and R ¹² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "s" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where r and s are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 6 or less, and R ¹¹ , R ¹² and R ¹³ are independently It is -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "s" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where r and s are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 6 or less, and R ¹¹ , R ¹² and R ¹³ are independently It is -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "s" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where r and t are each an integer from 0 to 3, and s is an integer from 0 to 3, preferably r is 0, s is 2 or 3, t is 2, and the sum of r, s, and t is 5 or less, and R ¹¹ , R ¹² and R ¹³ are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "t" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where r and t are each an integer from 0 to 3, s is an integer from 0 to 3, the sum of r, s and t is 5 or less, and R ¹¹ and R ¹² are independently -H or C ₁ - C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "t" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where r and s are each independently an integer of 0 to 3, preferably 0 to 2, the sum of r and s is 5 or less, and R ¹¹ , R ¹² and R ¹³ are independently -H or C ₁ - C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the integer "s" is a bond to a PEG fragment -[OCH ₂ - CH ₂ ] is a bond to _m -.

In some embodiments, X ¹ is

, where r is independently an integer of 0 to 4, preferably 0 to 2, more preferably 1 to 2, and R ¹¹ and R ¹² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the carbonyl group is a PEG fragment -[OCH ₂ -CH ₂ ] It is a bond to _m -.

In some embodiments, X ¹ is

, where r and s are each independently an integer of 0 to 4, preferably 0 to 2, more preferably 1 to 2, and R ¹¹ and R ¹² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the carbonyl group is a PEG fragment -[OCH ₂ -CH ₂ ] It is a bond to _m -.

In some embodiments, X ¹ is

, where r and s are each independently an integer of 0 to 4, preferably 0 to 2, the sum of r and s is 5 or less, and R ¹¹ , R ¹² and R ¹³ are independently -H or C ₁ - C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g. "Z" or ring A) and the wavy line closest to the carbonyl group is a PEG fragment -[OCH ₂ -CH ₂ ] It is a bond to _m -.

In some embodiments, X ¹ is

is selected from, where

r is, at each occurrence, independently 0 to 6, preferably 0, 1, 2 or 5,

s at each occurrence is independently 0 to 6, preferably 0, 2 or 4,

t is, at each occurrence, independently 0 to 6, preferably 0, 1, 2 or 4,

R ¹¹ and R ¹² at each occurrence are independently selected from H, -C ₁ -C ₂ alkyl, -SO ₃ H and -NH ₂ , more preferably -H, -SO ₃ H and -NH ₂ ; Even more preferably -H,

R ¹³ is -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" or "t" or a carbonyl group is a PEG It is a bond to the fragment -[OCH ₂ -CH ₂ ] _m -.

In some embodiments, X ¹ is

is selected from, where

r is, at each occurrence, independently 0 to 6, preferably 0, 1, 2 or 5,

s at each occurrence is independently 0 to 6, preferably 0, 2 or 4,

t is, at each occurrence, independently 0 to 6, preferably 0, 1, 2 or 4,

R ¹¹ and R ¹² at each occurrence are independently selected from -H and -C ₁ -C ₂ alkyl, preferably -H,

R ¹³ is -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" or "t" or a carbonyl group is a PEG It is a bond to the fragment -[OCH ₂ -CH ₂ ] _m -.

In some embodiments, X ¹ is

is selected from, where

r is at each occurrence independently 0 to 6, preferably 0, 1, 2 or 5, more preferably 0,

s at each occurrence is independently 0 to 6, preferably 0, 2, 3 or 4, more preferably 2 or 3,

t is, at each occurrence, independently 0 to 6, preferably 0, 1, 2 or 4, more preferably 2,

R ¹¹ and R ¹² at each occurrence are independently selected from -H and -C ₁ -C ₂ alkyl, preferably -H,

R ¹³ is -H. Preferably, the wavy line closest to the integer "r" is a bond to a divalent covalent moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" or "t" or a carbonyl group is a PEG It is a bond to the fragment -[OCH ₂ -CH ₂ ] _m -.

In some embodiments, X ¹ is

and

is selected from, where X ^A is -NHC(O)- or -C(O)NH-. Preferably, the wavy line on the left is a bond to a divalent covalent moiety (eg "Z" or ring A) and the wavy line on the right is a bond to the PEG fragment -[OCH ₂ -CH ₂ ] _m -.

In some embodiments, X ¹ is

is selected from Preferably, the wavy line on the left is a bond to a divalent covalent moiety (eg "Z" or ring A) and the wavy line on the right is a bond to the PEG fragment -[OCH ₂ -CH ₂ ] _m -.

In some embodiments, X ¹ is

is selected from Preferably, the wavy line on the left is a bond to a divalent covalent moiety (eg "Z" or ring A) and the wavy line on the right is a bond to the PEG fragment -[OCH ₂ -CH ₂ ] _m -.

In some embodiments, X ¹ is

is selected from Preferably, the wavy line on the left is a bond to a divalent covalent moiety (eg "Z" or ring A) and the wavy line on the right is a bond to the PEG fragment -[OCH ₂ -CH ₂ ] _m -.

In some embodiments, X ¹ is

is selected from Preferably, the wavy line on the left is a bond to a divalent covalent moiety (eg "Z" or ring A) and the wavy line on the right is a bond to the PEG fragment -[OCH ₂ -CH ₂ ] _m -.

In some preferred embodiments, X ¹ is -(CH ₂ ) _1-6 -, preferably X ¹ is -(CH ₂ ) _2-4 -, more preferably X ¹ is -(CH ₂ ) ₂ - .

X ² binding moiety

In some embodiments, the PEG fragment of the conjugate of the invention can be linked to the targeting fragment on the terminal end by a binding moiety. For example, ^the For example, a PEG fragment can be linked to a targeting fragment by standard means such as a peptide bond (e.g., to form an amide), nucleophilic addition, or other means known to those skilled in the art.

^{In one embodiment} _, ^{​ ​ ​} -, -O-, -S-, -C(O)-, amino acid residues, divalent phenyl moieties, divalent carbocyclic moieties, divalent heterocycle moieties and divalent heteroaryl moieties, each the divalent phenyl or divalent heteroaryl is optionally substituted with one or more R ²³ and each divalent heterocycle moiety is optionally substituted with one or more R ²⁴ ;

R ²¹ , R ²² and R ²³ are, at each occurrence, independently -H, -SO ₃ H, -NH ₂ , -CO ₂ H or C ₁ -C ₆ alkyl; each C ₁ -C ₆ alkyl is optionally substituted with one or more -OH, oxo, -CO ₂ H, -NH ₂ , C ₆ -C ₁₀ aryl or 5- to 8-membered heteroaryl;

R ²⁴ is, at each occurrence, independently -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo.

In some embodiments, R ²¹ , R ²² and R ²³ are, at each occurrence, each independently -H, -CO ₂ H or C ₁ -C ₆ alkyl. In some embodiments, R ²¹ , R ²² and R ²³ are each independently -H or C ₁ -C ₆ alkyl, preferably C ₁ -C ₂ alkyl.

In some embodiments, R ²¹ , R ²² , R ²³ and R ²⁴ are -H.

In some embodiments, R ²⁴ is independently -H, C ₁ -C ₆ alkyl, or oxo.

In some embodiments, the divalent heteroaryl moiety is a divalent heteroaryl group comprising one or more heteroatoms selected from O, N, S and P, preferably 1 or 2 atoms selected from O and N. In some embodiments, the divalent heteroaryl moiety is a divalent furan, pyrrole, imidazole, pyrazole, triazole, pyridine, pyrimidine, pyridazine, pyrazine, thiophene, oxazole, or isoxazole, and the divalent heteroaryl is one Or, preferably, it is optionally substituted with 1 or 0 R ²¹ .

_{In the embodiments} ^for represents.

In some embodiments, the divalent heterocycle moiety is a divalent heterocycle group comprising one or more heteroatoms selected from O, N, S and P, preferably 1 or 2 atoms selected from O and N. In some embodiments, the divalent heterocycle moiety is a divalent tetrahydrofuran, pyrrolidine, piperidine, or 4,5-dihydroisoxazole, each optionally substituted with one or more R ²⁴ . In some preferred embodiments, the divalent heterocycle moiety is succinimide. In some preferred embodiments, two Y ² are combined to form

Can form a binding moiety or a partial binding moiety.

In a further preferred embodiment, two Y ² in combination have the formula:

may form a binding moiety or partial binding moiety, where the wavy line next to the sulfur is the bond to the targeting fragment (L) and the wavy line next to the nitrogen is the bond to the PEG fragment (-[OCH ₂ CH ₂ ] _m- ) is a combination of

In a further preferred embodiment, two Y ² in combination have the formula:

_may form a binding moiety or _{partial binding} moiety of ) is a combination of

In a further preferred embodiment, Y ² has the formula

It may contain a binding moiety or a partial binding moiety.

In a further preferred embodiment, Y ² has the formula

may comprise a binding moiety or a partial binding moiety, where the wavy line next to the sulfur is the direction of connection to the targeting fragment.

_In ^{some embodiments , ​ ​} -, -O-, -S-, -C(O)-, amino acid residues and

is independently selected from, R ²¹ and R ²² is independently at each occurrence H, -CO ₂ H or C ₁ -C ₆ alkyl, and each C ₁ -C ₆ alkyl is one or more -OH, oxo, C ₆ -C ₁₀ aryl or 5- to is optionally substituted with 8-membered heteroaryl.

_In ^{some embodiments , ​} -O-, -S-, -C(O)-, amino acid residues and

is independently selected from,

R ²¹ is independently at each occurrence H, -CO ₂ H or C ₁ -C ₆ alkyl (preferably C ₁ alkyl), and each C ₁ -C ₄ alkyl is one or more C ₆ -C ₁₀ aryl or 5 - is optionally substituted with 8-membered heteroaryl.

_In ^{some embodiments , ​} -O-, -S-, -C(O)-, amino acid residues and

is independently selected from,

R ²¹ is independently at each occurrence H, -CO ₂ H or C ₁ -C ₄ alkyl (preferably C ₁ alkyl), and each C ₁ -C ₄ alkyl is one or more C ₆ -C ₁₀ aryl or 5 - is optionally substituted with 8-membered heteroaryl.

_In ^{some embodiments , ​} -O-, -S-, -C(O)-, amino acid residues and

is independently selected from,

R ²¹ is independently at each occurrence H, -CO ₂ H or C ₁ -C ₃ alkyl (preferably C ₁ alkyl), each C ₁ -C ₃ alkyl being optionally substituted with one or more phenyl or indole .

_In ^{some embodiments , ​} -O-, -S-, -C(O)-, amino acid residues and

is independently selected from,

R ²¹ is independently at each occurrence H, -CO ₂ H or C ₁ -C ₃ alkyl (preferably C ₁ alkyl), and each C ₁ -C ₃ alkyl is optionally substituted with one or more phenyl or 3-indole. It is replaced.

_In ^{some embodiments , ​} -O-, -S-, -C(O)-, amino acid residues and

is independently selected from, Y ² is uniquely -NH- when adjacent to a -C(O)- group to form a carbamate or amide,

R ²¹ is independently at each occurrence H, -CO ₂ H or C ₁ -C ₃ alkyl (preferably C ₁ alkyl), and each C ₁ -C ₃ alkyl is optionally substituted with one or more phenyl or 3-indole. It is replaced.

_In ^{some embodiments , ​} -O-, -S-, -C(O)-, amino acid residues and

is independently selected from, Y ² is uniquely -NH- when adjacent to a -C(O)- group to form an amide,

R ²¹ is independently at each occurrence H, -CO ₂ H or C ₁ -C ₃ alkyl (preferably C ₁ alkyl), and each C ₁ -C ₃ alkyl is optionally substituted with one or more phenyl or 3-indole. It is replaced.

In some embodiments, when Y ² is an amino acid residue, Y ² is a naturally occurring L-amino acid residue. In some embodiments, when Y ² is an amino acid residue, it can be oriented in any direction, i.e., -C(O)-CHR-NH- or -NH-CHR-C(O)-, wherein "R" is the side chain of a naturally occurring amino acid.

In some embodiments, X ² is

, where r is an integer of 1 to 8, preferably 1 to 4, more preferably 1 to 2, and R ²¹ and R ²² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H.

In some embodiments, X ² is

, where r and s are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 7 or less, and R ²¹ and R ²² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H.

In some embodiments, X ² is

, where r and s are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 7 or less, and R ²¹ , R ²² and R ²³ are independently It is -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H.

In some embodiments, X ² is

, where r is an integer of 0 to 3, preferably 1 to 3, more preferably 1 to 2, and s and t are each independently an integer of 0 to 2, preferably 0 to 1, r, s and t The sum of is not more than 6, and R ²¹ and R ²² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H.

In some embodiments, X ² is

, where r and s are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 6 or less, and R ²¹ , R ²² and R ²³ are independently It is -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line closest to the integer "s" is the binding to the targeting fragment (L) am.

In some embodiments, X ² is

, where r and s are each independently an integer of 0 to 4, preferably 1 to 3, more preferably 1 to 2, the sum of r and s is 6 or less, and R ²¹ , R ²² and R ²³ are independently It is -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line closest to the integer "s" is the binding to the targeting fragment (L) am.

In some embodiments, X ² is

, where r and t are each integers from 0 to 3, s is an integer from 0 to 3, preferably r is 0, s is 2 or 3, t is 2, and the sum of r, s, and t is 5. Hereinafter, R ²¹ , R ²² and R ²³ are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line closest to the integer "t" is the binding to the targeting fragment (L). am.

In some embodiments, X ² is

, where r and t are each independently an integer from 0 to 3, s is an integer from 0 to 3, the sum of r, s, and t is 5 or less, and R ²¹ and R ²² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line closest to the integer "t" is the binding to the targeting fragment (L). am.

In some embodiments, X ² is

, where r and t are each independently an integer of 0 to 3, preferably 0 to 2, the sum of r and s is 5 or less, and R ²¹ , R ²² and R ²³ are independently -H or C ₁ - C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line closest to the integer "s" is the binding to the targeting fragment (L) am.

In some embodiments, X ² is

, where r and s are each independently an integer of 0 to 4, preferably 0 to 2, more preferably 1 to 2, the sum of r and s is 5 or less, and R ²¹ and R ²² are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line closest to the carbonyl group is a bond to the targeting fragment (L).

In some embodiments, X ² is

, where r and s are each independently an integer of 0 to 4, preferably 0 to 2, the sum of r and s is 5 or less, and R ²¹ , R ²² and R ²³ are independently -H or C ₁ - C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is a bond to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line closest to the carbonyl group is a bond to the targeting fragment (L).

In some embodiments, X ² is

where r, s, t and u are each independently an integer from 0 to 6, preferably from 0 to 4, v is an integer from 0 to 10, w is an integer from 0 to 10, and AA is an amino acid. The residues, preferably naturally occurring amino acid residues, more preferably AA are Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, Leu, Met. , Phe, Tyr and Trp, and a is an integer of 0 to 10, preferably 0 to 6, more preferably 0 to 4,

R ²¹ , R ²² and R ²³ are independently -H, C ₁ -C ₆ alkyl or (-COOH), preferably -H, C ₁ -C ₂ alkyl or (-COOH), more preferably -H or ( -COOH). Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some preferred embodiments, (AA) _a comprises a tripeptide selected from Trp-Trp-Gly or Trp-Gly-Phe. In some preferred embodiments, (AA) _a is Trp-Trp-Gly-Phe (SEQ ID NO: 2).

In some embodiments, X ² is

is selected from, where r, s, t and u are each independently an integer from 0 to 6, preferably from 0 to 4, v is an integer from 0 to 10, and w is an integer from 0 to 10,

AA is an amino acid residue, preferably a naturally occurring amino acid residue, more preferably AA is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, is an amino acid selected from Leu, Met, Phe, Tyr and Trp, a is an integer of 0 to 10, preferably 0 to 6, more preferably 0 to 4,

R ²¹ , R ²² and R ²³ are independently -H, C ₁ -C ₆ alkyl or (-COOH), preferably -H, C ₁ -C ₂ alkyl or (-COOH), more preferably -H or ( -COOH). Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some preferred embodiments, (AA) _a is Trp-Trp-Gly-Phe (SEQ ID NO: 2).

In some embodiments, X ² is

is selected from, where r and s are each independently an integer from 0 to 4, preferably from 0 to 2, and w is an integer from 0 to 10,

R ²¹ , R ²² and R ²³ are independently -H, C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

is selected from, where r and s are each independently an integer from 0 to 4, preferably from 0 to 2, and w is an integer from 0 to 10,

R ²¹ , R ²² and R ²³ are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

is selected from, where r, s and t are each independently an integer from 0 to 4, preferably 0 to 2, and w is an integer from 0 to 10,

AA is an amino acid residue, preferably a naturally occurring amino acid residue, more preferably AA is Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, is an amino acid selected from Leu, Met, Phe, Tyr and Trp, a is an integer of 0 to 10, preferably 0 to 6, more preferably 0 to 4,

R ²¹ , R ²² and R ²³ are independently -H or C ₁ -C ₆ alkyl, preferably -H or C ₁ -C ₂ alkyl, more preferably -H. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In an even more preferred embodiment, (AA) _a is Trp-Trp-Gly-Phe (SEQ ID NO: 2).

In some embodiments, X ² comprises or alternatively is urea, carbamate, carbonate or ester. In a preferred embodiment, X ² is

is selected from Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In a preferred embodiment, X ² is

am. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In a further preferred embodiment, X ² is

and the L of the triple conjugate is a DUPA residue (HOOC(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-). Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In a further preferred embodiment, X ² is

And, the L of the triple conjugate is ^a DUPA residue (HOOC(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-), and the The end with the amide group is bonded to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -), and the end with the amine functionality is attached to the DUPA residue (HOOC(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH -CH(COOH)-(CH ₂ ) ₂ -CO-).

In some embodiments, X ² is

is selected from, where X ^B is -C(O)NH- or -NH-C(O)- and Y ² and R ²¹ are as defined above. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

is selected from, where X ^B is -C(O)NH- or -NH-C(O)- and Y ² and R ²¹ are as defined above. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

(SEQ ID NO: 10, where SEQ ID NO: 10 is defined as W1-Gly-Trp-Trp-Gly-Phe-W2, and W1 is And W2 is

im) or

is selected from, where Y ² and R ²¹ are as defined above. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

(SEQ ID NO: 14, where SEQ ID NO: 14 is defined as W9-Gly-Trp-Trp-Gly-Phe-W10, and W9 is And W10 is lim)

where R ²¹ is as defined above, preferably R ²¹ is -H or -CH ₂ -NH ₂ , more preferably -H. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

(SEQ ID NO: 11, where SEQ ID NO: 11 is defined as W3-Gly-Trp-Trp-Gly-Phe-W4, and W3 is And W4 is im) or

(SEQ ID NO: 14, where SEQ ID NO: 14 is defined as W9-Gly-Trp-Trp-Gly-Phe-W10, and W9 is And W10 is lim)

is selected from Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

(SEQ ID NO: 11, where SEQ ID NO: 11 is defined as W3-Gly-Trp-Trp-Gly-Phe-W4, and W3 is And W4 is lim),

(SEQ ID NO: 12, where SEQ ID NO: 12 is defined as W5-Gly-Trp-Trp-Gly-Phe-W6, and W5 is And W6 is lim),

(SEQ ID NO: 13, where SEQ ID NO: 13 is defined as W7-Gly-Trp-Trp-Gly-Phe-W8, and W7 is And W8 is lim),

,

(SEQ ID NO: 14, where SEQ ID NO: 14 is defined as W9-Gly-Trp-Trp-Gly-Phe-W10, and W9 is And W10 is im) or

is selected from Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, X ² is

am.

In some embodiments, X ² is

, where X ^B is -C(O)NH- or -NH-C(O)-. Preferably, the wavy line on the left is the binding to the PEG fragment (-[OCH ₂ -CH ₂ ] _m -) and the wavy line on the right is the binding to the targeting fragment (L).

In some embodiments, the composition is of Formula IA

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-1

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-2

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-3

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-3a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-3b

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IA-3c

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-3d

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-4

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IA-4a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-4b

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IA-4c

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-4d

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-5

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-6

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-7

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IA-7a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-8

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-8a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IA-9

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-9a

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-10

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IA-10a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IB

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IB-1

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of formula IB-1a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IB-2

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IB-2a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IC

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IC-1

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula ID

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula ID-1

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula ID-1a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula ID-2

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula ID-2a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula ID-3

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula ID-3a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula ID-4

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula ID-4a

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-1

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-2

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-3

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-3a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-4

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-4a

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-5

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-5a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-6

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-6a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-7

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-7a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-8

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-8a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-9

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-9a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-10

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-10a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-11

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-11a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-11b

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-12

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-12a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-12b

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-13

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-13a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-13b

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-13c

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-13d

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-14

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-14a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-14b

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-14c

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IE-14d

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IH

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IH-1

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IH-1a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IH-2

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IH-2a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IJ

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IJ-1

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IJ-1a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IJ-2

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition is of Formula IJ-2a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has Formula IJ-3

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IJ-4

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IK

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IK-1

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IK-2

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IK-3

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IK-4

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IK-3a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IK-4a

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IL

Preferably, n is from about 280 to about 700 with a dispersion degree of about 3 or less, more preferably from about 350 to about 630 with a dispersion degree of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has the formula IM

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has the formula IN

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has the formula IO

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has the formula IP

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has formula IQ

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has the formula IR

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In some embodiments, the composition has the formula IS

Preferably, n is about 280 to about 700 with a dispersion of about 3 or less, more preferably about 350 to about 630 with a dispersion of about 2 or less, and even more preferably about 1.2 or less. 400 to about 580, preferably m is from about 2 to about 80 with a dispersion of about 2 or less, preferably from about 2 to about 70 with a dispersion of about 1.8 or less, more preferably from about 2 to about 50 with a dispersion of about 1.5 or less. of repeating units, or alternatively m is the number of distinct repeating units, preferably m is 12 or 24.

In a further preferred embodiment, the conjugate of formula (I) is

and preferably where n is from about 280 to about 700 with a dispersion of about 3 or less, more preferably from about 350 to about 630 with a dispersion of about 2 or less, and even more preferably from about 400 to about 400 with a dispersion of about 1.2 or less. 580, preferably m is a repeating unit of about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. or alternatively m is the number of distinct repeat units, preferably m is 12 or 24.

In some embodiments, the conjugate of Formula I is

and preferably where n is from about 280 to about 700 with a dispersion of about 3 or less, more preferably from about 350 to about 630 with a dispersion of about 2 or less, and even more preferably from about 400 to about 400 with a dispersion of about 1.2 or less. 580, preferably m is a repeating unit of about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. or alternatively m is the number of distinct repeat units, preferably m is 12 or 24.

In some embodiments, the conjugate of Formula I is

and preferably where n is from about 280 to about 700 with a dispersion of about 3 or less, more preferably from about 350 to about 630 with a dispersion of about 2 or less, and even more preferably from about 400 to about 400 with a dispersion of about 1.2 or less. 580, preferably m is a repeating unit of about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. or alternatively m is the number of distinct repeat units, preferably m is 12 or 24.

In some embodiments, the conjugate of Formula I is

and preferably where n is from about 280 to about 700 with a dispersion of about 3 or less, more preferably from about 350 to about 630 with a dispersion of about 2 or less, and even more preferably from about 400 to about 400 with a dispersion of about 1.2 or less. 580, preferably m is a repeating unit of about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. or alternatively m is the number of distinct repeat units, preferably m is 12 or 24.

In some embodiments, the conjugate of Formula I is

and preferably where n is from about 280 to about 700 with a dispersion of about 3 or less, more preferably from about 350 to about 630 with a dispersion of about 2 or less, and even more preferably from about 400 to about 400 with a dispersion of about 1.2 or less. 580, preferably m is a repeating unit of about 2 to about 80 with a dispersion of about 2 or less, preferably about 2 to about 70 with a dispersion of about 1.8 or less, more preferably about 2 to about 50 with a dispersion of about 1.5 or less. or alternatively m is the number of distinct repeat units, preferably m is 12 or 24.

In some embodiments, the conjugate of Formula I is

is selected from

In some embodiments, the conjugate of Formula I is

is selected from

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In some embodiments, the composition has the formula:

wherein n is about 400 to 580 with a dispersion of about 1.2 or less.

In a preferred embodiment, the composition comprises Compound 1a, Compound 1b, Compound 4a, Compound 4b, Compound 7a, Compound 7b, Compound 10a, Compound 10b, Compound 14, Compound 17a, Compound 17b, Compound 18, Compound 19, Compound 22a, Compound 22b, Compound 28a, Compound 28b, Compound 31a, Compound 31b, Compound 38a, Compound 38b, Compound 43, Compound 47a, Compound 47b, Compound 51a, Compound 51b, Compound 56a, Compound 56b, Compound 62a, Compound 62b, Compound 70a, and conjugates comprising Compound 70b, Compound 72a, Compound 72b, Compound 75a, Compound 75b, Compound 78a, and/or Compound 78b.

In a preferred embodiment, the composition comprises Compound 1a, Compound 1b, Compound 4a, Compound 4b, Compound 7a, Compound 7b, Compound 10a, Compound 10b, Compound 14, Compound 17a, Compound 17b, Compound 18, Compound 19, Compound 22a, Compound 22b, Compound 28a, Compound 28b, Compound 31a, Compound 31b, Compound 38a, Compound 38b, Compound 43, Compound 47a, Compound 47b, Compound 51a, Compound 51b, Compound 56a, Compound 56b, Compound 62a, Compound 62b, Compound 70a, Compound 70b, Compound 72a, Compound 72b, Compound 75a, Compound 75b, Compound 78a, and/or Compound 78b.

In a preferred embodiment, the composition comprises a conjugate comprising Compound 1a and/or Compound 1b. In some embodiments, the composition includes a conjugate comprising Compound 4a and/or Compound 4b. In some embodiments, the composition includes a conjugate comprising Compound 7a and/or Compound 7b. In some embodiments, the composition includes a conjugate comprising Compound 10a and/or Compound 10b. In some embodiments, the composition includes a conjugate comprising Compound 14. In some embodiments, the composition includes a conjugate comprising Compound 17a and/or Compound 17b. In some embodiments, the composition includes a conjugate comprising Compound 18. In some embodiments, the composition includes a conjugate comprising Compound 19. In some embodiments, the composition includes a conjugate comprising Compound 22a and/or Compound 22b. In some embodiments, the composition includes a conjugate comprising Compound 28a and/or Compound 28b. In some embodiments, the composition includes a conjugate comprising Compound 31a and/or Compound 31b. In some embodiments, the composition includes a conjugate comprising Compound 38a and/or Compound 38b. In some embodiments, the composition includes a conjugate comprising Compound 43. In some embodiments, the composition includes a conjugate comprising Compound 47a and/or Compound 47b. In some embodiments, the composition includes a conjugate comprising Compound 51a and/or Compound 51b. In some embodiments, the composition includes a conjugate comprising Compound 56a and/or Compound 56b. In some embodiments, the composition includes a conjugate comprising Compound 62a and/or Compound 62b. In some embodiments, the composition includes a conjugate comprising Compound 70a and/or Compound 70b. In some embodiments, the composition includes a conjugate comprising Compound 72a and/or Compound 72b. In some embodiments, the composition includes a conjugate comprising Compound 75a and/or Compound 75b. In some embodiments, the composition includes a conjugate comprising Compound 78a and/or Compound 78b.

In a preferred embodiment, the composition comprises a conjugate, which is Compound 1a and/or Compound 1b. In a preferred embodiment, the composition comprises a conjugate, which is compound 4a and/or compound 4b. In a preferred embodiment, the composition comprises a conjugate, which is compound 7a and/or compound 7b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 10a and/or Compound 10b. In a preferred embodiment, the composition comprises a conjugate, which is compound 14. In a preferred embodiment, the composition comprises a conjugate, which is Compound 17a and/or Compound 17b. In a preferred embodiment, the composition comprises a conjugate, which is compound 18. In a preferred embodiment, the composition comprises a conjugate, which is compound 19. In a preferred embodiment, the composition comprises a conjugate, which is Compound 22a and/or Compound 22b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 28a and/or Compound 28b. In a preferred embodiment, the composition comprises a conjugate, which is compound 31a and/or compound 31b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 38a and/or Compound 38b. In a preferred embodiment, the composition comprises a conjugate, which is compound 43. In a preferred embodiment, the composition comprises a conjugate, which is Compound 47a and/or Compound 47b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 51a and/or Compound 51b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 56a and/or Compound 56b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 62a and/or Compound 62b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 70a and/or Compound 70b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 72a and/or Compound 72b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 75a and/or Compound 75b. In a preferred embodiment, the composition comprises a conjugate, which is Compound 78a and/or Compound 78b.

multiple complex

The composition of the present invention further includes a polyanion, preferably the polyanion is a nucleic acid, and preferably the polyanion and the conjugate form a multicomplex. In a preferred embodiment, the polyanion is non-covalently bound to the conjugate. This facilitates the dissociation of the polyanion, preferably the nucleic acid, from the targeting fragment, as indicated herein, after reaching the targeted cell or tissue and being internalized in the tumor cell or tumor tissue, preferably inducing production of the chemokine. The production of chemokines will attract immune cells to the tumor site.

The multicomplexes of the invention provide efficient delivery of polyanions, preferably nucleic acids, into cells bearing target cell surface receptors. As described herein, the targeting fragment comprised in the multicomplex of the invention is capable of binding to a target cell surface receptor.

In a preferred embodiment, the polyanion is a nucleic acid. In a preferred embodiment, the nucleic acid is dsRNA. In a very preferred embodiment, the dsRNA is polyinosinic acid:polycytidylic acid (poly(IC)). In a preferred embodiment, the nucleic acid is sRNA. In a very preferred embodiment, the ssRNA is mRNA.

Accordingly, in another aspect the invention provides a polycomplex comprising a conjugate as described herein and a polyanion, wherein the polyanion is preferably non-covalently bound to the conjugate. In a preferred embodiment, the conjugate is a conjugate of formula I ^* or a conjugate of formula I. In a preferred embodiment, the polyanion is a nucleic acid. In a preferred embodiment, the polyanion is a nucleic acid and the nucleic acid is RNA. In a preferred embodiment, the RNA is ssRNA or dsRNA. In a preferred embodiment, the RNA is ssRNA. In another preferred embodiment, the RNA is dsRNA. In a preferred embodiment, the ssRNA is mRNA. In a preferred embodiment, the dsRNA is polyinosinic acid:polycytidylic acid (poly(IC)). In a preferred embodiment, the RNA is mRNA or poly(IC). In a preferred embodiment, the RNA is mRNA. In a preferred embodiment, the RNA is polyinosinic acid:polycytidylic acid (poly(IC)).

In another aspect, the present invention relates to a multicomplex comprising Formula I ^* , preferably a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof, and a nucleic acid, wherein said nucleic acid is preferably non-covalently bound to the conjugate,

where A, R ¹ , R ² , X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1 , 1 ,}

In another aspect, the present invention relates to a multicomplex comprising Formula I ^* , preferably a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof, and a nucleic acid, wherein said nucleic acid is preferably non-covalently bound to the conjugate,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is the number of definite repeat units from 2 to 200, preferably from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a preferred embodiment, the nucleic acid is RNA. In a preferred embodiment, the RNA is ssRNA or dsRNA. In a preferred embodiment, the RNA is ssRNA. In another preferred embodiment, the RNA is dsRNA. In a preferred embodiment, the RNA is mRNA or poly(IC). In a preferred embodiment, the RNA is mRNA. In a preferred embodiment, the RNA is polyinosinic acid:polycytidylic acid (poly(IC)). In a preferred embodiment, the ssRNA is mRNA. In a preferred embodiment, the dsRNA is polyinosinic acid:polycytidylic acid (poly(IC)).

In another aspect, the present invention relates to a multicomplex comprising Formula I ^* , preferably a conjugate of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof, and a nucleic acid, wherein said nucleic acid is preferably non-covalently bound to the conjugate,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is the number of distinct repeat units of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ . In a preferred embodiment, the nucleic acid is RNA. In a preferred embodiment, the RNA is ssRNA or dsRNA. In a preferred embodiment, the RNA is ssRNA. In another preferred embodiment, the RNA is dsRNA. In a preferred embodiment, the RNA is mRNA or poly(IC). In a preferred embodiment, the RNA is mRNA. In a preferred embodiment, the RNA is polyinosinic acid:polycytidylic acid (poly(IC)). In a preferred embodiment, the ssRNA is mRNA. In a preferred embodiment, the dsRNA is polyinosinic acid:polycytidylic acid (poly(IC)).

As used herein, the term “RNA” refers to a nucleic acid comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues. “Ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2′-position of the β-D-ribofuranosyl group. As used herein, the term “RNA” includes double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA). The term “RNA” includes partially or completely purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, RNA transcribed in vitro from a template such as a DNA template, RNA transcribed in vivo , and replicon RNA; It specifically includes RNA that is self-replicating, or modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such modifications may include, for example, the addition of non-nucleotide substances to the end(s) of the RNA or internally. RNA may have modified naturally occurring or synthetic ribonucleotides. Nucleotides in RNA may also include non-standard nucleotides, such as nucleotides that do not occur naturally or chemically synthesized nucleotides or deoxynucleotides.

The term “single-stranded RNA (ssRNA)” generally refers to an RNA molecule to which no complementary nucleic acid molecule (typically a complementary RNA molecule) is associated. ssRNA may contain self-complementary sequences that allow portions of the RNA to refold and pair themselves to form double helices and secondary structural motifs including, but not limited to, base pairs, stems, stem loops, and overhangs. The size of the ssRNA strand can vary from 8 nucleotides up to 20,000 nucleotides.

The term “double-stranded RNA (dsRNA)” refers to RNA made up of two partially or fully complementary strands. dsRNA is preferably an RNA in which there are pairs of complete or partial (disconnected) RNAs hybridized together. This can be prepared, for example, by mixing partially or fully complementary strand ssRNA molecules. They can also be made by mixing defined fully or partially paired non-homopolymeric or homopolymeric RNA strands. The size of the dsRNA strands can vary from 8 nucleotides up to 20,000 nucleotides independently for each strand.

In a preferred embodiment, the RNA is ssRNA. In a preferred embodiment, the RNA is ssRNA consisting of one single stranded RNA. Single-stranded RNA can exist as a minus strand [(-) strand] or a plus strand [(+) strand]. [(+) Strand] is the strand that contains or encodes genetic information. Genetic information may be, for example, a nucleic acid sequence encoding a protein or polypeptide. When (+) strand RNA encodes a protein, the (-) strand RNA can act directly as a template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of ssRNA, the (+) strand and (-) strand are two separate RNA molecules. Positive-strand and negative-strand RNA molecules can associate with each other to form double-stranded RNA (“duplex RNA”).

In another aspect, the invention relates to a multicomplex comprising a conjugate of formula (I ^*) , preferably of formula (I), and RNA, wherein said RNA is preferably non-covalently bound to said conjugate,

where A, R ¹ , R ² , X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1 , 1 ,}

In a preferred embodiment, the size of the RNA strand may vary from 8 nucleotides up to 20,000 nucleotides.

In a preferred embodiment, the RNA is ssRNA or dsRNA. In a preferred embodiment, the ssRNA is mRNA. In a preferred embodiment, the dsRNA is polyinosinic acid:polycytidylic acid (poly(IC)). In a preferred embodiment, the RNA is mRNA or poly(IC). In a preferred embodiment, the RNA is mRNA. In a preferred embodiment, the RNA is polyinosinic acid:polycytidylic acid (poly(IC)).

In another aspect, the invention relates to a multicomplex comprising a conjugate of formula (I ^*) , preferably of formula (I), and an mRNA, wherein the mRNA is preferably non-covalently linked to the conjugate,

where A, R ¹ , R ² , X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1 , 1 ,}

In a preferred embodiment, the RNA is “messenger RNA” (mRNA). In a preferred embodiment, the term mRNA refers to an RNA transcript encoding a peptide or protein. mRNA can be modified by stabilizing modifications and capping. Typically, an mRNA includes a 5' untranslated region (5'-UTR), a protein coding region and a 3' untranslated region (3'-UTR). Preferably, the mRNA, especially synthetic mRNA, comprises a 5' cap, a UTR contiguous to the coding region and a 3' poly(A) end. In one embodiment, mRNA is produced by in vitro transcription using a DNA template that refers to a nucleic acid containing deoxyribonucleotides. The term “untranslated region” or “UTR” refers to the region of a DNA molecule in which the DNA is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) may be present 5' (upstream) (5'-UTR) of the open translation frame and/or 3' (downstream) (3'-UTR) of the open translation frame. The 3'-UTR, if present, is preferably located at the 3' end of the gene, downstream of the stop codon of the protein encoding region, but the term "3'-UTR" preferably never includes a poly(A) end. Accordingly, the 3'-UTR is preferably upstream of the poly(A) end (if present), eg directly adjacent to the poly(A) end. The 5'-UTR, if present, is preferably located at the 5' end of the gene, upstream of the start codon of the protein encoding region. The 5'-UTR is preferably downstream of the 5'-cap (if present), eg directly adjacent to the 5'-cap. The 5'- and/or 3' untranslated regions are functionally linked to the open translation frame according to the present invention, such that these regions are linked to the open translation frame in a manner that increases the stability and/or translation efficiency of the RNA comprising the open translation frame. can be associated with The term “poly(A) sequence” or “poly(A) end” refers to an uninterrupted or interrupted sequence of adenylate residues, typically located at the 3' end of an RNA molecule. Uninterrupted sequences are characterized by consecutive adenylate residues. While poly(A) sequences are not normally encoded in eukaryotic DNA but are attached by post-transcriptional template-independent RNA polymerase to the 3' ends of RNA released during eukaryotic transcription in the cell nucleus, the present invention provides Also encompasses the poly(A) sequence encoded by. Terms such as "5'-cap", "cap", "5'-cap structure" or "cap structure" are used synonymously and refer to nucleotide modifications, preferably at the 5' end of the mRNA, more preferably at the 5' end of the mRNA. This refers to the dinucleotide observed on the image. A 5'-cap may be a structure in which an (optionally modified) guanosine is attached to the first nucleotide of an mRNA molecule via a 5' to 5' triphosphate bond (or, for certain cap analogs, a modified triphosphate bond). The term cap may refer to a naturally occurring cap or a modified cap. RNA molecules may be characterized by adaptations of 5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence and/or codon usage. mRNA may be produced by chemical synthesis, in vivo or in vitro transcription, for example from DNA or other nucleic acid templates, or may be recombinantly produced or viral RNA. mRNA includes non-self-amplifying mRNA, such as the endogenous mRNA of mammalian cells, and self-amplifying mRNA. The mRNA is preferably an endogenous mRNA that must enter the cell from outside the cell, for example by direct passage through the cytoplasmic membrane or by endocytosis followed by endosomal escape. The mRNA preferably neither enters the nucleus nor is incorporated into the genome. In a preferred embodiment, the mRNA varies in size and ranges from at least 100 nucleotides up to 20,000 nucleotides.

Formation of the polycomplexes of the invention is typically caused by electrostatic interactions between the positive charges on the conjugate side of the invention and the negative charges on the polyanion, nucleic acid and RNA sides, respectively. This leads to complexation and spontaneous formation of multicomplexes. In one embodiment, the multicomplexes of the present invention refer to particles having a z-average diameter suitable for parenteral administration.

In a preferred embodiment, the RNA is a coding RNA, ie RNA encoding a peptide or protein. The RNA can express the encoded peptide or protein. In a very preferred embodiment, said RNA, ssRNA or encoding RNA is “messenger RNA” (mRNA).

In a preferred embodiment, the RNA is pharmaceutically active RNA. “Pharmaceutically active RNA” is a pharmaceutically active RNA that encodes a pharmaceutically active peptide or protein or is itself a pharmaceutically active RNA, e.g. it has one or more pharmaceutical activities, such as immunostimulatory activity, such as the activities described for pharmaceutically active proteins. .

The term “encoding” refers to an RNA, such as mRNA, that has either a defined nucleotide sequence or a defined amino acid sequence and the biological properties produced therefrom, such that it acts as a template for the synthesis of other polymers and macromolecules in biological processes. It refers to the essential characteristics of a specific sequence of nucleotides. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene in a cell or other biological system produces the protein. The terms "RNA encodes" or "encoding RNA", used interchangeably, mean that RNA, preferably mRNA, when present in an appropriate environment, such as within cells of a target tissue, drives the assembly of amino acids to form a peptide or protein. This means that it can be produced and encoded during the translation process. In one embodiment, RNA can interact with cellular translation machinery to allow translation of peptides or proteins. A cell may produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), secrete the encoded peptide or protein, or express it on a surface.

In the context of RNA, and specifically mRNA, the terms "expression" or "translation" relate to the process by which an mRNA strand drives the assembly of an amino acid sequence to create a peptide or protein, typically in the ribosomes of a cell. The term “expression” is used in its most general sense and includes the production of RNA and/or protein.

A “pharmaceutically active peptide or protein” or “therapeutic peptide or protein” is a peptide or protein that, when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the condition or disease state of the subject. In one embodiment, the pharmaceutically active peptide or protein has curative or palliative properties and improves, alleviates, alleviates, reverses, delays the onset, or reduces the severity of one or more symptoms of a disease or disorder. It can be administered to lower it. Pharmaceutically active peptides or proteins may have prophylactic properties and may be used to delay the onset of a disease or reduce the severity of such disease or pathological condition.

As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount administered to a subject, either as a single dose or as part of a series of doses, that produces the desired physiological response or desired therapeutic effect in the subject. This refers to the effective amount. Examples of desired therapeutic effects include, but are not limited to, improvement of symptoms or pathology; reducing the progression of symptoms or pathology in a subject suffering from an infection, disease, disorder and/or condition; and/or slowing, preventing or delaying the onset of symptoms or pathology of the infection, disease, disorder and/or condition in a subject susceptible to the infection, disease, disorder and/or condition. The therapeutically effective amount will vary depending on the nature of the formulation used and the type and condition of the recipient. Determination of the appropriate amount for any given composition falls to those skilled in the art through standard tests designed to assess appropriate therapeutic levels. Typical and preferred therapeutically effective amounts of the triconjugates and/or multicomplexes of the invention described herein range from about 0.05 to 1,000 mg/kg of body weight, specifically from about 5 to 500 mg/kg of body weight.

Accordingly, in another aspect the invention provides a multicomplex comprising a conjugate of formula (I ^*) , preferably formula (I), and RNA, wherein said RNA is preferably non-covalently linked to said conjugate, and said RNA has pharmaceutical activity It is RNA,

where A, R ¹ , R ^{2 ,} X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1} , ^{1 ,}

In another aspect, the invention relates to a multicomplex comprising a conjugate of formula (I ^*) , preferably formula (I), and an RNA, wherein the RNA is preferably non-covalently linked to the conjugate, and wherein the RNA is a pharmaceutically active peptide. or is a pharmaceutically active RNA encoding a protein,

where A, R ¹ , R ² , X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1 , 1 ,}

In a preferred embodiment, the RNA encoding the pharmaceutically active peptide or protein has a size of 100 to about 20,000 nucleotides.

In a preferred embodiment, the pharmaceutically active peptide or protein is or comprises an immunologically active compound, antigen or epitope. In a preferred embodiment, the pharmaceutically active peptide or protein is or comprises an immunologically active compound or antigen. In a preferred embodiment, the pharmaceutically active peptide or protein is or comprises an immunologically active compound.

The term “immunologically active compound” preferably refers to humoral immunity by inducing and/or inhibiting the maturation of immune cells, inducing and/or inhibiting cytokine biosynthesis, and/or stimulating antibody production by B cells. It relates to any compound that alters the immune response by altering it. In one embodiment, the immune response is an antibody response (usually including immunoglobulin G (IgG)), and/or T cells, dendritic cells (DC), macrophages, natural killer (NK) cells, natural Stimulation of cellular responses, including killer T (NKT) cells and γδ T cells, is involved. Immunologically active compounds may possess potent immunostimulatory activities, including but not limited to anti-viral and anti-tumor activities, and may also downregulate other aspects of the immune response, e.g. Modifying or, where appropriate, shifting the immune response away from the TH2 immune response, useful in treating TH2 mediated diseases.

The term “antigen” encompasses any substance that will elicit an immune response. Specifically, “antigen” refers to an antibody or any substance that specifically reacts with T lymphocytes (T cells). The term “antigen” includes any molecule containing at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that induces, optionally after processing, an immune response, preferably specific to the antigen, wherein the immune response can be both humoral as well as cellular. Preferably, the antigen is presented by a cell that produces an immune response in response to the antigen, preferably an antigen-presenting cell, in the context of MHC molecules. Antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens, or the antigens may be tumor antigens. In a preferred embodiment, the antigen is a surface polypeptide, i.e. a polypeptide naturally displayed on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen or tumor. Antigens can induce an immune response against cells, pathogens, bacteria, viruses, fungi, parasites, allergens, or tumors.

In one embodiment, the antigen is a self-antigen or a non-self antigen. In another embodiment, the non-self antigen is a bacterial antigen, viral antigen, fungal antigen, allergen, or parasitic antigen. The antigen preferably contains an epitope capable of inducing an immune response in the target organism. For example, an epitope can induce an immune response against bacteria, viruses, fungi, parasites, allergens, or tumors. In some embodiments, the non-self antigen is a bacterial antigen.

In some embodiments, the non-self antigen is a viral antigen. In some embodiments, the non-self antigen is a polypeptide or protein from a fungus. In some embodiments, the non-self antigen is a polypeptide or protein from a unicellular eukaryotic parasite.

In some embodiments, the antigen is an auto-antigen, specifically a tumor antigen. Tumor antigens and their determinants are known to those skilled in the art. In the context of the present invention, the term “tumor antigen” or “tumor-related antigen” relates to a protein that is specifically expressed in a limited number of tissues and/or organs under normal conditions or at certain stages of development, such as a tumor antigen Can be specifically expressed in gastrointestinal tissues, preferably gastric mucosa, reproductive organs such as testes, trophoblasts such as placenta or germline cells under normal conditions, or is expressed or abnormally expressed in one or more tumors or cancerous tissues. In this context, “limited number” preferably means less than 3, more preferably less than 2. Tumor antigens in the context of the present invention are, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e. proteins that are specifically expressed in certain cell types at certain stages of differentiation under normal conditions, i.e. in the testis and sometimes in the testis under normal conditions. Includes proteins specifically expressed in the placenta and germline-specific antigens. Preferably, the tumor antigen is associated with the cell surface of cancer cells and is preferably not or only slightly expressed in normal tissues. Preferably, the tumor antigen or abnormal expression of tumor antigens identifies cancer cells. The tumor antigen expressed by cancer cells of a subject, eg a patient suffering from a cancer disease, is preferably an auto-protein of the subject. In a preferred embodiment, the tumor antigen is a tissue or organ that is not essential under normal conditions, i.e., a tissue or organ that does not cause death of the subject when damaged by the immune system, or an organ of the body that is not at all or rarely accessible to the immune system. Or, it is specifically expressed in the structure. Preferably, the amino acid sequence of the tumor antigen is consistent between the tumor antigen expressed in the tumor tissue and the tumor antigen expressed in the cancer tissue.

In a preferred embodiment, the nucleic acid is a pharmaceutically active nucleic acid. “Pharmaceutically active nucleic acid” is a nucleic acid encoding a pharmaceutically active peptide or protein, or is itself a pharmaceutically active nucleic acid, e.g. it has one or more pharmaceutical activities such as those described for a pharmaceutically active protein, e.g. an immunostimulatory activity. have

In another aspect the invention provides a multicomplex comprising a conjugate of formula (I ^*) , preferably formula (I) and a nucleic acid, wherein the nucleic acid is preferably non-covalently linked to the conjugate, and wherein the nucleic acid is a pharmaceutically active nucleic acid. ,

where A, R ¹ , R ² , X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1 , 1 ,}

In another aspect, the invention relates to a multicomplex comprising a conjugate of formula (I ^*) , preferably formula (I), and a nucleic acid, wherein the nucleic acid is preferably non-covalently linked to the conjugate, and wherein the nucleic acid is a pharmaceutically active peptide. or a pharmaceutically active nucleic acid encoding a protein,

where A, R ¹ , R ^{2 ,} X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1} , ^{1 ,}

In a further aspect, the invention relates to compositions of the invention, conjugates of the invention, preferably formula (I ^*) or conjugates of formula (I), or multicomplexes of the invention described herein, and pharmaceutically acceptable pharmaceuticals thereof. A pharmaceutical composition comprising a salt is provided.

Negatively charged polyanions used to form polycomplexes.

In some embodiments, the triconjugates of the present invention can form multicomplexes with polyanions and anionic polymers. For example, at physiological pH (e.g., pH 7.4) the LPEI fragment of the triconjugate of the invention may be at least partially protonated and may retain a net positive charge. In contrast, polyanions, such as nucleic acids, can be at least partially deprotonated at physiological pH (e.g., pH 7.4) and can retain a net negative charge. Accordingly, in some embodiments, co-incubation of the triconjugates of the invention with a negatively charged polymer and a nucleic acid, preferably a polyanion such as RNA, will produce a multicomplex (e.g., held together by electrostatic interactions). .

In some embodiments, the nucleic acid may be cytotoxic and/or immunostimulatory in nature (e.g., polyinosinic acid:polycytidylic acid, also known as poly(IC)).

Accordingly, in a further aspect the invention provides a polycomplex comprising the composition described herein and a nucleic acid, preferably a polyanion such as polyinosinic acid:polycytidylic acid (poly(IC)). In some embodiments, the polyanion is a nucleic acid. In some embodiments, the polyanion is a nucleic acid, wherein the nucleic acid is RNA or DNA. In another embodiment, the polyanion is RNA. In another embodiment, the polyanion is dsRNA. In a further preferred embodiment, the polyanion is poly(IC).

In another aspect, the invention relates to a multicomplex comprising a conjugate of formula (I ^*) , preferably of formula (I), and a poly(IC), wherein the poly(IC) is preferably covalently bound to the conjugate,

where A, R ¹ , R ^{2 ,} X ¹ , X ² and L ^are individually related to ^{the respective parameters A, R 1 , R 2 , X 1} , ^{1 ,}

In another aspect, the present invention provides a conjugate of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof, and a polyinosinic acid:polycytidylic acid (poly(IC)). As a complex, the poly(IC) is preferably non-covalently bound to the conjugate,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is the number of definite repeat units from 2 to 200, preferably from 4 to 60;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In a preferred embodiment, ring A is cyclooctene, succinimide or 7- to 8-membered heterocycloalkenyl, and the heterocycloalkyl or heterocycloalkenyl is 1 or 2 heterocyclic groups selected from N, O and S. and each cyclooctene, heterocycloalkyl or heterocycloalkenyl is optionally substituted at any position with one or more R ^A1 , preferably R ^A1 is oxo or fluorine or two R ^A1 are in combination. Forming at least one fused phenyl ring, preferably one or two fused phenyl rings, each phenyl ring optionally substituted with one or more -SO ₃ H or -OSO ₃ H.

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the conjugate of formula (I) is

wherein R ¹ , X ^{1 ,} X ² and L ^are individually related to each ^{of the} parameters R ^{1 ,} or as defined herein insofar as they are comprehensively related in their entirety, and preferably as defined in any of the embodiments described herein.

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the targeting fragment comprises a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-), or preferably It consists of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In another aspect, the present invention provides a multicomplex comprising a conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof, and poly(IC), wherein the poly(IC) is preferably non-covalently bound to the conjugate,

here

is a single bond or a double bond;

n is any integer from 1 to 1,500;

m is the number of distinct repeat units of 36;

R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;

R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;

Ring A is 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;

X ¹ is a divalent covalent moiety;

X ² is a divalent covalent moiety;

L is a targeting fragment, preferably the targeting fragment is capable of binding to a cell, more preferably the targeting fragment is capable of binding to a cell surface receptor. In a preferred embodiment, R ¹ is -H. In a preferred embodiment, R ¹ is -CH ₃ .

In a preferred embodiment, ring A is cyclooctene, succinimide or 7- to 8-membered heterocycloalkenyl, and the heterocycloalkyl or heterocycloalkenyl is 1 or 2 heterocyclic groups selected from N, O and S. and each cyclooctene, heterocycloalkyl or heterocycloalkenyl is optionally substituted at any position with one or more R ^A1 , preferably R ^A1 is oxo or fluorine or two R ^A1 are in combination. Forming at least one fused phenyl ring, preferably one or two fused phenyl rings, each phenyl ring optionally substituted with one or more -SO ₃ H or -OSO ₃ H.

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the conjugate of formula (I) is

wherein R ¹ , X ^{1 ,} X ² and L ^are individually related to each ^{of the} parameters R ^{1 ,} or as defined herein insofar as they are comprehensively related in their entirety, and preferably as defined in any of the embodiments described herein.

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the conjugate of formula (I) is

^is a conjugate selected from, where R ¹ , R ^A1 , X ¹ , X ² and L ^are individually related to ^{the respective parameters R 1 , R A1 ,} X ^{1 ,}

In a preferred embodiment, the targeting fragment comprises a DUPA residue (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-), or preferably It is composed of this. In a further very preferred embodiment, the targeting fragment consists of a DUPA moiety (HOOC-(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-) and both chiral C-atoms have the ( S )-stereostructure as shown in Formula 1*.

In a preferred embodiment, the poly(IC) consists of RNA strands, wherein at least 50%, preferably at least 60% of each strand comprises at least 15 and at most 8,000 ribonucleotides, preferably at most 5,000 ribonucleotides. . In a preferred embodiment, the poly(IC) consists of RNA strands, wherein at least 50%, preferably at least 60% of each strand comprises at least 22 ribonucleotides, preferably at least 450 ribonucleotides. In certain embodiments, at least 50%, preferably at least 60%, of each strand has a ribonucleotide number within the range of 20 to 300.

In some embodiments, the poly(IC) consists of RNA strands each comprising at least 22 ribonucleotides, preferably at least 450 ribonucleotides. In certain embodiments, each strand has a number of ribonucleotides within the range of 20 to 300.

In another embodiment, a polycomplex comprising a conjugate as described herein, preferably a conjugate of Formula I ^* or Formula I above, and a nucleic acid, preferably a polyanion such as polyinosinic acid:polycytidylic acid (poly(IC)). provides. In some embodiments, the poly(IC) consists of RNA strands each comprising at least 22 ribonucleotides, preferably at least 450 ribonucleotides. In certain embodiments, each strand has a number of ribonucleotides within the range of 20 to 300.

Multicomplex synthesis and characterization

The present invention provides linear conjugates (e.g., LPEI, PEG and targeting fragments such as hEGF) complexed with polyanions (e.g., nucleic acids such as double-stranded RNA (dsRNA such as poly(IC))) such as cytotoxic agents. It relates to a multicomplex containing a linear conjugate). As shown in the examples below, multicomplexes can be prepared by incubating the triconjugates of the invention with polyanions and nucleic acids such as poly(IC). In some embodiments, the multicomplex is a triconjugate of the invention in a HEPES buffered glucose solution at pH 7 to 7.4 (e.g., at room temperature) or in an acetate solution at pH 4 to 4.5 with 5% glucose (e.g., at room temperature). The gate can be formed spontaneously (e.g., within 1 hour or within 30 minutes) by combining it with poly(IC).

Particle size distributions such as z-average diameter and ζ-potential of multicomplexes can be measured by dynamic light scattering (DLS) and electrophoretic resolution, respectively. DLS measures the light scattering intensity fluctuations of multicomplexes caused by Brownian motion, and calculates the hydrodynamic diameter (nm) using the Stokke-Einstein formula. Zeta potential (ζ-potential) measures the electrodynamic potential of a multicomplex.

In some embodiments, the z-average diameter and ζ-potential can be modified as a function of the N/P ratio, which is defined as the ratio of nitrogen atoms in LPEI to phosphorus atoms of poly(IC). In some preferred embodiments, the z-average diameter of the multicomplexes of the invention is less than about 300 nm, more preferably less than about 250 nm, and even more preferably less than about 200 nm. Without wishing to be bound by theory, multicomplexes with a z-average diameter of less than about 250 nm are believed to be well tolerated in vitro (e.g., high biodistribution and clearance), are stable, and do not readily form aggregates.

In some preferred embodiments, the N/P ratio of the multicomplex is at least 2, at least 2.4, at least 2.5, at least 3, at least 3.5, at least about 4, at least 4.5, at least 5, or at least 6. In some preferred embodiments, the N/P ratio is 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6. As shown herein, the above-mentioned N/P ratios can provide stable multicomplexes of acceptable size for the polyanion, preferably polycomplexes comprising nucleic acids.

In a preferred embodiment, the multicomposite of the invention has a monolithic or bimodal diameter distribution, preferably a monolithic diameter distribution. Preferably, the monolithic diameter distribution falls in the sub-micrometer range.

In a preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 350 nm. In a preferred embodiment, the multicomplex has a z-average diameter of less than 300 nm. In another preferred embodiment, the multicomplex has a z-average diameter of less than 250 nm. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 210 nm. In another preferred embodiment, the multicomplex has a z-average diameter of less than 200 nm. In another preferred embodiment, the multicomplex has a z-average diameter of less than 180 nm. In another preferred embodiment, the multicomplex has a z-average diameter of less than 150 nm. In another preferred embodiment, the multicomplex has a z-average diameter of 350 nm to 100 nm. In another preferred embodiment, the multicomplex has a z-average diameter of 300 nm to 100 nm. In another preferred embodiment, the multicomplex has a z-average diameter of 250 nm to approximately 100 nm. In another preferred embodiment, the multicomplex has a z-average diameter of approximately 200 nm to approximately 100 nm. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the multicomplex has a z-average diameter of less than 350 nm and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In a preferred embodiment, the multicomplex has a z-average diameter of less than 300 nm and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In a preferred embodiment, the multicomplex has a z-average diameter of less than 250 nm and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In a preferred embodiment, the multicomplex has a z-average diameter of less than 220 nm and the N/P ratio of the multicomplex is at least 2.4, more preferably at least 3, even more preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of less than 200 nm and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of less than 180 nm and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of less than 150 nm. In another preferred embodiment, the multicomplex has a z-average diameter of between 350 nm and 100 nm and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of between 300 nm and 100 nm and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of 250 nm to approximately 100 nm and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of approximately 200 nm to approximately 100 nm and the N/P ratio of the multicomplex is at least 3, preferably at least 4. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the composition of the invention has a polydispersity index (PDI) of less than or equal to 0.7. More preferably, the PDI is 0.5 or less, for example 0.5 to 0.05. Even more preferably, the PDI is 0.35 or less, for example 0.55 to 0.05. In another preferred embodiment, the PDI is 0.25 or less, for example 0.25 to 0.05. In another preferred embodiment, the PDI is 0.2 or less, for example 0.2 to 0.05. In another preferred embodiment, the PDI is less than 0.2, for example 0.19 to 0.05. In another more preferred embodiment, the PDI is between 0.2 and 0.1. In another preferred embodiment, the PDI is 0.25 to 0.1. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the composition of the invention has a polydispersity index (PDI) of less than or equal to 0.7 and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. More preferably, the PDI is 0.5 or less, for example 0.5 to 0.05. Even more preferably, the PDI is 0.35 or less, for example 0.35 to 0.05, and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In another preferred embodiment, the PDI is 0.25 or less, for example 0.25 to 0.05, and the N/P ratio of the multicomplex is at least 2.4, more preferably at least 3, even more preferably at least 4. In another preferred embodiment, the PDI is 0.2 or less, for example 0.2 to 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the PDI is less than 0.2, for example 0.19 to 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another more preferred embodiment, the PDI is between 0.2 and 0.1. In another preferred embodiment, the PDI is between 0.25 and 0.1 and the N/P ratio of the multicomplex is at least 3, preferably at least 4. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 350 nm, the PDI is less than or equal to 0.5 and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In a preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 350 nm, the PDI is less than or equal to 0.4 and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In a preferred embodiment, the multicomplex has a z-average diameter of less than about 300 nm, the PDI is less than or equal to 0.4, and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to about 250 nm, the PDI is less than or equal to 0.2, and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In a preferred embodiment, the multicomplex has a z-average diameter of about 220 nm, the PDI is less than or equal to 0.2, and the N/P ratio of the multicomplex is at least 2.4, more preferably at least 3, even more preferably at least 4. am. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 200 nm, the PDI is less than or equal to 0.2 and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 180 nm, the PDI is less than or equal to 0.2 and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 150 nm, the PDI is less than or equal to 0.2 and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of 350 nm to 100 nm, the PDI is less than or equal to 0.2, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of 300 nm to 100 nm, the PDI is less than or equal to 0.2, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of 250 nm to 100 nm, the PDI is less than or equal to 0.2, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the multicomplex has a z-average diameter of approximately 200 nm to approximately 100 nm, the PDI is less than or equal to 0.2, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the composition of the invention has a zeta potential of at least 18 mV, for example between 18 mV and 50 mV. In a preferred embodiment, the composition of the invention has a zeta potential of at least 18 mV, for example between 18 mV and 54 mV. In another preferred embodiment, the composition of the invention has a zeta potential of at least 18 mV, for example between 18 mV and 42 mV. In another preferred embodiment, the composition of the invention has a zeta potential of 20 mV to 50 mV. In another preferred embodiment, the composition of the invention has a zeta potential of 20 mV to approximately 45 mV. In another preferred embodiment, the composition of the invention has a zeta potential of 20 mV to approximately 42 mV. In another preferred embodiment, the composition of the invention has a zeta potential of 20 mV to approximately 40 mV. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the composition of the invention has a zeta potential of at least 18 mV, for example between 18 mV and 50 mV, and the N/P ratio of the multicomplex is at least 2, preferably at least 2.4. In a more preferred embodiment, the composition of the invention has a zeta potential of at least 18 mV, for example between 18 mV and 45 mV, and the N/P ratio of the multicomplex is at least 2.4, preferably at least 3, more preferably at least 4. In another preferred embodiment, the composition of the invention has a zeta potential of at least 18 mV, for example between 18 mV and 42 mV, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the composition of the invention has a zeta potential of 20 mV to 50 mV and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the composition of the invention has a zeta potential of from 30 mV to approximately 40 mV and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the composition of the invention has a zeta potential of from 18 mV to approximately 40 mV and the N/P ratio of the multicomplex is at least 3, preferably at least 4. In another preferred embodiment, the composition of the invention has a zeta potential of approximately 20 mV to approximately 40 mV and the N/P ratio of the multicomplex is at least 3, preferably at least 4. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the multicomplex has a z-average diameter of less than 350 nm, the N/P ratio of the multicomplex is at least 2, preferably at least 2.4, and the composition of the invention has a zeta potential of 18 mV to 50 mV. has In a preferred embodiment, the multicomplex has a z-average diameter of about 300 nm or less, the N/P ratio of the multicomplex is at least 2, preferably at least 2.4, and the composition of the invention has a zeta of 20 mV to 50 mV. It has potential. In a preferred embodiment, the multicomplex has a z-average diameter of about 250 nm or less, the N/P ratio of the multicomplex is at least 2, preferably at least 2.4, and the composition of the invention has a zeta of 20 mV to 50 mV. It has potential. In a preferred embodiment, the multicomplex has a z-average diameter of less than or equal to about 220 nm, the N/P ratio of the multicomplex is at least 2.4, preferably at least 3, more preferably at least 4, and the composition of the invention has It has a zeta potential of mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of less than 200 nm, the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the invention has a z-average diameter of 18 mV to 45 mV. It has zeta potential. In another preferred embodiment, the multicomplex has a z-average diameter of less than 180 nm, the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the invention has a z-average diameter of 18 mV to 45 mV. It has zeta potential. In another preferred embodiment, the multicomplex has a z-average diameter of less than 150 nm, the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the invention has a z-average diameter of 18 mV to 45 mV. It has zeta potential. In another preferred embodiment, the multicomplex has a z-average diameter of 350 nm to 100 nm, the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the invention has a z-average diameter of between 18 mV and 45 mV. It has a zeta potential of mV. In another preferred embodiment, the multicomplex has a z-average diameter of 300 nm to 100 nm, the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the invention has a z-average diameter of between 18 mV and 45 mV. It has a zeta potential of mV. In another preferred embodiment, the multicomplex has a z-average diameter of 250 nm to approximately 100 nm, the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the invention has a z-average diameter of between 18 mV and 18 mV. It has a zeta potential of 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of approximately 200 nm to approximately 100 nm, the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the invention has a z-average diameter of approximately 200 nm to approximately 100 nm. It has a zeta potential of from 45 mV. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

In a preferred embodiment, the multicomplex has a z-average diameter of less than 350 nm, the PDI is from 0.5 to 0.05, the N/P ratio of the multicomplex is at least 2, preferably at least 2.4, and the composition of the invention has It has a zeta potential of mV to 50 mV. In a preferred embodiment, the multicomplex has a z-average diameter of less than 300 nm, the PDI is from 0.5 to 0.05, the N/P ratio of the multicomplex is at least 2, preferably at least 2.4, and the composition of the invention has It has a zeta potential of mV to 50 mV. In a preferred embodiment, the multicomplex has a z-average diameter of less than or equal to about 250 nm, the PDI is from 0.35 to 0.05, the N/P ratio of the multicomplex is at least 2, preferably at least 2.4, and the composition of the present invention has It has a zeta potential of 18 mV to 50 mV. In a preferred embodiment, the multicomplex has a z-average diameter of less than or equal to about 220 nm, the PDI is less than or equal to 0.3, such as 0.3 to 0.05, and the N/P ratio of the multicomplex is at least 2.4, preferably at least 3, more preferably preferably at least 4, and the composition of the present invention has a zeta potential of 18 mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 200 nm, the PDI is less than or equal to 0.2, for example between 0.2 and 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4, The composition of the present invention has a zeta potential of 18 mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 180 nm, the PDI is less than or equal to 0.2, for example between 0.2 and 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4, The composition of the present invention has a zeta potential of 18 mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of less than or equal to 150 nm, the PDI is less than or equal to 0.2, for example between 0.2 and 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4, The composition of the present invention has a zeta potential of 18 mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of 350 nm to 100 nm, the PDI is less than or equal to 0.2, for example 0.2 to 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. and the composition of the present invention has a zeta potential of 18 mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of 300 nm to 100 nm, the PDI is less than or equal to 0.2, for example 0.2 to 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4. and the composition of the present invention has a zeta potential of 25 mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of 250 nm to approximately 100 nm, the PDI is 0.2 or less, for example 0.2 to 0.05, and the N/P ratio of the multicomplex is at least 3, preferably at least 4, and the composition of the present invention has a zeta potential of 18 mV to 45 mV. In another preferred embodiment, the multicomplex has a z-average diameter of approximately 200 nm to approximately 100 nm, the PDI is less than or equal to 0.2, for example between 0.2 and 0.05, and the N/P ratio of the multicomplex is at least 3, preferably is at least 4, and the composition of the present invention has a zeta potential of 18 mV to 45 mV. Preferably, the multicomposite has a monolithic diameter distribution, preferably within the sub-micrometer range.

1A, 1B, and 1C show the z-average diameter of the multicomplexes disclosed herein as a function of N/P ratio. Figure 1A shows that LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplex exhibiting an N/P ratio of 2.4 has a z-average diameter of greater than 200 nm (i.e., 306 nm). indicates a point. In contrast, Figures 1B and 1C show that LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplexes showing N/P ratios of 4 or 5.6 were less than 200 nm (i.e., 116 nm). and 107 nm). Without wishing to be bound by theory, the examples and figures herein demonstrate that the size of the multicomplexes disclosed herein can be controlled by adjusting the N/P ratio. Figure 3 demonstrates that triconjugates without targeting fragments can form multicomplexes of similar z-average diameter and dispersion as conjugates containing targeting fragments. Figure 3 shows DLS characterization of LPEI- l- [N ₃ :DBCO]-PEG ₂₃ -OMe:poly(IC) at an N/P ratio of 4. The multicomplex shown in Figure 3 contains a PEG fragment terminated with -OMe and does not contain a targeting fragment. However, the multicomplex shown in Figure 3 has a z-average diameter distribution and ζ-potential similar to targeting fragments such as hEGF (107 nm and 34.1 mV).

Figure 4 shows DLS characterization of LPEI- l- [N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) at an N/P ratio of 4. The multicomplex has a z-average diameter of 156 nm and a ζ-potential of 38.3 mV.

Figure 5 shows DLS characterization of multicomplexes formed with DUPA modified LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) at an N/P ratio of 4. As shown in Figure 5, the z-average diameter of the multicomplex is about 120 nm and the ζ-potential is 31.1 mV.

In some embodiments, the multicomplex has a z-average diameter of less than about 200 nm. In some embodiments, the N/P ratio of the multicomplex is from about 3 to about 10, and preferably the N/P ratio of the multicomplex is from about 4 to about 7. In some embodiments, the N/P ratio of the multicomplex is about 4, 5, or 7. In some preferred embodiments, the multicomplexes of the invention have a ζ- of about 15 mV to about 70 mV, about 20 mV to about 70 mV, preferably about 15 mV to about 50 mV, preferably about 15 mV to about 40 mV. It has potential.

Cytotoxic activity of multiple complexes

The present invention provides conjugates comprising LPEI, PEG and targeting fragments such as hEGF, DUPA, HER2 or folate, and dsRNA typically and preferably as cytotoxic and/or immunostimulatory agents such as nucleic acids comprising poly(IC). It relates to a multicomplex containing polyanions that can act.

The triconjugate:nucleic acid multicomplex disclosed herein has high efficacy and selectivity for delivering nucleic acids such as poly(IC) to cells with high surface expression of cell surface receptors such as EGFR or PSMA. As shown in the Examples below, the triconjugates of the invention serve as vectors for nucleic acids such as polyanions and poly(IC) herein. Moreover, the cytotoxic and/or immunostimulatory activity of the polycomplex can be tailored by selection of the appropriate polyanion. For example, poly(Glu), in contrast to poly(IC), does not exhibit immunostimulatory or cytotoxic effects and was therefore used as a comparative control in the cytotoxicity examples described herein.

Without wishing to be bound by theory, the linear conjugates of the present invention may contain targeting fragments that help increase the relative absorption of the triconjugate:poly(IC) multicomplex. For example, conjugates containing human epidermal growth factor (hEGF) (and the resulting multicomplexes) are more effective in cells with high expression of the human epidermal growth factor receptor (EGFR) compared to cells with lower levels of EGFR expression. Can be absorbed in high concentrations. Similarly, conjugates containing the targeting fragment 2-[3-(1,3-dicarboxypropyl)ureido]pentanoic acid (DUPA) (and the resulting multicomplexes) are capable of binding the prostate-specific membrane antigen (PSMA). Higher concentrations can be taken up in cells with high expression, and conjugates containing the targeting fragment folate (and the resulting multicomplexes) can be taken up at higher rates in cells with high expression levels of the folate receptor. there is. Those skilled in the art will appreciate that the conjugates of the invention can be effectively modified with a variety of targeting fragments to allow selective uptake of the conjugates into specific cell types.

In a preferred embodiment, the poly(IC)-containing multicomplex of the present invention exhibits high biological efficacy, as verified by the high cytotoxicity of the triconjugate:nucleic acid multicomplex of the present invention. In a preferred embodiment, the high cytotoxicity of the multicomplex is believed to be induced by poly(IC).

Furthermore. Examples herein show that the multicomplex of the present invention is used in A431 cells expressing higher levels of hEGFR (i.e., 10 ⁶ molecules/cell) than in cells expressing low levels of hEGFR (i.e., 10 ³ molecules/cell). This demonstrates significantly higher cytotoxicity and therefore a very high degree of selectivity. Accordingly, in a preferred embodiment, the multicomplex of the invention, preferably comprising a targeting fragment that selectively targets a cell surface receptor, selectively causes cell death in cells expressing a specific cell surface receptor at high levels. It triggers. In a preferred embodiment, the cytotoxicity of the triconjugate:nucleic acid multicomplex of the invention results from the delivery of a selected nucleic acid (e.g., poly(IC)). In a preferred embodiment, the cytotoxicity of the multicomplex of the invention can be increased by adding targeting fragments to the triconjugate of the invention.

As explained in the Examples, the multicomplex comprising LPEI- l -PEG:poly(IC) according to the present invention is a randomly branched multicomplex comprising prior art LPEI, PEG, targeting fragment and poly(IC). Compared to complexes, not only do they exhibit at least the same potency and similar cytotoxic activity against cells showing high surface expression of EGFR, but the multicomplexes of the present invention even exhibit biological activity, such as potency and selectivity derived from targeted nucleic acid delivery. indicates an increase in Moreover, the results in Figures 10A and 10B demonstrate that LPEI- l -PEG _n -hEGF:poly(IC) induces a strong and selective reduction in cell survival in cells overexpressing EGFR. Little or no significant cell death was observed in A431 cells when poly(IC) was replaced with poly(Glu) or when non-targeted multicomplexes were used.

Example 24 demonstrates that selective delivery of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(pIC) reduces survival of cells overexpressing PSMA. Cancer cell lines showing differential expression of PSMA (PC-3, low PSMA expression; and LNCaP, high PSMA expression) were incubated with LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) or LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) multicomplex was treated for 72 hours. Accordingly, Figure 11A shows LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in LNCaP cells. Cell survival is shown as a function of treatment. LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) was inactive (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL). , LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) induced a robust reduction in LNCaP cell survival with an IC ₅₀ of 0.02 μg/mL. Figure 11B shows the transformation of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in PC-3 cells. Cell survival is shown as a function of treatment. LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) showed non-specific cytotoxic activity at high concentrations. LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) was completely inactive (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL). ), LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) also demonstrated PC-3 cell survival with an IC ₅₀ value of 0.24 μg/mL.

11A and 11B show the LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) multicomplex treatment of the present invention compared to the control polyanion, poly(Glu) treatment in cells overexpressing PSMA. It shows that it selectively induces cancer cell death.

Figures 6A-11B demonstrate that the multicomplexes of the invention disclosed herein can be selective for treating heptacarcinomas, such as cancers that overexpress a specific cell surface receptor or receptors. For example, as shown in Figures 6A-10B, a multicomplex containing an hEGF targeting fragment selectively targets cells overexpressing EGFR. Similarly, as shown in Figures 11A and 11B, multicomplexes containing DUPA targeting fragments selectively target cells overexpressing PSMA. Those skilled in the art will recognize that the multicomplexes disclosed herein can be modified to include any suitable targeting fragment, including but not limited to the fragments described herein, to selectively target cell types overexpressing other cell surface receptors and/or antigens. You will understand.

Immunostimulatory activity of multiple complexes

As shown in Example 11 below, the immunostimulatory activity of LPEI- l -PEG ₂₄ -hEGF:poly(IC) was evaluated for high EGFR expression (A431) and low EGFR expression (MuCF7) using the IP-10 ELISA assay. Measurements were made in cell lines. As observed in Figure 17, IP-10 secretion was strongly and selectively increased in A431 cells in a dose-dependent manner. In MCF7 cells only a very slight increase was observed at the highest concentration. These results demonstrate that the multicomplexes described herein can be used to selectively induce an immune response (e.g., poly(IC) induced cytokine secretion) in cell types overexpressing specific cell surface receptors (e.g., EGFR). .

Targeted multicomplex targeted intervention

Figure 18 shows a Western blot image showing EGFR targeting engagement of LPEI- l -PEG ₂₄ -EGF:poly(IC) multicomplex.

NIH3T3 cells using both carrier LPEI- l -PEG ₂₄ -EGF (0.04 μg/mL) and multicomplex LPEI- l -PEG ₂₄ -EGF:poly(IC), (0.0615 μg/mL poly(IC) in multicomplex). Treatment induced EGFR protein phosphorylation (P-EGFR) after 30 minutes as a result of EGF ligand binding to EGFR. Protein levels are shown by Western blot imaging using serum starvation conditions as a negative control and hEGF treatment as a positive control. Tubulin demonstrates equal loading of total protein. Without wishing to be bound by theory, Figure 18 demonstrates that both triconjugates and multicomplexes described herein can effectively bind and target specific cell surface receptors such as EGFR.

Multiple complexes used to treat diseases

In one aspect, the invention provides compositions comprising the multicomplexes described herein for use in the treatment of a disease or disorder. In another aspect, the invention provides the use of a multicomplex described herein for the manufacture of a medicament for the treatment of a disease or disorder. In another aspect, the invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multicomplex described herein.

In one aspect, the invention provides a composition comprising the multicomplex described herein for use in the treatment of a disease or disorder, such as cancer. In another aspect, the invention provides the use of a multicomplex described herein for the manufacture of a medicament for the treatment of a disease or disorder, such as cancer. In another aspect, the invention provides a method of treating a disease or disorder, such as cancer, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a multicomplex described herein.

In some embodiments, a cancer may be characterized by cells that express or overexpress one or more cell surface receptors and/or antigens. Without wishing to be bound by theory, the triconjugates and/or multicomplexes of the present invention are capable of targeting specific cells by selecting an appropriate targeting fragment and linking the appropriate targeting fragment to a PEG fragment to form a targeted triconjugate as described above. Can be targeted to a type (eg, a cancer cell type). Cell surface receptors and/or antigens include, but are not limited to, EGFR; HER2; integrins (e.g., RGD integrin); Sigma-2 receptor; Trop-2; folate receptor; Prostate-specific membrane antigen (PSMA); p32 protein; Somatostatin receptors, such as somatostatin receptor 2 (SSTR2); Insulin-like growth factor 1 receptor (IGF1R); Vascular endothelial growth factor receptor (VEGFR); platelet-derived growth factor receptor (PDGFR); and/or fibroblast growth factor receptor (FGFR).

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of EGFR. In some preferred embodiments, cancers characterized by cells with increased expression of EGFR can be treated with a multicomplex comprising an EGFR targeting fragment, such as hEGF. In certain embodiments, the cancer characterized by cells overexpressing EGFR is adenocarcinoma, squamous cell carcinoma, lung cancer (e.g., non-small cell lung carcinoma), breast cancer, glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), kidney cancer. , colorectal cancer, ovarian cancer, cervical cancer, bladder cancer, or prostate cancer and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of HER2. In some preferred embodiments, cancers characterized by cells with increased expression of HER2 can be treated with a multicomplex comprising a HER2 targeting fragment, such as an anti-HER2 peptide (e.g., an anti-HER2 antibody or apibody). In some embodiments, the cancer characterized by cells overexpressing HER2 is breast cancer, ovarian cancer, gastrointestinal cancer (stomach cancer), uterine cancer (e.g., invasive forms of uterine cancer, such as uterine serous endometrial carcinoma), and/or metastases thereof. In certain embodiments, the cells overexpressing HER2 are treatment resistant cells (e.g., Herceptin/Trastuzumab resistant cells). Accordingly, the multicomplexes of the present invention can be used in the treatment of Herceptin/Trastuzumab resistant cancers, i.e. cancers comprising cells that do not respond or respond to a weak degree to exposure to Herceptin/Trastuzumab.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of a prostate-specific membrane antigen. In some preferred embodiments, cancers characterized by cells with increased expression of prostate-specific membrane antigen (PSMA) can be treated with a multicomplex comprising a PSMA targeting fragment, such as DUPA. In certain embodiments, the cancer characterized by cells overexpressing PSMA is prostate cancer and/or metastases thereof. In a preferred embodiment, the cancer is prostate cancer.

In some embodiments, cancer associated neovascularization is characterized by increased expression (e.g., overexpression) of PSMA (see, e.g., Van de Wiele et al ., Histol Histopathol. (2020), 35(9): 919-927 ). In some preferred embodiments, cancers characterized by neovascularization with increased expression of thyroid-specific membrane antigen (PSMA) can be treated with a multicomplex comprising a PSMA targeting fragment, such as DUPA. In some preferred embodiments, the cancer characterized by association with PSMA-overexpressing neovessels is glioblastoma, breast cancer, bladder cancer, and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of folate receptors. In some preferred embodiments, cancers characterized by cells with increased expression of folate receptors can be treated with multicomplexes comprising folate and/or folic acid as targeting fragments. In certain embodiments, the cancer characterized by cells overexpressing folate receptors is gynecological cancer, breast cancer, cervical cancer, uterine cancer, colorectal cancer, renal cancer, nasopharyngeal cancer, ovarian cancer, endometrial cancer, and/or metastases thereof. .

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of a somatostatin receptor, such as somatostatin receptor 2 (SSTR2). In some embodiments, cancers characterized by increased expression of SSTR2 can be treated with a multicomplex comprising somatostatin and/or a somatostatin receptor targeting fragment, such as octreotide. In certain embodiments, the cancer characterized by increased expression of somatostatin receptor (e.g., SSTR2) is colorectal cancer and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of integrins (e.g., RGD integrins such as α _γ β ₆ integrins or α _γ β ₈ integrins). In some embodiments, cancers characterized by increased expression of integrins, such as RGD integrins, are treated with a multicomplex comprising an integrin targeting fragment, such as a ligand comprising arginine-glycine-aspartic acid (RGD) (e.g., a cyclic RGD ligand). It can be. In some preferred embodiments, the integrin targeting fragment may be a peptide such as SFITGv6, SFFN1, SFTNC, SFVTN, SFLAP1, SFLAP3, A20FMDV2 (e.g., Roesch et al ., J. Nucl. Med., 2018, 59 (11) : 1679-1685). In some embodiments, the integrin targeting fragment can be an anti-integrin antibody, such as an α _γ β ₆ integrin antibody, anti-integrin diabody, or knotin. In some embodiments, the integrin targeting fragment can be latent transforming growth factor β (TGFβ). In some embodiments, the cancer cell characterized by increased expression of an integrin, such as RGD integrin, is a solid tumor, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colon cancer, oral squamous cell carcinoma, astrocytoma, It may include head and neck squamous cell carcinoma and/or metastases thereof.

In some embodiments, cancer may be characterized by cells residing in a low pH microenvironment. In some embodiments, cancers characterized by a low pH microenvironment can be treated with a multicomplex comprising a low-pH insertion peptide (pHLIP) as a targeting fragment. In some embodiments, the cancer characterized by cells residing in a low pH microenvironment includes breast cancer and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression of the asialo glycoprotein receptor. In some embodiments, cancers characterized by increased expression of the asialo glycoprotein receptor can be treated with a multicomplex comprising an asialo glycoprotein receptor targeting fragment, such as asialoorosomucoid. In certain embodiments, the cancer characterized by increased expression of the asialo glycoprotein receptor is liver cancer, gallbladder cancer, gastrointestinal cancer, and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression of the insulin receptor. In some embodiments, cancers characterized by increased expression of the insulin receptor can be treated with a multicomplex comprising an insulin receptor targeting fragment, such as insulin. In certain embodiments, the cancer characterized by cells overexpressing the insulin receptor is breast cancer, prostate cancer, endometrial cancer, ovarian cancer, liver cancer, bladder cancer, lung cancer, colon cancer, thyroid cancer, and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells (e.g., monocytes) that have increased expression of the mannose-6-phosphate receptor. In some embodiments, cancers characterized by increased expression of the mannose-6-phosphate receptor can be treated with a multicomplex comprising a mannose-6-phosphate receptor targeting fragment, such as mannose-6-phosphate. In some embodiments, the cancer characterized by overexpression of mannose-6-phosphate receptor is leukemia.

In some embodiments, the cancer may be characterized by cells that have increased expression of mannose receptors. In some embodiments, cancers characterized by increased expression of mannose receptors can be treated with multicomplexes comprising mannose receptor targeting fragments, such as mannose. In some embodiments, the cancer characterized by increased expression of mannose receptors includes gastric cancer and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression of glycosides, such as siaryl Lewis ^X antigen. In some embodiments, ^cancers characterized by increased expression of siaryl ^Lewis

In some embodiments, the cancer may be characterized by cells that have increased expression of N-acetyllactosamine. In some embodiments, cancers characterized by increased expression of N-acetyllactosamine can be treated with multicomplexes comprising N-acetyllactosamine targeting fragments.

In some embodiments, the cancer may be characterized by cells that have increased expression of galactose. In some embodiments, cancers characterized by increased expression of galactose can be treated with multicomplexes comprising galactose targeting fragments. In some embodiments, the cancer characterized by increased expression of galactose includes colon carcinoma and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression of the sigma-2 receptor. In some embodiments, the cancer characterized by increased expression of the sigma-2 receptor is a sigma-2 receptor such as N,N-dimethyltryptamine (DMT), sphingolipid-derived amines, and/or steroids (e.g., progesterone). It can be treated with multicomplexes containing agonists. In some embodiments, the cancer characterized by increased expression of the sigma-2 receptor includes pancreatic cancer, lung cancer, breast cancer, melanoma, prostate cancer, ovarian cancer, and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression of the mitochondrial protein p32. In some embodiments, cancers characterized by increased expression of p32 can be treated with a multicomplex comprising an anti-p32 antibody or a p32 targeting ligand, such as a p32 binding LyP-1 tumor homing peptide. In some embodiments, the cancer characterized by increased expression of p32 includes glioblastoma, breast cancer, melanoma, endometrial cancer, adenocarcinoma, colon cancer, and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression of Trop-2. In some embodiments, cancers characterized by increased expression of Trop-2 can be treated with multicomplexes comprising Trop-2 targeting fragments, such as anti-Trop-2 antibodies and/or antibody fragments. In some embodiments, the cancer characterized by increased expression of Trop-2 is breast cancer, squamous cell carcinoma, esophageal squamous cell carcinoma (SCC), pancreatic cancer, hilar cholangiocarcinoma, colorectal cancer, bladder cancer, cervical cancer, ovarian cancer, and thyroid cancer. , non-small cell lung cancer (NSCLC), hepatocellular cancer, small cell lung cancer, prostate cancer, head and neck cancer, renal cell cancer, endometrial cancer, glioblastoma, gastric cancer and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of the insulin-like growth factor 1 receptor. In some preferred embodiments, cancers characterized by cells with increased expression of the insulin-like growth factor 1 receptor are to be treated with a multicomplex comprising an insulin-like growth factor 1 receptor targeting fragment, such as insulin-like growth factor 1. You can. In certain embodiments, the cancer characterized by cells overexpressing the insulin-like growth factor 1 receptor is breast cancer, prostate cancer, lung cancer, and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of the VEGF receptor. In some preferred embodiments, cancers characterized by cells with increased expression of the VEGF receptor can be treated with a multicomplex comprising a VEGF receptor targeting fragment, such as VEGF.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of platelet-derived growth factor receptor. In some preferred embodiments, cancers characterized by cells with increased expression of platelet-derived growth factor receptor can be treated with a multicomplex comprising a platelet-derived growth factor receptor targeting fragment, such as platelet-derived growth factor. In some preferred embodiments, the cancer characterized by cells with increased expression of platelet-derived growth factor receptor includes breast cancer and/or metastases thereof.

In some embodiments, the cancer may be characterized by cells that have increased expression (e.g., overexpression) of fibroblast growth factor receptor. In some preferred embodiments, cancers characterized by cells with increased expression of fibroblast growth factor receptor can be treated with a multicomplex comprising a fibroblast growth factor receptor targeting fragment, such as fibroblast growth factor.

equivalent

While the present invention has been described in connection with the specific embodiments described above, various alternatives, modifications and other modifications thereof will be apparent to those skilled in the art. Such alternatives, modifications and alterations are intended to fall within the scope and spirit of the invention.

Example

The invention is further illustrated by the following examples and synthetic schemes, which should not be considered to limit the scope or spirit of the invention to the specific procedures described herein. It will be understood that the examples are provided to illustrate specific implementations and are not intended to be limiting on the scope of the invention herein. It is further to be understood that various other embodiments, modifications, and equivalents thereof may be suggested to those skilled in the art without departing from the spirit of the invention and/or the appended claims.

Abbreviations used in the examples below and elsewhere herein:

Unless otherwise stated, the following polymer nomenclature is used herein. Linear (i.e., unbranched) polymers are denoted by “ l ”, and random (i.e., branched) polymers are denoted by “ r ”. Conjugates are further identified using the abbreviations for each fragment of the conjugate (e.g., PEG or LPEI) and/or the targeting group (e.g., hEGF) and the orientation in which they are linked. The subscripts following each fragment in the conjugate, when used, indicate the number of monomer units (e.g., LPEI or PEG units) in each fragment. A binding moiety linking the LPEI and PEG fragments, specifically a divalent covalent moiety Z of formula I ^* (e.g., 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3 ]triazole) are each defined by the binding moiety and the reactive group that forms the divalent covalent moiety Z of formula I ^* . For example, a conjugate abbreviated as "LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF" has the terminal end of the PEG fragment bound to hEGF while the azide contained in the LPEI fragment is contained in the PEG fragment. It is an unbranched (i.e. linear) conjugate containing LPEI linked to a 24-unit PEG chain via a 1,2,3-triazole formed by the reaction of DBCO.

Analytical methods, materials and instruments

Unless otherwise stated, reagents and solvents were obtained from commercial suppliers. Starting materials are either commercially available or made according to procedures known or as described in the reported literature. α-Hydrogen-ω-azido-poly(iminoethylene) (H-(NC ₂ H ₅ ) _n -N ₃ ; LPEI-N ₃ ) Ultroxa ^® (MW = 22 kDa; dispersity ≤ 1.25) is Obtained from Broxa (Belgium). DBCO-amine (compound 35) was purchased from Broadfarm (USA) (product number BP-22066; C ₁₈ H ₁₆ N ₂ O; MW 276.3), and NHS-PEG ₃₆ -OPSS was purchased from Quanta Biodesign (USA) ( Purchased from product number 10867; MW 1969.3). DBCO-PEG ₄ -TFP (Product No. PEG6740; C ₃₇ H ₃₈ F ₄ N ₂ O ₈ ; MW 714.7), DBCO-PEG ₁₂ -TFP (Product No. JSI-A1201-068; C ₅₃ H ₇₀ F ₄ N ₂ O ₈ ; MW 1067.12), DBCO-PEG ₂₄ -TFP (Product No. PEG6760; C ₇₇ H ₁₁₈ F ₄ N ₂ O ₂₈ ; MW 1595.75), DBCO-PEG ₂₄ -MAL (Product No. JSI-A2405-004; C ₇₆ H ₁₂₂ N ₄ O ₂₉ ; MW 1555.79) were all obtained from Iris Biotech (Germany). Low-molecular weight (LMW) poly(IC) was purchased from Dalton Pharma Services (Canada). Poly(Glu) (MW range: 50-100 kDa) was obtained from Sigma Aldrich. DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys ((C ₅₇ H ₇₁ N ₁₁ O ₁₆ S; MW 1198.3; SEQ ID NO: 4), DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Malay Mid (C ₆₀ H ₇₂ N ₁₂ O ₁₆ ; MW 1217.3; SEQ ID NO: 5), hEGF peptide and MCC-hEGF (C ₂₈₂ H ₄₀₉ N ₇₉ O ₈₆ S ₇ ; MW 6435) were synthesized from CBL Patras (Greece). Cys-GE-11 peptide (sequence: Cys-Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile; CYHWYGYTPQNVI, SEQ ID NO: 6) was purchased from GenScript Biotech (Netherlands). ) was cut and synthesized from Abcam (anti-ErbB2/HER2 epibody ^® molecule; product number ab31889) with folic acid (product number F7876) and N ¹⁰ -methyl-4-amino-4-dehyde. Oxyphteric acid (product number 861553) was purchased from Sigma Aldrich, and cysteamine 4-methoxytrityl resin (Novobiochem®; product number 8.56087.0001) was purchased from Merck _. -NH ₂ (product number SC-8301) was purchased from Seachem, and tris-GalNAc ₃ -Ala-PEG ₃ -NH ₂ (C ₇₃ H ₃₂ N ₁₂ O ₃₂ ; MW 1689.9) was purchased from Sussex Research Laboratories (Canada). ) (Product No. MV100017 ⁾ The cell lines used here were A431 (Product No. CRL-1555), MCF7 (Product No. HTB-22), and LNCaP (Product No. CRL-1740). and PC-3 (product number CRL-1435). The acetate buffer was 50 mM sodium acetate (aqueous), pH 4-4.5, and the HEPES buffer was 20 mM HEPES (aqueous), pH 7. It was 7.4.

UV spectrophotometry of samples containing hEGF

Determination of hEGF content in reagent solutions and conjugated samples was performed with a microplate reader (Spectramax Paradigm, Molecular Devices) at 280 nm using Brand ^® Puregrade UV transparent microplates. The UV absorbance of 100 mL sample solution was measured in the buffer solution, and the absorbance of the sample was corrected by subtracting the absorbance of the buffer solution alone (blank sample). ε (280 nm) of hEGF was calculated using the following formula.

The concentration of total hEGF was calculated using the formula below.

UV spectrophotometry of samples containing HER2

Measurement of HER2 (e.g. DBCO-PEG ₂₄ -HER2 or LPEI-PEG ₂₄ -HER2 content in the sample) was performed on a ThermoFisher NanoDrop One C device by UV spectrophotometry at 280 nm. A 2 mL sample was analyzed, and the absorbance of the sample was corrected by subtracting the absorbance of 2 mL of the appropriate buffer alone (blank sample). ε (280 nm) of HER2 was 16,600 cm ^-1 ·M ^-1 . The concentration of total HER2 was calculated using the formula below.

UV spectrophotometry of samples containing DBCO

Determination of DBCO content in reagent solutions and conjugated samples was performed with a microplate reader (Spectramax Paradigm, Molecular Devices) at 309 nm using Brand ^® Puregrade UV transparent microplates. The UV absorbance of the 100 mL buffered solution was measured, and the absorbance of the sample was corrected by subtracting the absorbance of the buffer solution alone (blank sample). ε (309 nm) of DBCO was 12,000 cm ^-1 ·M ^-1 . The concentration of total DBCO was calculated using the formula below.

RP-HPLC coupled mass spectrometry

Samples were analyzed by LC-MS using an Agilent 1260 Infinity II HPLC system or an Agilent UHPLC 1290 system.

The Agilent 1260 Infinity II HPLC system was connected to an Agilent iFunnel 6550B qTOF equipped with an Agilent Jetstream electrospray ionization (AJS ESI) source. Samples were separated at 40°C on a Phenomenex Airless photopore column XB-C8 - 3.6 μm, 100 × 2.1 mm (P/N: 00D-4481-AN). 1 to 5 μL was injected and eluted with an eluent gradient at a flow rate of 0.3 mL/min as shown in Table 1, where solvent A was 100% H ₂ O with 0.1% HCOOH and solvent B was 100% HCOOH with 0.1% HCOOH. It was ACN. The AJS ESI source is equipped with a capillary voltage of 3,000 V and a nozzle voltage of 1,000 V, a drying gas temperature of 200°C, a flow rate of 14 L/min, an atomizing gas pressure of 20 psig, and an external gas temperature of 325°C, 12 L/min. It was operated at a flow rate of . MS data were acquired in positive ion mode at 4 Ghz high-resolution mode between 1 and 12 min over a standard mass range of 100 to 3200 m/z. The fragment maker and octupole RF voltages were set at 380 and 750 V, respectively.

The Agilent UHPLC 1290 system included an Agilent 1290 dual pump (G4220A), an Agilent 1290 HiP sampler (G4226A), an Agilent 1290 column compartment (G1316C), an Agilent 1290 DAD UV module (G4212A), and an Agilent quadrupole LC/MS (6130). , a Phenomenex Biogen column XB-C8 (3.6 μm, 150 × 2.1 mm (00F-4766-AN)) equipped with a precolumn filter of the same material (AJ0-9812) was used at 4°C. 5 μL of sample was injected. The flow rate was 0.4 mL/min. Signals were monitored at 210 nm, 215 nm, 240 nm and 280 nm. The mobile phases were (A) H ₂ O with 0.1% (vol.) HCOOH and (B) ACN. The eluent gradients used are given in Table 2.

Analytical RP-HPLC .

RP-HPLC experiments were performed on the same material (AJ0) on an Agilent UHPLC 1290 system containing an Agilent 1290 dual pump (G4220A), Agilent 1290 HiP sample collector (G4226A), Agilent 1290 column compartment (G1316C), and Agilent 1290 DAD UV module (G4212A). -9812) was carried out at 40°C using a Phenomenex Biogen ^™ column XB-C8 (3.6 μm, 150 × 2.1 mm (00F-4766-AN)) equipped with a precolumn filter. 20 μL of sample was injected. The flow rate was 0.4 mL/min. Signals were monitored at 210 nm, 214 nm, 220 nm, 230 nm, 240 nm and 280 nm. The mobile phases were (A) H ₂ O + 0.1% TFA (by volume) and (B) ACN + 0.1% TFA (by volume). The eluent gradients used are given in Table 2.

For manufacturing RP-HPLC

Preparative RP-HPLC experiments were performed with a Waters Preparation System or a Puriflash RP Preparation System. The Waters system included a Waters 515 HPLC pump, Waters 2545 dual gradient module, Waters 2777C sample collector, Waters Fraction Collector III and Waters 2487 dual λ absorbance detector module, and a Phenomenex SecurityGuard PREP cartridge Core-Shell C18 pre-column ( A Phenomenex Kinetex 5 mm The flow rate was 35 mL/min and the signal was monitored at 240 nm. The fraction collector was purified at ~8 mL/tube for 0.1 to 30 min, depending on the following profile, eluent A: H ₂ O with 0.1% TFA (vol.) and eluent B: ACN (vol.) with 0.1% TFA (vol.). Volume was collected. The eluent gradients used are given in Table 4.

The Puriflash system included an interfilament Puriflash 1 Series system including injector, pump, detector and fraction collector, and a Phenomenex SecurityGuard PREP cartridge core-shell C18 pre-column (C18 15 × 21.2 mm, G16-007037). ) was used. A Phenomenex Kinetex 5 mm XB-C18 column (100Å, 100 At the time of injection (00 to 04 seconds), the flow rate was 10 mL/min, then 35 mL/min until the end of development. The signal was monitored at 210 nm. The mobile phases were Eluent A: H ₂ O with 0.1% TFA (vol.) and Eluent B: ACN (vol.) with 0.1% TFA (vol.). The eluent gradients used are given in Table 5.

copper assay

The copper assay provides the mg/mL concentration of total LPEI present in solution (Ungaro et al ., J. Pharm. Biomed. Anal., 31: 143-9 (2003)). A stock solution (10×) of the copper reagent was prepared by dissolving 23.0 mg of CuSO ₄ in 10.0 mL acetate buffer (100 mM; pH 5.4). This stock solution was stored at 4°C. Prior to analysis, this reagent was diluted 10-fold in acetate buffer (100 mM pH 5.4) and used immediately. As a control, an LPEI solution of known concentration ( in vivo jetPEI; 150 mM nitrogen concentration; Polyplus 201-50G) was used. A 6.7 μL aliquot of in vivo jetPEI solution was prepared in a plastic tube and frozen as a control sample, freshly thawed and diluted 15-fold with Milli-Q water (93.3 μL) before use.

As shown in Table 6, the solutions of the experimental and control samples were distributed into UV-qualified 96-well plates (Brand Plate Puregrade) and measured in triplicate.

In addition, blank samples consisting of 100 μL water and 100 μL CuSO ₄ reagent were measured in triplicate, and the average absorbance of the blank samples was subtracted from the absorbance values recorded for the experimental and control samples. The solution was left to react at room temperature for 20 minutes, and then the absorbance was measured at 285 nm with a microplate reader (Spectramax Paradigm, Molecular Devices). Individual measurements were valid if the absorbance values fell within the calibration range, otherwise they were further diluted. Individual measurements are not valid if the coefficient of variation of the measurements is greater than 10.0%, but instead repeated measurements were made. The measurement was valid if the control value was within 10% of 150 mM. The concentration was calculated according to the formula below using a correction slope k = 0.0179.

Freeze drying

Freeze-drying was performed with a freeze-drying device from Krait (Alpha 2-4 LP Plus). Due to the presence of acetonitrile in some samples, the samples were cooled to -196°C for approximately 3 minutes under liquid nitrogen prior to lyophilization.

Samples were freeze-dried at -82°C (concentrator temperature) and 100 mbar (75 Torr). The freeze-drying time was adjusted based on the properties of the freeze-dried compound.

General buffer exchange method

For the preparation of the triconjugate in HEPES buffer, the resuspended TFA lyophilisate solution was adjusted to pH 6.5 using NaOH and then the buffer was exchanged to 20 mM HEPES, pH 7.2.

For the preparation of the triconjugate in acetate buffer, the resuspended TFA lyophilisate solution was adjusted to pH 4.5 using NaOH and then buffer exchanged to 20 mM acetate, pH 4.3.

Additionally, detailed buffer exchange procedures specific to the compounds are provided below.

Tangential flow filtration ( TFF) 2 kDa purification

DBCO-PEG ₃₆ -DUPA (Compound 37) * To remove TFA from the TFA salt, use Masterplex ^® Pharmamed ^® tubing (Product No. 06508-15) with a molecular weight cutoff (MWCO) of 2 kDa and a surface area of 200 cm ² A Sartorius Slice cassette (Satorius Stedym / Inline Model 1082 / Cylog) consisting of a peristaltic pump, together with a Hydrosart membrane (Satorius Stedym / Sartocon Slice 200 / Product No. 3051441901E-SG / Lot No. 90279123). ) was performed on tangential flow filtration. Membranes were stored in 20-24% aqueous EtOH.

The following TFF parameters were used: TMP: 2.0 bar, feed flow rate: 428 mL/min, permeate flow rate: 28 g/min.

For step-by-step TFF, (1) 169 mL of DBCO-PEG ₃₆ -DUPA (Compound 37) solution was supplemented with 81 mL of 15 mM acetate, pH 5.5. The solution was filtered by TFF to 50 mL. (2) The resulting 50 mL was supplemented with 250 mL of 15 mM acetate, pH 5.5. The solution was filtered by TFF to 50 mL. (3) The resulting 50 mL was supplemented with 250 mL of 15 mM acetate, pH 5.5. The solution was filtered by TFF to 50 mL. (4) The resulting 50 mL was supplemented with 250 mL of 15 mM acetate, pH 5.5. The solution was filtered by TFF to 50 mL. (5) The resulting 50 mL was supplemented with 250 mL of 15 mM acetate, pH 5.5. The solution was filtered by TFF to 50 mL. (6) The resulting 50 mL was supplemented with 250 mL of 15 mM acetate, pH 5.5. The solution was filtered by TFF to 50 mL.

Tangential flow filtration (TFF) 10 kDa purification

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA (Compound 31a and Compound 31b) * To remove TFA from the TFA salt, use Masterplex ^® Pharmamed ^® tubing (Product No. 06508-15) and 10 kDa A Sartorius Slice cassette (consisting of a peristaltic pump) together with a Hydrosart membrane (Sartorius Stedim / Sartocon Slice 200 / Cat. No. 3051441901E-SG / Lot No. 01181123) with a molecular weight cut-off (MWCO) and a surface of ²⁰⁰ cm 2 Tangential flow filtration was performed on a Sartorius Stedim / Inline Model 1082 / Cylog. Membranes were stored in 20-24% aqueous EtOH.

The following TFF parameters were used: TMP: 1.6 bar, feed flow rate: 517 mL/min, permeate flow rate: 155 g/min.

For step-by-step TFF, (1) 30 mL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA (Compound 31a and 31b)*TFA salt solution was supplemented with 220 mL of 20 mM HEPES, pH 7.2 . The solution was filtered by TFF to 50 mL. (2) The resulting 50 mL was supplemented with 250 mL of 20 mM HEPES, pH 7.2. The solution was filtered by TFF to 50 mL. (3) The resulting 50 mL was supplemented with 250 mL of 20 mM HEPES, pH 7.2. The solution was filtered by TFF to 50 mL. (4) The resulting 50 mL was supplemented with 250 mL of 20 mM HEPES, pH 7.2. The solution was filtered by TFF to 50 mL. (5) The resulting 50 mL was supplemented with 250 mL of 20 mM HEPES, pH 7.2. The solution was filtered by TFF to 50 mL.

Multicomplex size measurement and characterization

Triconjugates (e.g., LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF) are complexed with nucleic acids (e.g., poly(IC)) to form multicomplexes (e.g., LPEI- l -[N ₃ :DBCO]) -PEG ₂₄ -hEGF:poly(IC)) was formed. The multicomplexes were characterized by measuring the molar ratio (referred to herein as the N/P ratio) in the nitrogen of LPEI to the phosphorus of poly(IC). Multicomplex size and ζ-potential were measured by DLS and ELS according to Hickey et al. (Hickey et al ., J. Control. Release, 2015, 219: 536-47). The size of the multicomplex was measured, for example, using a Zetasizer Nano ZS instrument (Malvern Instruments) operating at 633 nm at 25°C and equipped with a backscattering detector (173°) in HBG buffer (20 mM HEPES, 5% glucose, pH 7.2). was measured by DLS using (Korea, UK). Each sample was measured in triplicate. Briefly, the multicomplex in HBG buffer was transferred into a crystal cuvette and used with a particle RI of 1.59 and an absorbance of 0.01, a viscosity of 0.98 mPa, and an RI of 1.330 in HGB at 25°C. Measurements were performed in triplicate using a 173° backscattering detection angle previously equilibrated to 25°C for 60 seconds, with five deployments and automatic deployment durations each, without delay between measurements. Data were analyzed using a general purpose model at normal resolution. Calculations for particle size and PDI were determined according to the ISO standard document ISO 22412:2017. The ζ-potential of the multicomplex was determined as described by the instrument supplier (https://www.malvernpanalytical.com/en/products/technology/light-scattering/electrophoretic-light-scattering) (e.g., HBG at 25°C). in buffer) by phase-analytical light scattering (PALS) and/or electrophoretic light scattering (ELS).

Example 1

LPEI- L -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of hEGF (Compound 1a and Compound 1b)

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF was synthesized as a mixture of regioisomers 1a and 1b in two steps according to the following reaction scheme. In the first step, human epidermal growth factor (hEGF) was incubated with dibenzoazacyclooctyne-24(ethylene glycol)-propionyl 2,3,5,6-tetrafluorophenyl ester (DBCO-PEG ₂₄₎ in 20 mM HEPES buffer. -TFP; compound 2) was combined to produce DBCO-PEG ₂₄ -hEGF (compound 3). In the second step, DBCO-PEG ₂₄ -hEGF was conjugated to LPEI-N ₃ to produce LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF (Compound 1a and Compound 1b).

Step 1: DBCO-PEG ₂₄ -Synthesis of hEGF (compound 3)

Human epidermal growth factor (hEGF acetate salt, 152.6 mg, 24.5 mmol; MW = 6216.01 g/mol; CBL Patras, Greece) was weighed in a 250 mL round bottom flask. 75 mL of 20 mM HEPES (pH 7.4) was added to the hEGF powder to obtain a 2 mg/mL solution of hEGF protein. The solution was shaken with magnetic stirring for 10 minutes until complete dissolution of the protein. The pH was adjusted to 7.5 using 150 mL of 1 M NaOH and 60 mL of 5 M NaOH. The purity of the solution was determined by UV spectrophotometry at 280 nm, and the effective concentration of protein was confirmed to be 0.23 mM (17.2 mmol).

Dibenzoazacyclooctyne-24(ethylene glycol)-propionyl 2,3,5,6-tetrafluorophenyl ester (DBCO-PEG ₂₄ -TFP; Compound 2; 100.2 mg, 62.8 mmol, MW = 1,595.75 g/mol ; Iris Biotech, Germany) was weighed into a 15 mL Falcon tube and dissolved in 6.0 mL DMSO to form a 10 mM stock solution. The purity of the DBCO-PEG ₂₄ -TFP solution was determined by UV spectrophotometry at 309 nm after 40-fold dilution with DMSO. The effective concentration of DBCO-PEG ₂₄ -TFP was confirmed to be 9.32 mM (89%, 55.9 mmol).

DBCO-PEG ₂₄ -TFP (Compound 2; 3.68 mL, 34.3 mmol, 2.0 equiv. stock solution) was added to the hEGF solution under magnetic stirring at room temperature. After 2.5 hours, an additional 0.92 mL of DBCO-PEG-TFP (Compound 2) stock solution (8.6 mmol, 0.5 equiv) was added to the reaction mixture. The solution was left to react for an additional 30 minutes. The reaction mixture was transferred into a 50 mL falcon tube and left at 4°C for 2 hours prior to purification.

The reaction mixture (79 mL) was purified by four runs using a Waters preparative chromatography system. Before each run, the solution was supplemented with acetonitrile to 10% ACN to have the same composition as the eluent at the start of the preparative chromatography. Pools of fractions were collected for lyophilization. A total of approximately 273 mL of isolated DBCO-PEG ₂₄ -hEGF (Compound 3) was collected in a 50 mL Falcon tube (3.4-fold dilution). Four pools were mixed and the combined samples were analyzed by C8-RP-HPLC and stored at -80°C under argon prior to lyophilization.

Isolated DBCO-PEG ₂₄ -hEGF (Compound 3) was cooled in liquid nitrogen for approximately 3 minutes prior to lyophilization. Fluffy lyophilisate (70 mg, 46% yield of hEGF, 89% yield of DBCO, [(M+6H) ⁶⁺ ]/6 = 1274.42, measured monoisotopic mass [Da] 7,640.47, calculated monoisotope Mass [Da] 7,640.47) was recovered and stored at -80°C under argon.

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of hEGF (Compound 1a and Compound 1b)

DBCO-PEG ₂₄ -hEGF lyophilisate (Compound 3; ~43 mg) was weighed into a 15 mL falcon tube and dissolved in 5.4 mL of 20 mM HEPES (pH 6.5; 8 mg/mL solution). After dissolution, the pH reached 3.9 and was adjusted to pH 4.5 using 3 μL of 5 M NaOH. Because the solution was cloudy, the precipitate was redissolved using 15 μL of 1 M HCl and the solution became clear again. The final pH of the solution was 3.7. The solution was filtered using a 0.45 μm nylon filter (13 mm nylon membrane from Exapure, Germany) to provide ˜4.7 mL of DBCO-PEG ₂₄ -hEGF (Compound 3) solution. The effective concentration of DBCO-PEG ₂₄ -hEGF (Compound 3) was determined by UV spectrophotometry at 309 nm after 20-fold dilution with H ₂ O. The assay gave a compound content of ~86% at a concentration of 0.89 mM (4.2 μmol).

LPEI-N ₃ (199.5 mg) was weighed into a 50 mL Falcon tube and dissolved in 10 mL MilliQ water pH 2.2 (20 mg/mL solution). 350 μL of 1 M HCl was added to help dissolve LPEI-N ₃ . The solution was sonicated for approximately 3 minutes and heated to -70°C until LPEI-N ₃ was completely dissolved. The pH was measured to be 7.8 and adjusted to pH 4.6 using 800 μL of 1 M HCl + 300 μL of 1 M NaOH. The concentration of LPEI-N ₃ was measured by copper assay, and purity of ~69% was confirmed. The effective concentration of the solution was 0.55mM.

In a 50 mL Falcon tube, mix DBCO-PEG ₂₄ -hEGF (Compound 3) solution (4.7 mL, 4.2 μmol), LPEI-N ₃ solution (7.6 mL, 4.2 μmol) and NaCl solution (400 μL, 4.8 M); The reaction was left to react on a Stuart rotator at 20 rpm at room temperature. Samples were collected regularly for analytical HPLC monitoring of the reaction at 240 nm and 309 nm. After 95 hours no significant further conversion was evident and the reaction was stopped. Based on the decrease in peak area, 55 to 60% of DBCO-PEG ₂₄ -hEGF (Compound 3) was consumed. Approximately 12.5 mL of solution was recovered and measured to be pH 4.9. The solution was stored at -80°C under argon prior to purification.

The reaction mixture (approximately 12.5 mL) was brought to room temperature and treated with 1.4 mL of acetonitrile and 15 μL TFA. Prior to purification, the solution was filtered through a 0.45 μM filter using Puriflash RP preparative chromatography. Fractions containing pure product were lyophilized, weighed, and analyzed by RP-HPLC, copper assay, and UV spectrophotometry at 280 nm. The retention time of LPEI -l -[N ₃ :DBCO]-PEG ₂₄ -hEGF (Compound 1a and 1b) was 5.6 to 5.8 minutes in analytical RP-HPLC analysis. A mixture of 29 mg of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF (Compound 1a and 1b) trifluoroacetate was isolated without additional impurities at an LPEI:hEGF ratio of 1:1, respectively (the overall yield 12%).

Step 3: Exchange of TFA salts with HEPES buffer

To exchange TFA for HEPES, 11.5 mg of lyophilized LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF (Compound 1a and 1b) trifluoroacetate (w _LPEI = 26%, total LPEI ~ 3 mg) was dissolved in 1.0 mL of 20 mM HEPES (pH 7.2) in a 2 mL Eppendorf tube. Initially the pH was 3.5 and adjusted to pH 7.2 using 8 μL of 5 M NaOH and 9 μL of 1 M HCl. An additional 483 μL of 20 mM HEPES (pH 7.2) was added to give a final volume of approximately 1.5 mL. The concentration of total LPEI was approximately 2 mg/mL. Three centrifugal filters were charged with 450 μL (total 1350 μL) of LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF trifluoroacetate. Each tube was centrifuged once at 14,000 g for 30 minutes. The supernatant was discarded and the pellet was resuspended in 20 mM HEPES buffer (pH 7.2) at 25°C. The tube was centrifuged again at 14,000 g for 30 minutes and the supernatant was discarded. The pellet was resuspended in 20 mM HEPES buffer (pH 7.2) and recentrifuged two additional times. Approximately 1.3 mL of LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF (Compound 1a and 1b) solution was recovered as the HEPES salt at a total LPEI concentration of 2.1 mg/mL.

Step 4: Exchange of TFA salts with acetate buffer

To exchange TFA for acetate, 12.5 mg of lyophilized LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF (Compound 1a and 1b) trifluoroacetate (w _LPEI = 26%, total LPEI ~ 3 mg) was dissolved in 1.3 mL of 50 mM acetate buffer (pH 7.2) in a 2.0 mL Eppendorf tube. Initially the pH was 4.0 and adjusted to pH 4.5 using 3.5 μL of 5 M NaOH. The concentration of total LPEI was approximately 2 mg/mL. Four centrifugal filters were charged with 325 μL (1300 μL total) of LPEI -l- [N ₃ :DBCO]-PEG ₂₄ -hEGF trifluoroacetate. Each tube was centrifuged once at 14,000 g for 30 minutes. The supernatant was discarded and the pellet was resuspended in 20 mM acetate buffer (pH 4.5) at 4°C. The tube was centrifuged again at 14,000 g for 30 minutes and the supernatant was discarded. The pellet was resuspended in 50 mM acetate buffer (pH 4.5) and recentrifuged two additional times. Approximately 1.4 mL of LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF (Compound 1a and 1b) solution was recovered as the acetate salt at a total LPEI concentration of 2.3 mg/mL.

Example 2

LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -Synthesis of hEGF (Compound 4a and Compound 4b)

LPEI- l- [N ₃ :DBCO]-PEG ₁₂ -hEGF was synthesized as a mixture of regioisomers 4a and 4b in two steps according to the following reaction scheme. In the first step, human epidermal growth factor (hEGF) was incubated with dibenzoazacyclooctyne-12(ethylene glycol)-propionyl 2,3,5,6-tetrafluorophenyl ester (DBCO-PEG ₁₂₎ in 20 mM HEPES buffer. -TFP; combined with compound 5) to produce DBCO-PEG ₁₂ -hEGF (compound 6). In the second step, DBCO-PEG ₁₂ -hEGF (compound 6) was conjugated to LPEI-N ₃ to produce LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF (compound 4a and 4b).

Step 1: DBCO-PEG ₁₂ -Synthesis of hEGF (compound 6)

56 mg of dibenzoazacyclooctyne-12(ethylene glycol)-propionyl 2,3,5,6-tetrafluorophenyl ester (DBCO-PEG ₁₂ -TFP; Compound 5; 96.3% assay; 51 μmol pure product) was weighed in a 5 mL Eppendorf tube and dissolved in 2.6 mL DMSO (~20 mM stock solution, pure product). DBCO-PEG ₁₂ -TFP was dissolved by mixing the solution by hand.

148 mg (crude mass) of hEGF (lot no. 5263, 87.1% peptide content; 21 μmol) was weighed in a 100 mL round bottom flask and dissolved in 75 mL 20 mM HEPES, pH 7.4. The solution was shaken by magnetic stirring for approximately 10 min to dissolve the protein, and the pH of the solution was adjusted to pH 7.5 using 100 μL 5 M NaOH and 16 μL 6 M HCl.

2.08 mL of DBCO-PEG ₁₂ -TFP (Compound 5) stock solution (2 equiv, 42 μmol) was added slowly to the hEGF solution with stirring. After approximately 15 minutes, 8.6 mL of ACN was added to the reaction mixture (10% of final volume). After approximately 10 minutes, an additional 0.52 mL of DBCO-PEG ₁₂ -hEGF stock solution (0.5 monomeric compound 5; 10 μmol) was added to the reaction mixture and stirred for an additional 3 hours. The slightly cloudy reaction mixture was centrifuged at 15,000 g for 5 minutes prior to purification. 86 mL of the reaction mixture (10% acetonitrile) was purified in one run using a Puriflash RP-preparative column system. A pool of fractions was collected and lyophilized (47 mg, 31% yield of compound 6; [(M+5H) ⁵⁺ ]/5 = 1186.37, (measured monoisotopic mass [Da] 7112.16, calculated monoisotopic mass Mass [Da] 7112.18)).

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF (Compound 4a and Compound 4b)

DBCO-PEG ₁₂ -hEGF lyophilisate (Compound 6; 46 mg, 6.5 μmol) was dissolved in a mixture of 20 mL of 50 mM acetate, pH 4.0, and 2.2 mL acetonitrile (final volume of 10% acetonitrile). The solution was pH 4.2 and was adjusted to pH 4.0 using 6 μL 6 M HCl. The concentration of DBCO-PEG ₁₂ -hEGF (Compound 6) in solution was 2.3 mg/mL.

LPEI-N ₃ (204 mg) was weighed in a 15 mL falcon tube and dissolved in 10 mL 50 mM acetate, pH 4.0. The solution was heated to about 70° C. for about 2 minutes, and 360 μL of 6 M HCl was added to help dissolve the LPEI-N ₃ and adjusted to pH 4.0 (19.7 mg/mL). The concentration of LPEI-N ₃ (MW = 22 kDa) was determined by copper assay and a purity of approximately 85% was determined. The effective concentration of the solution was 16.8 mg/mL (7.9 μmol of LPEI-N ₃ in solution).

The LPEI-N ₃ solution (7.9 μmol, 1.2 equiv) was transferred to a 100 mL round bottom flask equipped with a magnetic stirrer, and the DBCO-PEG ₁₂ -hEGF (Compound 6) solution (6.5 μmol, 1.0 equiv) was added. The reaction mixture was stirred at room temperature and protected from light for approximately 45 hours. Samples were collected regularly for monitoring and diluted 10-fold with acetonitrile/H ₂ O (1:9) prior to injection. The reaction mixture (about 35 mL) was adjusted to contain about 6% (by volume) acetonitrile and purified using a Puriflash PEM injection system coupled to a preparative HPLC column. Pools of fractions containing pure product were lyophilized, weighed, and analyzed by RP-HPLC, copper assay, and UV spectrophotometry at 280 nm. A mixture of 47 mg of LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF (compound 4a and 4b) was isolated without additional impurities at an LPEI:hEGF ratio of 1:1, respectively (overall yield of LPEI 7 %). The retention time of LPEI- l- [N ₃ :DBCO]-PEG ₁₂ -hEGF was 5.5 to 6.2 minutes in analytical RP-HPLC analysis.

Example 3

LPEI- l -[N ₃ :DBCO]-PEG ₄ -Synthesis of hEGF (Compound 7a and Compound 7b)

LPEI- l- [N ₃ :DBCO]-PEG ₄ -hEGF was synthesized as a mixture of regioisomers 7a and 7b in two steps according to the following reaction scheme. In the first step, human epidermal growth factor (hEGF) was incubated with dibenzoazacyclooctyne-4(ethylene glycol)-propionyl 2,3,5,6-tetrafluorophenyl ester (DBCO-PEG ₄₎ in 20 mM HEPES buffer. -TFP; combined with compound 8) to produce DBCO-PEG ₄ -hEGF (compound 9). In the second step, DBCO-PEG ₁₂ -hEGF (compound 9) was conjugated to LPEI-N ₃ to produce LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF (compound 7a and 7b).

Step 1: DBCO-PEG ₄ -Synthesis of hEGF (compound 9)

A solution (83.6 mL) of hEGF (150 mg, peptide content 87.1%, 24 μmol pure peptide, 1.0 equiv, 0.29 mM) in 20 mM HEPES pH 7.5/ACN (9:1) was incubated with DBCO-PEG ₄ -TFP (compound 8; 43 μmol, 1.8 equivalent, 20 mM) (2.2 mL). The reaction mixture was incubated in a 100 mL round bottom flask at room temperature under magnetic stirring and monitored by RP-C ₈ -HPLC. After 25 minutes, the reaction mixture was supplemented with acetonitrile (10 mL), and after 1.5 hours, the mixture was supplemented with additional DBCO-PEG ₄ -TFP (12 μmol, 0.5 equiv, 20 mM). After a total of 3 hours, the reaction mixture was stored at 4°C overnight. The reaction mixture was adapted to 10% ACN and DBCO-PEG ₄ -hEGF was isolated following RP-C ₁₈ preparative HPLC and lyophilization of the fraction pools. The solid (59 mg) was recovered and analyzed by HPLC - ESI ⁺ qTOF mass spectrometry. The solid included DBCO-PEG ₄ -hEGF (calculated monoisotopic mass: 6,759.95 Da; measured: 6,760.02 Da).

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₄ -Synthesis of hEGF (Compound 7a and Compound 7b)

LPEI-N ₃ solution (10 mL, 6.4 μmol, 1.0 equiv, 0.64 mM) in 50 mM acetate buffer pH 4.0 was mixed with DBCO-PEG ₄ -hEGF solution (6.6 μmol, 1.0 equivalent, 0.30 mM) (22.3 mL) was added slowly. The reaction mixture was incubated in a 100 mL round bottom flask at room temperature under magnetic stirring, blocked from light, and monitored by RP-C ₈ -HPLC. After 45 hours of reaction, LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF was isolated using RP-C ₁₈ preparative HPLC as a mixture of regioisomers 7a and 7b. The pool of fractions was lyophilized (47 mg, fluffy white solid) and characterized by RP-C ₈ -HPLC, copper assay and spectrophotometry at 280 nm for determination of hEGF content. The lyophilisate showed a weight percentage of LPEI of 26% and an LPEI to hEGF ratio of 1/1.0.

Step 3: LPEI- l -[N ₃ :DBCO]-PEG ₄ Preparation of -hEGF-HEPES salt

LPEI- l- [N ₃ :DBCO]-PEG ₄ -hEGF (Compounds 7a and 7b) TFA salt (22.9 mg, w _LPEI = 26%, 6.0 mg total LPEI) was dissolved in 2.5 mL 20 mM HEPES pH 7.2. Six centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each charged with 420 μL of LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF solution and centrifuged once at 14,000 g for 30 min. Centrifugation was performed three times after addition of 400 μL of 20 mM HEPES, pH 7.2. Approximately 449 μL of LPEI- l- [N ₃ :DBCO]-PEG ₄ -hEGF-HEPES salt solution was recovered and supplemented with 1.2 mL of 20 mM HEPES, pH 7.2. The concentration of the solution was determined by copper assay (total LPEI 3.6 mg/mL, LPEI/hEGF ratio = 1/1.0).

Step 4: LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF-acetate salt preparation

LPEI- l- [N ₃ :DBCO]-PEG ₄ -hEGF (Compounds 7a and 7b) TFA salt (13.2 mg, w _LPEI = 26%, total LPEI 3.6 mg) was dissolved in 1.7 mL 50 mM acetate pH 4.3. Four centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each charged with 425 μL of LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF solution and centrifuged once at 14,000 g for 30 min. Centrifugation was performed three times followed by the addition of 400 μL of 50 mM acetate, pH 4.3. Approximately 211 μL of LPEI- l- [N ₃ :DBCO]-PEG ₄ -hEGF-acetate salt solution was recovered and supplemented with 1.2 mL of 50 mM acetate, pH 4.3. The concentration of the solution was determined by copper assay (total LPEI 2.2 mg/mL, LPEI/hEGF ratio = 1/1.0).

Example 4

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of DUPA (Compound 10a and Compound 10b)

LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA was synthesized as a mixture of regioisomers 10a and 10b in two steps according to the following reaction scheme. In the first step, DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12; SEQ ID NO: 4) (prepared analogously as described in international patent applications WO2015/173824 A1 and WO2019/063705 A1) DBCO-PEG ₂₄ -DUPA (Compound 13) was prepared by linking to dibenzoazacyclooctyne-24(ethylene glycol)-maleimide (DBCO-PEG ₂₄ -MAL; Compound 11) by Michael addition. In the second step, DBCO-PEG ₂₄ -DUPA (Compound 13) was conjugated to LPEI-N ₃ to prepare LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA (Compound 10a and Compound 10b).

Step 1: DBCO-PEG ₂₄ -Synthesis of DUPA (Compound 13)

Weigh 18.06 mg (crude mass) of DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (compound 12; 15 μmol theoretical pure peptide content) in a 50 mL falcon tube and dissolve in 9 mL of _HO. / dissolved in 25% ACN (2.0 mg/mL stock solution). The solution was sonicated for 15 seconds to help dissolve DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12). The solution was adjusted to pH 3.5 using 8.5 μL of 6 M HCl.

21.38 mg (crude mass) of DBCO-PEG ₂₄ -MAL (Compound 11; 13 μmol pure product) was weighed in a 1.5 mL Eppendorf tube and dissolved in 650 μL of DMSO (20 mM pure product). To a 50 mL Falcon tube containing Compound 12 solution (15 μmol, 1.5 equiv), 500 μL of DBCO-PEG ₂₄ -MAL (Compound 11) stock solution (10 μmol, 1.0 equiv) was added. The reaction mixture was protected from light and incubated on a Stuart rotator (20 rpm) for approximately 20 minutes (RT). The reaction was monitored by C8-RP-HPLC and continued until complete conversion of DBCO-PEG ₂₄ -MAL (compound 11). Characterization of DBCO-PEG ₂₄ -DUPA (compound 13) produced by the reaction was analyzed by LC-MS (C8-RP-HPLC coupled to ESI-qTOF MS) ((M+2H)2+]/2 = 1377.16, determined. It was verified by the calculated monoisotope mass [Da] 2752.30 and the calculated monoisotope mass [Da] 2752.30). The reaction was not quenched or purified and was used directly in step 2.

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of DUPA (Compound 10a and Compound 10b)

201.8 mg (crude mass) of LPEI-N ₃ was weighed in a 15 mL falcon tube and dissolved in 8 mL of 50 mM acetate buffer, pH 4.0. The pH of the solution was adjusted to pH 3.5 using 375 μL of 6 M HCl, heated to 70°C, and sonicated for approximately 3 minutes to completely dissolve the LPEI particles. The solution was assayed using the copper assay and a total LPEI concentration of 17.8 mg/mL (0.811 mM) was determined (74% assay of LPEI-N ₃ ).

8.3 mL of LPEI-N ₃ solution (7 μmol, 1.0 equiv) was transferred to a 50 mL falcon tube and mixed with 6.5 mL of the DBCO-PEG ₂₄ -DUPA (Compound 13) preparation from Step 1 (7 μmol, 1.0 equiv). . Because the reaction mixture became cloudy, 2 mL of acetonitrile was added (final volume of approximately 22% ACN). The solution was degassed with argon for approximately 30 seconds.

The mixture of LPEI-N ₃ and DBCO-PEG ₂₄ -DUPA was incubated on a Stuart rotator (20 rpm) for approximately 70 hours (RT), blocked from light, and monitored by RP-C8-HPLC. After about 3 hours, a white precipitate was observable in the solution and the reaction mixture had a sweet fruity aroma.

Prior to preparative separation, the reaction mixture (˜16 mL) was diluted with 20 mL of H ₂ O containing 0.1% TFA to reduce the acetonitrile percentage to approximately 10%. The solution was centrifuged at 15,000 g for 5 minutes, and the supernatant was purified using a Puriflash preparative RP-HPLC system.

Pools of fractions containing pure Compound 10a and Compound 10b were lyophilized, weighed, and analyzed by RP-HPLC, copper assay, and UV spectrophotometry at 280 nm. 28 mg of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA (Compound 10a and Compound 10b) were each isolated without additional impurities at an LPEI:DUPA ratio of 1:1 (7% overall yield of LPEI). . In analytical RP-HPLC analysis, the retention time of LPEI -l -[N ₃ :DBCO]-PEG _24- DUPA (Compound 10a and 10b) was 5.4 to 6.4 minutes with a maximum of 5 minutes.

Example 5

LPEI- l -[N ₃ :BCN]-PEG ₁₂ -Synthesis of hEGF (compound 14)

LPEI- l- [N ₃ :DBCO]-PEG ₁₂ -hEGF (Compound 14) was synthesized in two steps according to the following reaction scheme. In the first step, human epidermal growth factor (hEGF) was coupled to endo -BCN-PEG ₁₂ -NHS ester (compound 15) in 20 mM HEPES buffer to produce endo -BCN-PEG ₁₂ -hEGF (compound 16). In the second step, endo -BCN-PEG ₁₂ -hEGF (Compound 16) is conjugated to LPEI-N ₃ to produce LPEI- l -[N ₃ :BCN]-PEG ₁₂ -hEGF (Compound 14) (Compound 14). produced.

Step 1: Endo -BCN-PEG ₁₂ -Synthesis of hEGF

Endo -BCN-PEG ₁₂ -NHS (Compound 15; 21.8 mg, 23.9 μmol, 97.7% assay) was weighed in a 5 mL Eppendorf tube and dissolved in 2.4 mL of DMSO (10 mM stock solution, pure product). The solution was manually shaken to aid dissolution. hEGF (157 mg, 22.0 μmol, 87.1% peptide content) was weighed in a 100 mL round bottom flask and dissolved using 75 mL of 20 mM HEPES, pH 7.4. The solution was shaken with magnetic stirring for approximately 10 minutes and adjusted to pH 7.4 using 60 μL of 5 M NaOH. Endo -BCN-PEG ₁₂ -NHS (Compound 15) stock solution (2.2 mL, 22.0 μmol, 1.0 eq) was slowly added to the magnetically stirred hEGF solution (22.0 μmol, 1.0 eq). After ~4 hours the reaction mixture was diluted with 10% ACN prior to Puriflash purification. The pool of fractions from preparative chromatography was analyzed by C8-RP-HPLC and lyophilized to give 43 mg of endo -BCN-PEG ₁₂ -hEGF (Compound 16). The resulting lyophilisate of Compound 16 was dissolved in 5.0 mL of 85% v/v 50 mM acetate (pH 4.0) containing 15% v/v ACN, further purified using three NAP-25 columns, and hydrolyzed. The decomposed endo -BCN-PEG ₁₂ -OH impurity was removed (identified by RP-C8-HPLC-MS (single quadrupole, positive ionization)).

Stage 2: LPEI- l- [N ₃ :BCN]-PEG ₁₂ -Synthesis of hEGF

2.9 mL of LPEI-N ₃ (2.9 mL, 2.2 μmol, 1.5 equiv) from a 0.77 mM stock solution was incubated with Endo -BCN- pre-dissolved in 85% v/v 50 mM acetate, pH 4.0 and 15% v/v ACN. PEG ₁₂ -hEGF (Compound 16; 7 mL, 1.5 μmol peptide content, 1.0 equiv) was added slowly to the solution. The mixture was shaken on a heat shaker (40°C) for a total of 95 hours and protected from light. After ~70 hours, an additional 0.85 mL (0.65 μmol, 0.4 equiv) of LPEI-N ₃ stock solution was added to the reaction mixture and adjusted to pH 4.0 using 5 M NaOH. Preparative chromatography was performed using an Agilent 1260 Infinity II preparative system to isolate the trifluoroacetate salt of compound 14, which was subsequently lyophilized.

Step 3: LPEI- l- [N ₃ :BCN]-PEG ₁₂ Preparation of -hEGF (Compound 14) acetate salt

The lyophilized LPEI- l- [N ₃ :BCN]-PEG ₁₂ -hEGF-TFA salt (~50 mg) produced above was mixed and dissolved in 4.5 mL of 50 mM acetate (pH 4.5). The pH was adjusted to 4.3 using 5 M NaOH. Ten centrifugal filters (Amicon Ultra - 0.5 mL, Merck Millipoa) were each filled with 450 μL of LPEI- l- [N ₃ :BCN]-PEG ₁₂ -hEGF TFA salt solution. They were centrifuged once at 14,000 g for 30 minutes to remove the buffer, and then subjected to 450 μL of 50 mM acetate, pH 4.3, three times at 4°C. Approximately 574 μL of concentrated LPEI- l- [N ₃ :BCN]-PEG ₁₂ -hEGF (Compound 14) acetate salt solution was recovered after buffer exchange and supplemented with 3.0 mL of 50 mM acetate, pH 4.3. The copper assay was performed on the final LPEI -l- [N ₃ :BCN]-PEG ₁₂ -hEGF (Compound 14) acetate salt solution (~3.5 mL) to determine a total LPEI concentration of 2.1 mg/mL (LPEI/hEGF ratio) = 1/0.9).

Example 6

LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ Synthesis of (Compound 17a and Compound 17b)

LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ was synthesized as regioisomers 17a and 17b in one step according to the following reaction scheme. DBCO-PEG ₂₃ -OCH ₃ (Compound 18) was coupled to LPEI-N ₃ and purified through a 10 kDa filter using small-scale size exclusion centrifugation.

Step 1: LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ Synthesis of (Compound 17a and Compound 17b)

DBCO-PEG ₂₃ -OCH ₃ (Compound 18, 3.25 mg, 2.4 μmol, 98.9% assay) was weighed in a 1.5 mL Eppendorf tube and dissolved in 116 μL of DMSO (21 mM pure product). LPEI-N ₃ (14.4 mg, MW = 22 kDa) was weighed in a 1.5 mL Eppendorf tube and dissolved in 400 μL of 50 mM acetate buffer (pH 4.0). 6 M HCl (19 μL) was added to aid dissolution and the pH was adjusted to 3.5. The concentration of total LPEI was determined by copper assay (25.1 mg/mL, 1.14 mM).

Transfer the LPEI-N ₃ solution (400 μL, 0.46 μmol, 1.0 equiv) to a 1.5 mL Eppendorf tube and add the DBCO-PEG ₂₃ -OCH ₃ (Compound 18) solution (29 μL, 0.60 μmol, 1.3 equiv) to the reaction mixture. was added, and the resulting solution was left at 40°C for about 3 days.

The reaction mixture was purified through Amicon centrifugal filter (10 kDa MWCO) against 50 mM acetate buffer (pH 4.0). The purified LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ solution was further diluted with 2.8 mL of 50 mM acetate buffer (pH 4.0). The total LPEI content of the LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ (Compound 17a and 17b) solutions (~3 mL) was measured by copper assay and found to be 1.3 mg/mL total LPEI. . Based on the copper assay, the overall reaction and purification yield was 39%.

Comparative Example 1

LPEI-OH and DBCO-PEG ₂₃ -OCH ₃ (Compound 18) There is no ring portion between

To demonstrate the chemical specificity of the click-coupling reaction between the azide modified LPEI fragment and the activated alkyne modified PEG fragment, non-azide containing LPEI was incubated at pH 4 under the conditions described in Example 6 above. Treated with DBCO-PEG ₂₃ -OCH ₃ (Compound 18).

Step 1: DBCO-PEG with LPEI-OH ₂₃ -OCH ₃ processing of

11.1 mg (crude mass) of non-azide modified LPEI (α-methyl-ω-hydroxy-poly(iminoethylene), CH ₃ (NC ₂ H ₅ ) _n -OH, 21 kDa, Chemcon, Product number 9002-98-6) was weighed in a 1.5 mL Eppendorf tube and dissolved in 400 μL of 50 mM acetate, pH 4.0. 26 μL of 6 M HCl was added to help dissolve and the pH was adjusted to 4. The concentration measured by copper assay was 25.7 mg/mL (1.22 mM pure product). 400 μL of LPEI solution (0.49 μmol, 1.0 equiv) was transferred to a 1.5 mL Eppendorf tube and 29 μL of DBCO-PEG ₂₃ -OCH ₃ (Compound 18) solution (0.60 μmol, 1.3 equiv) was added to the reaction mixture. . The solution was incubated at 40°C for approximately 67 hours and monitored for product formation using analytical RP-HPLC. No product was evident at pH 4.

No reaction was observed over 18 hours at room temperature using analytical RP-HPLC monitoring. At higher pH 5, evidence of the product was observed by analytical RP-HPLC, which characterized the hydroamination reaction products from the coupling of activated alkynes with LPEI polyamines (F. Pohlki & S. Doye The catalytic hydroamination of alkynes, Soc. Rev., 32: 104-114 (2003).

Example 7

LPEI- l -[N ₃ :MAL]-PEG _2K -Synthesis of DUPA (Compound 19)

LPEI- l -[N ₃ :MAL]-PEG _2K -DUPA (Compound 19) is a polydisperse fragment with a molecular weight of about 2,000, and is synthesized in two steps according to the following reaction formula. In the first step, DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12; SEQ ID NO: 4) (see Example 4) is combined with half the equivalent of MAL-PEG _2K -MAL (Compound 20) MAL-PEG _2K -DUPA (Compound 21) was prepared. In the second step, MAL-PEG _2K -DUPA (compound 21) was reacted with LPEI-N ₃ according to the procedure taught by Zhu et al ., Macromol. Res., 24: 793-799 (2016). The 1,3-dipolar cycloaddition reaction is undertaken to produce LPEI- l -[N ₃ :MAL]-PEG _2K -DUPA (compound 19).

Step 1: MAL-PEG _2K -Synthesis of DUPA (Compound 21)

DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12; SEQ ID NO: 4) was combined with 0.5 equivalents of MAL-PEG _2K -MAL (Compound 20) according to the procedure of Example 4 to produce MAL-PEG. Prepare _2K -DUPA (Compound 21).

Stage 2: LPEI- l -[N ₃ :MAL]-PEG _2K -Synthesis of DUPA (Compound 19)

MAL-PEG _2K -DUPA (compound 21) was prepared as a 1,3-dipole with LPEI-N ₃ according to the procedure taught by Zhu et al ., Macromol. Res., 24: 793-799 (2016). The cycloaddition reaction is undertaken to produce LPEI- l -[N ₃ :MAL]-PEG _2K -DUPA (Compound 19).

Example 8

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of folate (compound 22a and compound 22b)

LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -folate was synthesized as a mixture of regioisomers 22a and 22b in a multi-step procedure according to the following reaction scheme. In the first step, folic acid (compound 24) was synthesized using a solid-phase synthesis approach similar to the method described by Atkinson et al ., J. Biol. Chem., 276(30): 27930-35 (2001). The gamma-Glu residue was functionalized with a cysteamine spacer. The resulting folate-thiol (compound 26) was coupled to dibenzoazacyclooctyne-24(ethylene glycol)-maleimide (DBCO-PEG ₂₄ -MAL; compound 11) by Michael addition. In the next step, DBCO-PEG ₂₄ -folate (compound 27) is added to LPEI-N ₃ by [2 + 3] cycloaddition reaction to produce LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -folate (compound 27). Compound 22a and compound 22b) were produced.

Step 1: Folic Acid Loading onto Solid Resin

20 mL of DMSO was heated to 50° C. in a 50 mL Erlenmeyer flask, and folic acid (Compound 24; 881.4 mg, 2.0 mmol, 5.0 equiv) was added slowly under magnetic stirring. Dry cysteamine 4-methoxytrityl resin (Compound 23; 397.3 mg, 0.4 mmol, 1.0 equiv, 1.01 mmol/g) was added to a 50 mL Erlenmeyer flask, the previously prepared folic acid solution was added to the resin, and then DIEA (1018 μL, 6.0 mmol, 15.0 equiv) and PyBOP (1084.0 mg, 2.0 mmol, 5.0 equiv) were added. The reaction mixture was stirred at room temperature for 4 h, then transferred to a glass column, filtered through a glass frit, and mixed with DMSO (7 × 10 mL), DMF (5 × 10 mL), DCM (5 × 10 mL), and MeOH (5 × 10 mL). × 10 mL). The presence of free amines was verified by a TNBSA (picrylsulfonic acid) color test on the sample resin.

Step 2: Cleavage of folate-thiol from resin

Add 10 mL of DCM/TFA/TIS (92/3/5 v/v/v) to the folate modified resin (compound 25) from Step 1 in a glass column and stir the mixture for 30 minutes with occasional stirring of the flask. It was left unattended. The resin was filtered and washed (10 mL DCM/TFA (95/5 v/v)), and the filtrate and wash were recovered and concentrated under reduced pressure. After concentration, the mixture was separated into two phases and the light phase was removed. The crude product was precipitated by adding 30 mL of cold diethyl ether and washed twice with diethyl ether. The crude product of folate-SH (compound 26) was dried under reduced pressure overnight and verified by mass spectrometry. The thiol content of crude compound 26 was determined by Ellman's test, which gave a positive result for free thiol. Mass spectrometry (ESI): C ₂₁ H ₂₄ N ₈ O ₅ S [MH] ^- 499.54, observed 499.2.

Step 3: DBCO-PEG ₂₄ -Synthesis of folate (compound 27)

Folate-thiol (Compound 26) (16.0 mg, 29.4 μmol, 1.7 equiv) from Step 2 was dissolved in 8 mL DMSO (2.0 mg/mL stock solution) in a round bottom flask. The solution was sonicated and compound 26 was completely dissolved and diluted with 72 mL of 20 mM HEPES (pH 7.4). DBCO-PEG ₂₄ -MAL (Compound 11; see Example 4) (29.1 mg, 17.5 μmol, 93.6% black, 1.0 equiv) was weighed in a 1.5 mL Eppendorf tube and added to 875 μL of DMSO (20 mM pure product stock solution). ) was dissolved in. Into an 80 mL round bottom flask containing folate-thiol (Compound 26) solution (29.4 μmol, 1.7 equiv), slowly add DBCO-PEG ₂₄ -MAL (Compound 11) stock solution (13 μmol, 1.0 equiv) under magnetic stirring. Added. The reaction mixture was kept at room temperature and protected from light for about 1 hour. DBCO-PEG ₂₄ -folate (compound 27) was purified by preparative chromatography using the Puriflash system and verified by mass spectrometry. Mass spectrometry (ESI): [M+3H] ³⁺ 2,056.32, observed 686.2.

Step 4: LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of folate (compound 22a and compound 22b)

LPEI-N ₃ stock (203.9 mg) was weighed in a 15 mL falcon tube and dissolved in 8 mL of 50 mM acetate buffer (pH 4.0). The solution was acidified, heated to 70°C, sonicated to completely dissolve the LPEI particles, and adjusted to pH 4.0 using a total of 340 μL of 6 M HCl. A copper assay was performed on the solution to determine the total LPEI content of the LPEI-N ₃ solution. The LPEI-N ₃ solution (8.3 mL, 6.7 μmol, 1.0 equiv) was transferred to a 50 mL falcon tube and mixed with 1.5 mL of DBCO-PEG ₂₄ -folate solution (Compound 27; 7 μmol, 1.0 equiv). The reaction mixture was degassed with argon and incubated on a thermal shaker for approximately 20 hours, protected from light.

Crude LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -folate was purified by preparative chromatography using the Puriflash system and isolated as a mixture of regioisomers 22a and 22b. Fraction pools were measured for total LPEI content using a copper assay and for folate content spectrophotometrically (360 nm, ε = 6,765 M ^-1 cm ^-1 ). Yield: LPEI content 19 mg (copper assay); LPEI/folate ratio 1:1.

Example 9

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of HER2-Apibody (Compound 28 and Compound 28b)

LPEI -l- [N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody was prepared in a Michael addition reaction to DBCO-PEG ₂₄ -MAL (Compound 11) using a commercially available cysteine terminally modified Apibody (Compound 29) , was synthesized as a mixture of regioisomers 28a and 28b using a procedure similar to that described for LPEI -l -[N ₃ :DBCO]-PEG ₂₄ -DUPA in Example 4. The resulting DBCO-PEG ₂₄ -HER2-Api body (compound 30) was coupled to LPEI-N3 through a [2 + 3] cycloaddition reaction to form LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Ae. Pice (Compound 28a and Compound 28b) were produced.

Step 1: DBCO-PEG ₂₄ -Synthesis of HER2

HER2 epibody (Compound 29; 4 mg, 0.29 μmol, MW = 14 kDa) was weighed in a 5 mL Eppendorf tube. To reduce potential disulfide bonds in the HER2 apibody, a 0.5 M DTT stock solution was prepared and HER2 apibody was added to a final HER2 apibody concentration of 20 mM. The reaction mixture was incubated at room temperature for approximately 5 hours. After reduction, DTT was removed with a Sephadex G-25 column using 20 mM HEPES (pH 7.4) as elution buffer. Approximately 3.6 mg of purified HER2 epibody was recovered after NAP purification. The yield after NAP purification was estimated to be 90%.

DBCO-PEG ₂₄ -MAL (Compound 11) stock solution was prepared by weighing 4.4 mg (crude mass) of Compound 11 in a 1.5 mL Eppendorf tube and adding 132 μL of DMSO to make a 20 mM stock solution. DBCO-PEG ₂₄ -MAL (Compound 11; 15 μL, 0.31 μmol, 1.2 eq) stock solution was added slowly to the purified HER2-Apibody solution (0.26 μmol, 1.0 eq). The reaction mixture was incubated on a Stuart rotator for approximately 2 hours at room temperature and the reaction was monitored by RP-C8-HPLC at 280 nm and 309 nm.

The reaction mixture was purified using an Amicon filter (10 kDa MWCO) to remove excess DBCO-PEG ₂₄ -MAL (Compound 11) from the DBCO-PEG ₂₄ -HER2-Apibody conjugate (Compound 30). Fourteen centrifugal filters (Amicon Ultra - 0.5 mL, Merck Millipore) were each filled with 429 μL of reaction mixture. They were centrifuged once at 14,000 g for 30 minutes to exchange buffer and remove residual DBCO-PEG ₂₄ -MAL (compound 11) and then run three times at 20°C in 50 mM acetate buffer, pH 4.0. A concentrated solution of DBCO-PEG ₂₄ -HER2-Apibody (Compound 30; 243 μL) was recovered after buffer exchange and supplemented with 1.0 mL of 50 mM acetate, pH 4.0. A total of ∼1.24 mL of purified DBCO-PEG ₂₄ -HER2-Apibody (Compound 30) solution was obtained after the NAP purification step. The purified solution was analyzed by RP-C ₈ -HPLC and spectrophotometry at 309 nm using a NanoDrop One C device, and a DBCO concentration of 118 μM was determined (~0.15 μmol).

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -Synthesis of HER2

LPEI-N ₃ (7.4 mg 0.34 μmol, based on LPEI 72% (Cu assay)) was weighed in a 15 mL falcon tube and dissolved in 0.4 mL of 50 mM acetate buffer (pH 4.0). The solution was acidified, heated to 70°C, sonicated to completely dissolve the LPEI particles, adjusted to pH 4.0 using a total of 15 μL of 6 M HCl, and degassed with argon. LPEI-N ₃ (333 μL, 0.28 μmol, 2.0 equiv) from the stock solution was added slowly to the DBCO-PEG ₂₄ -HER2 (Compound 29) solution (0.14 μmol, 1 equiv). The reaction mixture was incubated on a Stuart rotator for approximately 72 hours. Additional LPEI-N ₃ (215 μL, 0.14 μmol, 1.0 equiv) from the stock solution was added to the reaction mixture, and the solution was incubated on a heat shaker at 35° C. for approximately 24 hours, and the 240 nm, 280 nm, and 309 nm was monitored by RP-C ₈ -HPLC with an ELSD detector. Prior to preparative chromatography, the acetonitrile percentage of the reaction mixture was adjusted to 10% (final volume) using 189 μL of ACN and 1% TFA (final volume) using 19 μL of TFA. Supplement the solution (~1.7 mL) with 1.0 mL of 90% v/v H ₂ O (0.1% TFA) / 10% v/v ACN (0.1% TFA) and inject the total sample volume into the Agilent Prep-HPLC system. did. The pool of fractions was lyophilized to yield 4 mg LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -HER2-Apimer as a mixture of regioisomers 28a and 28b (overall yield of LPEI = 6.9%; anti-HER2 Overall yield of blood = 14%; LPEI:DUPA ratio = 1/1.4).

Step 3: Preparation of HEPES salt form

Lyophilized LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody (Compounds 28a and 28b) was dissolved in 0.8 mL of 20 mM HEPES pH 7.2 in a 1.5 mL Eppendorf tube. The pH was adjusted to pH 7.2 using 5 M NaOH/1 M HCl. Two centrifugal filters (Amicon Ultra - 0.5 mL, Merck Millipoa) were each filtered with 400 μL of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody (Compound 28a and Compound 28b) solution. Charged. They were centrifuged once at 14,000 g for 30 minutes at 4°C to remove the buffer, and then processed three times in 20 mM HEPES, pH 7.2. A concentrated solution (~146 μL) of LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody HEPES salt was recovered after buffer exchange and supplemented with 170 μL 20 mM HEPES, pH 7.2. The copper assay was performed with the final HEPES salt solution (~0.3 mL) and a concentration of 1.7 mg/mL of total LPEI was determined.

Example 10

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of DUPA (Compound 31a and Compound 31b)

LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA was synthesized as a mixture of regioisomers 31a and 31b according to the following reaction scheme. In the first step, HOOC-PEG ₃₆ -NH ₂ (Compound 32) is coupled to N-succinimidyl3-maleimidepropionate (Compound 33) by amine formation to form HOOC-PEG ₃₆ -MAL (Compound 34). produced. In the next step, HOOC-PEG ₃₆ -MAL (compound 34) was coupled to DBCO-NH ₂ (compound 35) by amine formation to produce DBCO-PEG ₃₆ -MAL (compound 36). In the next step, DBCO-PEG ₃₆ -MAL (compound 36) was coupled to DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (compound 12; SEQ ID NO: 4) by Michael reaction to form DBCO-PEG ₃₆ - DUPA (Compound 37) was produced. In the next step, DBCO-PEG ₃₆ -DUPA (compound 37) is coupled to LPEI-N ₃ by [2 + 3] cycloaddition to produce LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA as regioisomer 31a. and 31b.

Step 1: HOOC-PEG ₃₆ -Synthesis of MAL (compound 34)

Stock solutions were prepared as follows: HOOC-PEG ₃₆ -NH ₂ (Compound 32) was weighed (364.4 mg, 218 μmol, 1.0 equiv) in a 50 mL falcon tube and 5.0 mL of DCM was added to make a 44 mM stock solution. was obtained. N-Succinimidyl 3-maleimidepropionate (Compound 33) was weighed (83.0 mg, 312 μmol) in a 5.0 mL Eppendorf tube and 3.0 mL of DCM was added to obtain a 104 mM stock solution.

In a falcon tube containing HOOC-PEG ₃₆ -NH ₂ , add DIEA (55.6 μL, 327 μmol, 1.5 equiv) and 2.308 mL (240 μmol, 1.1 equiv) of N-succinimidyl 3-maleimidepropionate stock solution. Added. The reaction mixture was incubated on a Stuart rotator (RT, 15 rpm, light blocked) and monitored by RP-C ₈ -HPLC. After 30 minutes, all HOOC-PEG ₃₆ -NH ₂ was reacted. After a total of 2 hours the reaction mixture (~7.3 mL) was purified by precipitation. 30 mL of n-hexane was added and the mixture was vortexed for a few seconds and centrifuged (10 minutes, 4,400 rpm). The yellow oil was recovered and dried overnight (25° C., 10 mbar). 458 mg (crude mass) of off-white material (crude HOOC-PEG ₃₆ -MAL; compound 34) was recovered and analyzed by RP-C8-HPLC; Analyzed by qTOF mass spectrometry (calculated monoisotopic mass: 1,825.02 Da; measured 1,825.02 Da).

Step 2: DBCO-PEG ₃₆ -Synthesis of MAL (compound 36)

A stock solution of HOOC-PEG ₃₆ -MAL was prepared by dissolving 458 mg (crude mass) of HOOC-PEG ₃₆ -MAL (Compound 34) in 4.0 mL DCM. For stoichiometric calculations, the crude mass was assumed to be pure HOOC-PEG ₃₆ -MAL (246 μmol, 1.0 equiv). A stock solution of DBCO-NH ₂ (Compound 35) was prepared by weighing 84.0 mg of DBCO-NH ₂ (246 μmol) in a 5.0 mL Eppendorf tube followed by adding 1.0 mL of DMF to obtain a 304 mM stock solution. A stock solution of HATU was prepared by weighing 82.5 mg of HATU (217 μmol) in a 5.0 mL Eppendorf tube. 1.0 mL of DMF was added to obtain a 217 mM stock solution.

To HOOC-PEG ₃₆ -MAL (Compound 34), 1.0 mL (221 μmol, 0.9 equiv) of HATU stock solution was added. The solution was stirred on a Stuart rotator for approximately 1 minute. DIEA (75 μL, 442 μmol, 2.0 eq) was added and the solution was stirred for approximately 3 minutes, followed by the addition of DBCO-NH ₂ (Compound 35; 728 μL, 221 μmol, 0.9 eq) stock solution. The reaction mixture was incubated on a Stuart rotator (15 rpm, RT, photoprotected) and monitored by RP-C ₈ -HPLC. After 1 hour of incubation, additional DBCO-NH ₂ solution (80 μL, 25 μmol, 0.1 equiv) was added to the reaction mixture to ensure complete consumption of HOOC-PEG ₃₆ -MAL. After 3 hours the reaction mixture (~5.9 mL) was purified by precipitation. n-Hexane (30 mL) was added to the reaction mixture, vortexed, and centrifuged (10 minutes, 4,400 rpm). The supernatant was discarded and 20 mL of cold diethyl ether was added. The precipitate was recovered and dried in a vacuum drying oven overnight (25°C, 10 mbar). DBCO-PEG ₃₆ -MAL (Compound 36) was recovered as a pale yellow solid (542 mg) and analyzed for purity by RP-C ₈ -HPLC and qTOF mass spectrometry (calculated monoisotopic mass: 2,083.13 Da) ; measured value 2,083.14 Da).

Step 3: DBCO-PEG ₃₆ -Synthesis of DUPA

DBCO-PEG ₃₆ -MAL (Compound 36) stock solution was prepared by weighing 548 mg in a 50 mL falcon tube and dissolving in 10 mL DMSO (26.3 mM stock solution). A stock solution of DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12; SEQ ID NO: 4) was prepared by weighing 318 mg in a 250 mL round bottom flask equipped with a magnetic stirrer. Acetate buffer (15 mM, 159 mL, pH 5.2) was added and the mixture was shaken for several minutes until compound 12 was completely dissolved. The solution was adjusted to pH 5.5 using 350 μL of 5 M NaOH. DBCO-PEG ₃₆ -MAL stock solution (10 mL, 263 μmol, 1.0 eq) was added slowly to the compound 12 solution (265 μmol, 1.0 eq), and the reaction mixture was stirred with protection from light. The reaction was monitored by RP-C8 HPLC. After 1 hour, excess compound 12 was removed by TFF (2 kDa MWCO membrane). The solution (~169 mL) was ultracentrifuged using TFF corresponding to 15 mM acetate buffer (pH 4.8). The recovered solution (~55 mL) was lyophilized using a lyophilizer for approximately 48 hours, and the lyophilisate was analyzed by RP-C8-HPLC. Residual impurities were removed by precipitation. 500 mg of lyophilized material was dissolved in 6 mL DMF in a 50 mL falcon tube. To the slightly cloudy solution, cold diethyl ether (30 mL) was added to form a precipitate, which was collected, washed with cold diethyl ether (30 mL), and dried in a vacuum oven overnight (25°C; 10 mbar) at 270 °C. mg of DBCO-PEG ₃₆ -DUPA (Compound 37) was obtained. qTOF mass spectrometry (calculated monoisotopic mass: 3,280.60 Da; measured: 3,280.64 Da)

Step 4: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of DUPA

1013 mg (crude mass) of LPEI-N ₃ was weighed into a 50 mL falcon tube and dissolved in 35.0 mL of 50 mM acetate buffer, pH 4.0. The solution was acidified and sonicated for 10 minutes to completely dissolve the LPEI-N ₃ and adjusted to a final pH of 4.0. A total LPEI amine concentration of 22.1 mg/mL (1.0 mM) was determined by copper assay (corresponding to an LPEI-N ₃ content of 82% of the crude mass). A stock solution of DBCO-PEG ₃₆ -DUPA (Compound 37) was prepared by dissolving 219 mg of DBCO-PEG ₃₆ -DUPA in a 50 mL Falcon tube with 20.0 mL of 50 mM acetate buffer. The solution was adjusted to pH 4.0 by adding 1 M HCl. The concentration of DBCO was determined spectrophotometrically at 309 nm using Nanodrop One C and was measured to be 2.0 mM. DBCO-PEG ₃₆ -DUPA solution (~21 mL, 40 μmol) was slowly added to a magnetically stirred solution of LPEI solution (37 mL, 38 μmol, 1.0 equiv). The mixture was stirred at room temperature for 72 hours and protected from light. The reaction mixture (~60 mL) was supplemented with acetonitrile (final volume of 10% ACN) and TFA (final volume of 1% TFA). The solution became cloudy, but became clear after adjusting the pH to 3.5 using 5 M NaOH. Purification was performed by preparative RP-C ₁₈ -HPLC. A pool of fractions of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA was recovered as a mixture of regioisomers 31a and 31b. Fractions were lyophilized to give 830 mg lyophilisate as the TFA salt, 34% LPEI weight content by Cu assay. Pools of fractions containing purified product were analyzed by RP-HPLC, copper assay and UV spectrophotometry at 280 nm. An LPEI:DUPA molar ratio of 1:1 was determined.

Step 5: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Preparation of DUPA (Compound 31a and Compound 31b) HEPES salts

To exchange TFA for HEPES, 421 mg (crude mass) of lyophilized LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA-TFA salt (w _LPEI = 34%, ~143 mg total LPEI) was dissolved in 30 mL of 20 mM HEPES, pH 7.2, in a 50 mL falcon tube. The pH was adjusted to 6.0 using 11 μL of 5 M NaOH and 7 μL of 6 M HCl. TFF was performed on 20 mM HEPES, pH 7.2, at a total dilution of 10,757 times. Approximately 45 mL of LPEI -l- [N3:DBCO]-PEG ₃₆ -DUPA The HEPES salt solution was recovered after TFF. Copper assay and RP-C8-HPLC were performed on the final LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA (Compound 31a and 31b) HEPES salt solution (~45 mL) to obtain a total LPEI concentration of 2.7 mg/mL. (LPEI/DUPA ratio = 1/1.1) was measured. The recovery yield after TFF was calculated to be 85% based on the total LPEI content.

Step 6: LPEI- l -[N3:DBCO]-PEG ₃₆ -Preparation of DUPA (Compound 31a and Compound 31b) acetate salt

Lyophilized LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA-TFA salt (4.9 mg, w _LPEI = 34%, ~1.7 mg total LPEI) was dissolved in 0.8 mL of 50 mM acetate in a 1.5 mL Eppendorf tube. , dissolved in pH 4.3. The pH was adjusted to 4.5 using 3.0 μL of 5 M NaOH. Two centrifugal filters (Amicon Ultra - 0.5 mL, 3 kDa MWCO) were each filled with 400 μL of LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA-TFA salt solution. They were centrifuged once at 14,000 g for 30 minutes to remove the buffer, and then three times at 4°C in 400 μL of 50 mM acetate, pH 4.3. A concentrated solution (177 μL) of LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA-acetate salt was recovered after buffer exchange and supplemented with 0.45 mL of 50 mM acetate, pH 4.3. Copper assay and analytical RP-C ₈ -HPLC were performed on LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA (Compound 31a and 31b) acetate salt solution (~0.6 mL), total LPEI 2.0 The mg/mL concentration was determined.

Example 11

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA (Compound 38a and Compound 38b) Synthesis

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA was synthesized as a mixture of regioisomers 38a and 38b according to the following reaction scheme. In the first step, HOOC-PEG ₃₆ -NH ₂ (compound 32) is condensed with Mal-L-Dap(Boc)-OH (compound 39) to give HOOC-PEG ₃₆ -(Boc)-MAL (compound 40) did. Compound 40 was subsequently condensed with DBCO-NH ₂ (compound 35) and deprotected to give DBCO-PEG ₃₆ -(NH ₂ )-MAL (compound 41). Compound 41 was reacted with DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (compound 12) through Michael addition and cyclized with LPEI-N ₃ to produce compounds 38a and 38b.

Step 1: HOOC-PEG ₃₆ Synthesis of -(Boc)-MAL (Compound 40)

Solution Mal-L-Dap(Boc)-OH (N-α-maleimido-N-β-t-butyloxycarbonyl-L-2,3-diaminopropionic acid DCHA salt; Compound 39) in DCM (0.17 mL). ; 50 μmol, 1.1 equiv, 294 mM) was mixed with a solution of HATU (45 μmol, 0.9 equiv, 217 mM) in DMF (0.207 mL). 17 μL of DIEA (100 μmol, 2.0 equivalent) was added to the resulting mixture. Finally, HOOC-PEG ₃₆ -NH ₂ (Compound 32, 50 μmol, 1.0 equiv, 248 mM) was added as a solution in DCM (0.20 mL). The reaction mixture was incubated at room temperature on a Stuart rotator and the reaction was monitored by RP-C ₈ -HPLC. After 1.5 hours, an additional 0.2 equivalents of Mal-L-Dap(Boc)-OH were added. After an additional hour and a half, 5.0 mL of n-hexane was added to induce precipitation and the reaction mixture was centrifuged. The precipitate was washed with 4.5 mL of cold diethyl ether. A solid (77 mg) containing crude HOOC-PEG ₃₆ -(Boc)-MAL (Compound 40) was recovered and analyzed by HPLC - ESI ⁺ qTOF mass spectrometry (calculated monoisotopic mass: 1,940.08 Da; Measured value: 1,940.10 Da). Crude compound 40 was used in the next step without further purification.

Step 2: DBCO-PEG ₃₆ -(NH ₂ )-Synthesis of MAL (compound 41)

Add HATU (35 μmol, 0.9 equiv, 208 mM) in DMF (169 μL) to solution HOOC-PEG _36- (Boc)-MAL (Compound 40; 39 μmol, 1.0 equiv, 98 mM) in DCM (400 mL). did. The solution was mixed on a Stuart rotator for 1 min, followed by the addition of DIEA (13 μL, 78 μmol, 2.0 eq.) and solution DBCO-NH ₂ (compound 35; 20 μmol, 0.5 eq., 370 mM) in DMF (53 μL). did. The reaction mixture was incubated with Ceylon on a Stuart rotator and monitored by RP-C ₈ -HPLC. At 20 minutes of reaction, an additional amount of DBCO-NH ₂ (8 μmol, 0.2 equiv) in DMF (22 μL) was added. After a total of 45 minutes, 4.5 mL of cold diethyl ether was added. The precipitate was further purified using 4.5 mL of cold diethyl ether. Crude DBCO-PEG ₃₆ -(Boc)-MAL was isolated as a yellow solid (92 mg) and analyzed by HPLC - ESI ⁺ qTOF MS (calculated monoisotopic mass: 2,198.20 Da; measured: 2,198.20 Da) Dissolved in 2.7 mL DCM and 40 μL TFA without further purification.

Deprotection of the Boc group of DBCO-PEG ₃₆ -(Boc)-MAL was monitored by RP-C ₈ -HPLC. Upon completion, n-hexane (2.5 mL) was added and the precipitate was washed with 4.5 mL of cold diethyl ether. The recovered solid material (DBCO-PEG ₃₆ -(NH ₂ )-MAL; Compound 41) was analyzed by HPLC - ESI ⁺ qTOF mass spectrometry (calculated monoisotopic mass: 2,098.14 Da; measured: 2,098.14 Da) .

Step 3: DBCO-PEG ₃₆ -[(NH ₂ )Synthesis of MAL-S]-DUPA (Compound 42)

Solution DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12; SEQ ID NO: 4) (20 μmol, 0.5 eq., 142 mM) in DMF (141 μL) (59 μmol, 3.0 eq.) was added to 400 μL of DBCO-PEG ₃₆ -(NH ₂ )-MAL (Compound 41; 39 μmol, 1.0 equiv. 98 mM) solution and 10 μL of DIEA (59 μmol, 3.0 equiv.). The reaction mixture was incubated at room temperature on a Stuart rotator and monitored by RP-C8-HPLC. After 1 hour, cold diethyl ether (4.5 mL) was added and the product precipitated. The precipitate was washed with 4.5 mL of cold diethyl ether, dissolved in 1.0 mL DMSO, and supplemented with a mixture of 1% TFA/H ₂ O : 1% TFA ACN (14 mL, 9 : 1 v/v). The solution was made transparent by adjusting the pH to 6.0. The DBCO-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA (Compound 42) solution was purified using RP-C ₁₈ preparative HPLC, and the fraction pool was lyophilized. The lyophilisate was analyzed by RP-HPLC-ELSD and RP-HPLC - ESI ⁺ qTOF mass spectrometry (DBCO-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA calculated monoisotopic mass: 3,313.64 Da ( maleimide ring opening); measured: 3313.66 Da).

Step 4: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA (Compound 38a and Compound 38b) Synthesis

LPEI-N ₃ solution (2.3 mL, 2.3 μmol, 1.5 equiv, 1.0 mM) in 50 mM acetate buffer pH 4.0 was mixed with 4.0 mL of solution DBCO-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA (Compound 42; 1.5 μmol, 1.0 equivalent, 0.37 mM) was added slowly. After 70 hours, the reaction mixture was supplemented with 0.78 mL acetonitrile and 78 μL TFA. LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA was isolated by RP-C ₁₈ preparative HPLC as a mixture of regioisomers 38a and 38b. The pool of fractions was lyophilized to give 38 mg of a fluffy white solid and characterized by RP-C ₈ -HPLC, copper assay and spectrophotometry at 280 nm for determination of UPA content. The lyophilisate showed a 32% weight percentage of LPEI and an LPEI to DUPA ratio of 1:1.1.

Step 5: LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA (Compound 38a and Compound 38b) Preparation of HEPES salt

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA (Compound 38a and 38b) TFA salt (21.9 mg, w _LPEI = 32%, total LPEI 7.0 mg) Dissolved in 1.2 mL of 20 mM HEPES pH 7.5. Separate three centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) each with 400 μL of LPEI -l -[N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )-MAL-S]-DUPA solution. Charged and centrifuged once at 14,000 g for 30 minutes, followed by three additions of 400 μL of 20 mM HEPES, pH 7.2. Approximately 261 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA-HEPES salt solution was recovered and supplemented with 2.4 mL of 20 mM HEPES, pH 7.2. The concentration of the solution was determined to be 2.2 mg/mL total LPEI by copper assay.

Example 12

LPEI- l -[N ₃ :BCN]-PEG ₃₆ -Synthesis of DUPA (Compound 43)

LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA (Compound 43) was synthesized according to the following reaction scheme. Endo -BCN-PEG ₃₆ -MAL (Compound 45) was prepared by condensing HOOC-PEG ₃₆ -MAL (Compound 34) with Endo -BCN-PEG ₂ -NH ₂ (Compound 44). In the next step, compound 45 was condensed with compound 12, and the resulting endo -BCN-PEG ₃₆ -DUPA (compound 46) was reacted with LPEI-N ₃ to provide compound 43.

Step 1: Endo-BCN-PEG ₃₆ -Synthesis of MAL (compound 45)

A solution (165 μL) of HATU (20 μmol, 0.9 equiv, 123 mM) in DCM (400 μL) and DIEA (7.7 μL, 45 μmol, 2.0 eq) was added to HOOC-PEG _36- MAL (Compound 34; see Example 10; 23 μmol, 1.0 equivalent, 58 mM) was added to the solution. Endo -BCN-PEG ₂ -NH ₂ (Compound 44; 18 μmol, 0.8 equiv, 145 mM) was added to the reaction mixture as a solution (124 μL) in DCM and monitored by RP-C ₈ -HPLC. Additional amounts of endo -BCN-PEG ₂ -NH ₂ (2×0.2 equivalents) were added at 10 min intervals. After an additional hour, n-hexane (4.5 mL) was added to the reaction mixture. The resulting precipitate was separated by centrifugation, washed with 4.5 mL of cold diethyl ether and dried under vacuum. Crude endo-BCN-PEG ₃₆ -MAL (compound 45; 61 mg) was isolated and analyzed by RP-C ₈ -HPLC coupled to ESI ⁺ -qTOF mass spectrometry (calculated monoisotopic mass: 2,131.21 Da) ; Measured value: 2,131.22 Da), used in the next step without further purification.

Step 2: Endo -BCN-PEG ₃₆ -Synthesis of DUPA (Compound 46)

Solution DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12; SEQ ID NO:4) (21 μmol, 1.1 equiv) in DMF (239 μL) was reacted with Endo -BCN-PEG ₃₆ -MAL (Compound 45; 400 μmol, 1.0 eq., 48 mM) and DIEA (7 μL, 42 μmol, 2.0 eq.). After 1 hour, cold diethyl ether (4.5 mL) was added. The precipitated solid was filtered, washed with cold diethyl ether, and dried to give 70 mg of endo -BCN-PEG ₃₆ -DUPA (Compound 46). Samples were analyzed by HPLC ESI ⁺ qTOF mass spectrometry ( endo -BCN-PEG ₃₆ -DUPA: calculated monoisotopic mass: 3,328.69 Da; measured: 3,328.72 Da).

Step 3: LPEI- l -[N ₃ :BCN]-PEG ₃₆ -Synthesis of DUPA (Compound 43)

Endo- BCN-PEG ₃₆ -DUPA (Compound 46; 3.8 μmol, 1.5 mM, 1.0 equiv) in acetate buffer (50 mM, 2.5 mL, pH 4.0) was solution in acetate buffer (50 mM, 4.2 mL, pH 4.0). It was slowly added to LPEI-N ₃ (4.1 μmol, 1.1 equivalent, 22 mg/mL). The mixture was shaken on a Stuart rotator at room temperature for approximately 70 hours and protected from light. To the reaction mixture was added 3.0 mL of 50 mM acetate buffer, pH 4.0, followed by acetonitrile (1.0 mL) and TFA (100 μL). The resulting mixture was filtered (0.45 μm PA membrane) and purified using RP-C ₁₈ preparative chromatography. A pool of fractions containing LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA (compound 43) was lyophilized to give 61 mg of lyophilized product, which was analyzed using analytical RP-C ₈ HPLC, copper assay, and Characterized by spectrophotometry at 280 nm for determination of DUPA content. The product had a 31% weight percentage of LPEI as determined by the Cu assay.

Step 4: LPEI- l -[N ₃ :BCN]-PEG ₃₆ -Preparation of DUPA (Compound 43) HEPES salt

24.8 mg of LPEI -l -[N ₃ :BCN]-PEG ₃₆ -DUPA (Compound 43) TFA salt (w _LPEI = 31%, ~7.7 mg total LPEI) was dissolved in 1.2 mL of 20 mM HEPES, pH 7.2. . The solution was adjusted to pH 7.3. Three centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI -l -[N ₃ :BCN]-PEG ₃₆ -DUPA solution. They were centrifuged once at 14,000 g for 30 minutes and then three times at 20°C after addition of 400 μL of 20 mM HEPES, pH 7.2. Approximately 263 μL of a concentrated solution of LPEI -l -[N ₃ :BCN]-PEG ₃₆ -DUPA HEPES salt was recovered after buffer exchange and supplemented with 2.4 mL of 20 mM HEPES, pH 7.2. The concentration of the solution was determined to be 2.3 mg/mL total LPEI by copper assay.

Step 5: LPEI -l -[N ₃ :BCN]-PEG ₃₆ -Preparation of DUPA (Compound 43) acetate salt

5.5 mg of LPEI -l -[N ₃ :BCN]-PEG ₃₆ -DUPA (Compound 43) TFA salt (w _LPEI = 31%, ~1.7 mg total LPEI) was dissolved in 0.8 mL of 50 mM acetate, pH 4.0. . Two centrifugal filters (Amicon Ultra - 0.5 mL, 3 kDa MWCO) were each filled with 400 μL of LPEI -l -[N ₃ :BCN]-PEG ₃₆ -DUPA solution. These were centrifuged once at 14,000 g for 30 minutes, followed by three additions of 400 μL of 50 mM acetate, pH 4.3. Approximately 144 μL of LPEI -l -[N ₃ :BCN]-PEG ₃₆ -DUPA acetate salt solution was recovered and supplemented with 0.6 mL of 50 mM acetate, pH 4.3. The concentration of the solution was determined to be 2.2 mg/mL total LPEI by copper assay.

Example 13

LPEI- l -[N ₃ :SCO]-PEG ₃₆ -Synthesis of DUPA (Compound 47a and Compound 47b)

LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA was synthesized as a mixture of regioisomers 47a and 47b according to the following reaction scheme. SCO-PEG ₃₆ -MAL (Compound 49) was prepared by condensing HOOC-PEG ₃₆ -MAL (Compound 34) with SCO-PEG ₃ -NH ₂ (Compound 48). Compound 49 was reacted with compound 12 through Michael addition, and the resulting SCO-PEG ₃₆ -DUPA (compound 50) was reacted with LPEI-N ₃ to synthesize compounds 47a and 47b.

Step 1: SCO-PEG ₃₆ -Synthesis of MAL (compound 49)

Solution HATU (25 μmol, 0.9 equiv, 147 mM) in DMF (69 μL) was added to HOOC-PEG ₃₆ -MAL (compound 34; 28 μmol, 1.0 equiv, 70 mM) in DCM, followed by DIEA (9.6 μL, 56 μmol, 2.0 equivalent) was added. To the reaction mixture was added solution SCO-PEG ₃ -NH ₂ (Compound 48; 22 μmol, 0.8 equiv, 137 mM) in DCM (166 μL). The reaction was placed on a Stuart rotator and reaction progress was monitored by RP-C ₈ -HPLC. After 10 minutes, HATU (0.1 equiv) and two additional lots of SCO-PEG ₃ -NH ₂ (0.2 equiv and 0.1 equiv) were added to the reaction mixture. After a total of 1.5 hours, 4.5 mL of n-hexane was added. The precipitated solid was washed with 4.5 mL of cold diethyl ether and dried. SCO-PEG ₃₆ -MAL (Compound 49) was isolated as a yellow solid (69 mg) and characterized by analytical RP-C ₈ -HPLC and ESI ⁺ qTOF mass spectrometry (calculated monoisotopic mass: 2,149.2 Da) ; measured value: 2,149.2 Da).

Step 2: SCO-PEG ₃₆ -Synthesis of DUPA (Compound 50)

Solution DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (Compound 12; SEQ ID NO: 4) (15 μmol, 0.5 eq., 100 eq.) in DMF (150 μL) and DIEA (10 μL, 62 μmol, 2.0 eq.) mM) was added to the solution SCO-PEG ₃₆ -MAL (Compound 49; 31 μmol, 1 equiv, 78 mM) in DMF. The reaction mixture was placed on a Stuart rotator. After 10 minutes, an additional amount of compound 12 (30 μL, 3 μmol, 0.1 equiv) was added. After 1 hour, cold diethyl ether was added, and the resulting precipitate was washed with 4.5 mL of cold diethyl ether and dried. Solid (98 mg) was resuspended in 0.5 mL DMSO and diluted with 7.5 mL H ₂ O (+ 1% TFA)/ACN (+ 1% TFA) (9:1 v/v) to prepare Prep-pRP-C _18. -Purified by HPLC. A pool of fractions of SCO-PEG ₃₆ -DUPA (Compound 50) was lyophilized and analyzed by HPLC-ESI ⁺ qTOF mass spectrometry (SCO-PEG ₃₆ -DUPA calculated monoisotopic mass: 3,346.70 Da; measured: 3,346.71 Da).

Step 3: LPEI- l -[N ₃ :SCO]-PEG ₃₆ -Synthesis of DUPA (Compound 47a and Compound 47b)

LPEI-N ₃ solution (4.2 mL, 5 μmol, 1.0 equiv) in 50 mM acetate buffer, pH 4.0 was mixed with 5.0 mL of solution SCO-PEG ₃₆ -DUPA (Compound 50) (5 μmol, 1.0 equivalent (1 mM) was added. The mixture was incubated on a Stewart rotator at room temperature for approximately 90 hours and photoprotected. For preparative RP-C ₁₈ HPLC purification, acetonitrile (1 mL) and TFA (100 μL) were added to the reaction mixture. The pool of fractions was lyophilized to provide 66 mg of LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA as a mixture of regioisomers 47a and 47b. The lyophilized solid was characterized by analytical RP-C ₈ HPLC, copper assay and spectrophotometry at 280 nm. The 32% weight percentage of LPEI was determined by copper assay on lyophilized solids.

Step 4: LPEI- l -[N ₃ :SCO]-PEG ₃₆ -Preparation of DUPA (compounds 47a and 47b) HEPES salts

23.2 mg of LPEI -l -[N ₃ :SCO]-PEG ₃₆ -DUPA (Compound 47a and Compound 47b) TFA salt (w _LPEI = 26%, total LPEI 6.0 mg) was dissolved in 1.2 mL of 20 mM HEPES, pH 7.4. dissolved. Three centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI -l -[N ₃ :SCO]-PEG ₃₆ -DUPA solution and centrifuged at 14,000 g for 30 min. This was performed three times after adding 400 μL of 20 mM HEPES, pH 7.2. Approximately 276 μL of LPEI -l -[N ₃ :SCO]-PEG ₃₆ -DUPA HEPES salt solution was recovered and supplemented with 2.4 mL of 20 mM HEPES, pH 7.2. The concentration of the solution was determined to be 2.1 mg/mL total LPEI by copper assay.

Example 14

LPEI- l -[N ₃ :DBCO]CONH-PEG ₃₆ -Synthesis of DUPA (Compound 51a and Compound 51b)

LPEI- l -[N ₃ :DBCO]CONH-PEG ₃₆ -DUPA was synthesized as a mixture of regioisomers 51a and 51b according to the following reaction scheme. DBCO-PEG ₃₆ -[CONH]-MAL (Compound 54) was prepared by condensing DBCO-PEG36-TFP (Compound 52) with NH2-MAL (Compound 53). The resulting DBCO-PEG ₃₆ -[CONH]-MAL (compound 54) was condensed with compound 12 and reacted with LPEI-N ₃ to provide compounds 51a and 51b.

Step 1: DBCO-PEG ₃₆ Synthesis of -[CONH]-MAL (Compound 54)

Solution DBCO-PEG ₃₆ -TFP (compound 52; 24 μmol, 1.0 eq, 60 mM) in DCM (0.40 mL) was dissolved in solution NH ₂ -MAL (compound 53; 26 μmol, 1.1 eq 480 mM) in DMF (55 μL). and DIEA (8 μL, 48 μmol, 2.0 equiv). The reaction mixture was incubated at room temperature on a Stuart rotator and the reaction was monitored by RP-C ₈ -HPLC. After 2 hours, n-hexane (4.5 mL) was added to precipitate the product. The precipitate was washed with 4.5 mL of cold diethyl ether. The recovered material was analyzed by RP-HPLC - ESI ⁺ qTOF mass spectrometry. The solid contained DBCO-PEG ₃₆ -[CONH]-MAL (compound 54; calculated monoisotopic mass: 2,097.15 Da; measured: 2,097.16 Da).

Step 2: DBCO-PEG ₃₆ Synthesis of -[CONH]-DUPA (Compound 55)

Solution DBCO-PEG _36- [CONH]-MAL (compound 54; 24 μmol, 1.0 equiv, 120 mM) in DMF (0.20 mL) was dissolved in DUPA-Aoc-Phe-Gly-Trp-Trp-Gly in DMF (137 μL). -Cys (Compound 12; SEQ ID NO: 4) (17 μmol, 0.7 equivalent 123 mM). The reaction mixture was incubated at room temperature on a Stuart rotator and protected from light. After 15 minutes, an additional amount of DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (39 μL, 5 μmol, 0.2 equiv) was added. At 40 minutes of reaction, an additional amount of DUPA-Aoc-Phe-Gly-Trp-Trp-Gly-Cys (14 μL, 1.7 μmol, 0.07 equiv) was added. After an additional hour of mixing, cold diethyl ether (4.5 mL) was added. The precipitate was washed with cold diethyl ether (4.5 mL). The precipitate was dissolved in DMSO (0.5 mL) and supplemented with H ₂ O (6.75 mL) and acetonitrile (0.75 mL). DBCO-PEG ₃₆ -[CONH]-DUPA (Compound 55) was isolated following RP-C ₁₈ preparative HPLC and lyophilization of the fraction pool. The lyophilisate was analyzed by RP-HPLC-ELSD and RP-HPLC - ESI ⁺ qTOF mass spectrometry (solid DBCO-PEG ₃₆ -[CONH]-DUPA (Compound 55; 36 mg), calculated monoisotopic mass: 3,294.64 Da; measured value: 3,294.65 Da).

Step 3: LPEI- l -[N ₃ :DBCO]CONH-PEG ₃₆ -Synthesis of DUPA (Compound 51a and Compound 51b)

Solution LPEI-N ₃ (4.2 mL, 5 μmol, 1.0 eq., 1.2 mM) was mixed with 2.4 mL of solution DBCO-PEG ₃₆ -[CONH]-DUPA (Compound 55; 5 μmol, 1.0 eq.) in 50 mM acetate buffer, pH 4.0. 2.0 mM) was added slowly. The mixture was incubated at room temperature on a Stuart rotator and monitored by RP-C ₈ -HPLC. After 70 hours, the reaction mixture was supplemented with acetonitrile (0.73 mL) and TFA (74 μL) and isolated using RP-C ₁₈ preparative HPLC. The pool of fractions was lyophilized to yield LPEI -l -[N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA (87 mg) as a mixture of regioisomers 51a and 51b as a fluffy white solid. The lyophilisate was characterized by RP-C ₈ -HPLC, copper assay and spectrophotometry at 280 nm for determination of DUPA content. The lyophilisate showed a 30% weight percentage of LPEI and an LPEI to DUPA ratio of 1:1.1.

Step 4: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ Preparation of -[CONH]-DUPA (Compound 51a and Compound 51b) HEPES salt

LPEI- l- [N ₃ :DBCO]-PEG _36- [CONH]-DUPA (Compound 51a and Compound 51b) TFA salt (20.8 mg, w _LPEI = 30%, 6.2 mg total LPEI) in 1.2 mL of 20 mM Dissolved in HEPES, pH 7.2. Three centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA solution and 1,4000 g Centrifugation was performed once for 30 minutes, and then 400 μL of 20 mM HEPES, pH 7.2, was added three times. Approximately 246 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA-HEPES salt solution was recovered and supplemented with 2.4 mL of 20 mM HEPES, pH 7.2. The concentration of the solution was determined to be 2.1 mg/mL total LPEI by copper assay.

Example 15

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of [S-MAL]-DUPA (Compound 56a and Compound 56b)

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA was prepared as a mixture of regioisomers 56a and 56b according to the following reaction scheme. DBCO-PEG ₃₆ -SH (Compound 59) was prepared by condensing DBCO-NH ₂ (Compound 35) with NHS-PEG ₃₆ -OPSS (Compound 57) and subsequent reduction. Next, compound 59 was condensed with DUPA-MAL (compound 60) and reacted with LPEI-N ₃ to provide compound 56a and compound 56b.

Step 1: DBCO-PEG ₃₆ -Synthesis of SPDP (Compound 57)

Solution NHS-PEG ₃₆ -OPSS (compound 57; 49 μmol, 1.0 eq, 123 mM) in DCM (0.40 mL) was dissolved in DBCO-NH ₂ (compound 35; 54 μmol, 1.1 eq, 357 mM) in DMF (151 μL). and DIEA (17 μL, 100 μmol, 2.0 equiv). The reaction mixture was incubated at room temperature on a Stuart rotator and the reaction was monitored by RP-C ₈ -HPLC. After 15 minutes, an additional amount of DBCO-NH ₂ (5 μmol, 0.1 equiv, 357 mM) was added. After a total of 30 minutes, 4.5 mL of n-hexane was added. The resulting precipitate was filtered, centrifuged, and washed with 4.5 mL of cold diethyl ether. Solid DBCO-PEG ₃₆ -OPSS (Compound 58) was analyzed by HPLC - ESI+ qTOF mass spectrometry (calculated monoisotopic mass: 2,129.10 Da; measured: 2,129.12 Da) and used without further purification in the next step.

Step 2: DBCO-PEG ₃₆ Synthesis of -SH (Compound 59)

Solution DBCO-PEG ₃₆ -OPSS (compound 58; 4.8 μmol, 1.0 equiv, 12 mM, assuming 100% purity) in DMSO (0.40 mL) was dissolved in solution TCEP (5.8 μmol, 1.2 equivalents (127 mM) were mixed. The reaction mixture was incubated at room temperature on a Stuart rotator and the reaction was monitored by RP-C ₈ -HPLC. The reaction mixture containing DBCO-PEG ₃₆ -SH (compound 59) was used in the next step without further purification.

Step 3: DBCO-PEG ₃₆ -Synthesis of [S-MAL]-DUPA (Compound 61)

Solution DUPA-MAL (Compound 60; 4.0 μmol, 1.0 eq 2.5 mM) in 20 mM HEPES pH 7.4 (1.6 mL) was added to DBCO-PEG ₃₆ -SH (Compound 59; 364 μL, 4.0 μmol, 1.0 eq) prepared in step 2. ) was added to the solution, and the reaction mixture was incubated at room temperature on a Stuart rotator and monitored by RP-C ₈ -HPLC. After 15 minutes, an additional amount of DUPA-MAL (320 μL, 0.3 μmol, 0.1 equiv) was added. After a total of 30 minutes, DBCO-PEG ₃₆ -[S-MAL]-DUPA (compound 61) was isolated following preparative RP-C ₁₈ HPLC and lyophilization of a pool of fractions. Freeze-drying was analyzed by RP-HPLC-ELSD and RP-HPLC - ESI ⁺ qTOF mass spectrometry (DBCO-PEG ₃₆ -[S-MAL]-DUPA (7 mg), calculated monoisotopic mass: 3,236.62 Da; Measured value: 3,236.65 Da).

Step 4: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of [S-MAL]-DUPA (Compound 56a and Compound 56b)

LPEI-N ₃ solution (2.5 mL, 2.0 μmol, 1.0 eq) in 50 mM acetate buffer, pH 4.0 was mixed with 4.0 mL of solution DBCO-PEG _36- [S-MAL]-DUPA (compound) in 50 mM acetate buffer, pH 4.0. 61; 2.5 μmol, 1.2 equivalent, 1 mM). The mixture was incubated at room temperature on a Stuart rotator and protected from light. After 20 hours, the reaction mixture was supplemented with acetonitrile (0.78 mL) and TFA (79 μL). LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA was isolated as a mixture of regioisomers 56a and 56b using RP-C ₁₈ preparative HPLC and analytical RP-C ₈ -HPLC. , copper assay and spectrophotometry at 280 nm for determination of DUPA content. The lyophilisate showed a weight percentage of LPEI of 28% and an LPEI to DUPA ratio of 1:1.08.

Step 5: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ Preparation of -[S-MAL]-DUPA (Compound 56a and Compound 56b) HEPES salt

LPEI- l- [ _N3 :DBCO]-PEG _36- [S-MAL]-DUPA (Compound 56a and Compound 56b) TFA salt (24.9 mg, w _LPEI = 28%, total LPEI 7.0 mg) was dissolved in 0.8 mL of 20 Dissolved in mM HEPES, pH 7.2. Two centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA solution and 14,000 g Centrifugation was performed once for 30 minutes, and then 400 μL of 20 mM HEPES, pH 7.2, was added three times. Recover approximately 269 μL of LPEI- l- [N ₃ :DBCO]-PEG _36- [S-MAL]-DUPA (Compound 56a and Compound 56b) HEPES salt solution and supplement with 2.4 mL of 20 mM HEPES, pH 7.2. did. The concentration of the solution was determined to be 2.5 mg/mL total LPEI by copper assay.

Example 16

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ Synthesis of -[MAL-S]-MTX (Compound 62a and Compound 62b)

Compound 62a and compound 62b were synthesized as a mixture of regioisomers 62a and 62b according to the following reaction scheme. Thiol modified methotrexate MTX-SH (Compound 68) was prepared using solid phase synthesis. Compound 68 was condensed with DBCO-PEG ₃₆ -MAL (compound 36) through Michael addition, and the resulting DBCO-PEG ₃₆ -MTX was reacted with LPEI-N ₃ to obtain compounds 62a and 62b.

Step 1: Synthesis of Fmoc-Glu-(OtBu)-cysteamine-4-methoxytrityl resin (Compound 64)

Solution Fmoc-Glu-(OtBu) (compound 63; 242 μmol, 5 eq., 242 mM) in DMF (1 mL) was mixed with solution HATU (246 μmol, 1 eq. 246 mM) and DIEA (42 μL) in DMF (1 mL). , 250 μmol, 5 equivalents) was added. After 3 minutes, the reaction mixture was added to cysteamine 4-methoxytrityl resin (Compound 23; 51.1 mg, 50 μmol, 1.0 equiv). The reaction mixture was incubated at room temperature on a shaker. After 1 hour, the reaction mixture was filtered and Fmoc-Glu-(OtBu)-cysteamine-4-methoxy trityl resin (Compound 64) was washed with DMF (3 × 10 mL), DCM (3 × 10 mL) and Washed with MeOH (3×10 mL).

Step 2: Synthesis of Glu-(OtBu)-cysteamine-4-methoxytrityl resin (Compound 65)

A solution of 25% piperidine in DMF (5 mL) was added to Fmoc-Glu-(OtBu)-cysteamine-4-methoxytrityl resin (Compound 64) prepared in Step 1, and the reaction mixture was allowed to cool for about 10 minutes. It was stirred by hand for several minutes. The resin was filtered and washed with DMF (3 × 10 mL), DCM (3 × 10 mL), and MeOH (3 × 10 mL) to obtain Glu-(OtBu)-cysteamine-4-methoxytrityl resin (Compound 65). ) was obtained.

Step 3: Synthesis of MTX-4-methoxytrityl resin (Compound 67)

Solution N ¹⁰ -methyl-4-amino-4-deoxyphteric acid (MADOPA; Compound 66; 154 μmol, 3 equiv, 17 mM) in DMF/DMSO (2:1) (9 mL) was dissolved in DMF (1 mL). ) and mixed with solutions HATU (146 μmol, 3 equiv, 146 mM) and DIEA (25 μL, 147 μmol, 3 equiv). The reaction mixture was mixed for 3 minutes and then added to 50 μmol (1 equivalent) of Glu-(OtBu)-cysteamine-4-methoxy trityl resin (Compound 65) prepared in Step 2. The reaction mixture was transferred to a glass column with a glass frit, filtered, and washed with DMSO (3 × 10 mL), DMF (3 × 10 mL), DCM (3 × 10 mL), and MeOH (3 × 10 mL) with MTX. -4-Methoxy trityl resin (Compound 67) was obtained.

Step 4: Synthesis of MTX-SH (Compound 68)

TFA/TIS/H ₂ O (95:2.5:2.5) solution (4 mL) was added to MTX-4-methoxy trityl resin (Compound 67) prepared in Step 3. The reaction mixture was incubated on a shaker at room temperature for 1 hour. The resin was filtered, the filtrate was collected and concentrated under nitrogen flow for 15 minutes to evaporate the TFA. Cold diethyl ether (10 mL) was added. The resulting precipitate was washed with cold diethyl ether (4.5 mL). The tan solid material containing MTX-SH (Compound 68) was recovered and analyzed by HPLC - ESI ⁺ single quadrupole mass spectrometry (calculated mass [M+1] ⁺ : 514.20 Da, [M+2] ⁺ : 257.80 Da; measured mass [M+1] ⁺ : 515.0 Da, [M+2] ⁺ : 258.00 Da).

Step 5: DBCO-PEG ₃₆ -Synthesis of MTX (Compound 69)

Solution MTX-SH (compound 68; 8 μmol, 0.9 equiv, 1.1 mM in thiol) in DMSO/20 mM HEPES, pH 7.4 (1:9) (7.0 mL) was mixed with solution DBCO-PEG ₃₆ in DMSO (220 μL). Mixed with MAL (Compound 36; 9 μmol, 1.0 equiv, 41 mM). The reaction mixture was incubated at room temperature on a Stuart rotator, protected from light, and monitored by RP-C8-HPLC. After 1.5 hours, acetonitrile (0.8 mL) was added to the reaction mixture. DBCO-PEG ₃₆ -MTX (Compound 69; 14 mg) was isolated following RP-C ₁₈ preparative HPLC and lyophilization of the fraction pool and analyzed by HPLC - ESI ⁺ qTOF mass spectrometry (calculated monoisotopic mass : 2,596.32 Da; measured value: 2,596.35 Da).

Step 6: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ Synthesis of -[MAL-S]-MTX (Compound 62a and Compound 62b)

Solution LPEI-N ₃ (4.2 mL, 5.0 μmol, 0.9 equiv, 1.2 mM) in 50 mM acetate buffer, pH 4.0 was mixed with 5.0 mL of solution DBCO-PEG ₃₆ -MTX (Compound 69; 5.4) in 50 mM acetate buffer, pH 4.0. μmol, 1.0 equivalent, 1.1 mM) was added slowly. The reaction mixture was incubated at room temperature on a Stuart rotator, protected from light, and monitored by RP-C8-HPLC. After 20 hours of reaction. The mixture was supplemented with acetonitrile (1.0 mL) and TFA (100 μL). LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-MTX was isolated as a mixture of regioisomers 62a and 62b using RP-C ₁₈ preparative HPLC. Fractions were lyophilized to yield 90 mg of a fluffy off-white solid, which was characterized by RP-C ₈ -HPLC, copper assay and spectrophotometry at 305 nm for determination of methotrexate content. The lyophilisate showed a 34% weight percentage of LPEI and an LPEI to methotrexate ratio of 1:1.0.

Step 7: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ Preparation of -[MAL-S]-MTX (Compound 62a and Compound 62b) HEPES salt

LPEI- l- [ _N3 :DBCO]-PEG _36- [MAL-S]-MTX (Compound 62a and Compound 62b) TFA salt (23.8 mg, w _LPEI = 34%, total LPEI 8.1 mg) was dissolved in 0.8 mL of 20 Dissolved in mM HEPES, pH 7.2. Two centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-MTX solution and 14,000 g Centrifugation was performed once for 30 minutes, and then 400 μL of 20 mM HEPES, pH 7.2, was added three times. Approximately 250 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-MTX-HEPES salt solution was recovered and supplemented with 2.3 mL of 20 mM HEPES, pH 7.2. The concentration of the solution was determined to be 2.6 mg/mL total LPEI by copper assay.

Example 17

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of hEGF (Compound S 70a and Compound 70b)

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF was prepared as a mixture of regioisomers 70a and 70b according to the following reaction scheme. DBCO-PEG ₃₆ -TFP (Compound 52) was condensed with hEGF, and the resulting DBCO-PEG ₃₆ -hEGF (Compound 71) was reacted with LPEI-N ₃ to obtain Compound 70a and Compound 70b.

Step 1: DBCO-PEG ₃₆ -Synthesis of hEGF (compound 71)

Solution DBCO-PEG ₃₆ -TFP (compound 52; 128 μmol, 1.4 eq, 64 mM) in DMSO (2.0 mL) was dissolved in solution hEGF (92 μmol, 1.0 eq, 2.6 mM) in 20 mM HEPES, pH 7.5 (35 mL). It was added slowly. The reaction mixture was stirred in a round bottom flask and the reaction was monitored by RP-C8-HPLC. After 1 hour, an additional amount of DBCO-PEG ₃₆ -TFP (140 μL, 9 μmol, 0.1 equiv, 64 mM) was added. After a total of 1.5 hours, acetonitrile (4 mL) was added to the reaction mixture and the pH was adjusted to 3.5. DBCO-PEG ₃₆ -hEGF (Compound 71) was isolated following RP-C ₁₈ preparative HPLC. The pool of fractions was lyophilized to yield 310 mg of a fluffy white solid, which was analyzed by RP-HPLC-ELSD and RP-HPLC - ESI ⁺ qTOF mass spectrometry (DBCO-PEG ₃₆ -hEGF, calculated monoisotope Element mass: 8,168.79 Da; measured value: 8,168.80 Da).

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of hEGF (Compound 70a and Compound 70b)

Solution LPEI-N ₃ (24 mL, 23 μmol, 1.0 eq 0.94 mM) in 50 mM acetate buffer, pH 4.0 was mixed with solution DBCO-PEG ₃₆ -hEGF (Compound 71; 16 mL, 22 μmol) in 50 mM acetate buffer, pH 4.0. , 1.0 equivalent) was added. The reaction mixture was stirred in a round bottom flask and monitored by RP-C8-HPLC. After a total of 72 hours, acetonitrile (4 mL) and TFA (400 μL) were added to the reaction mixture. LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF was isolated as a mixture of regioisomers 70a and 70b using RP-C ₁₈ preparative HPLC. Fraction pools were lyophilized (505 mg) and characterized by RP-C ₈ -HPLC, copper assay and spectrophotometry at 280 nm for determination of hEGF content.

The lyophilisate was dissolved in 50 mM acetate, pH 4.5, and processed by TFF (10 kDa MWCO membrane) to remove TFA residues. Solution LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -hEGF (Compound 70a and Compound 70b) Recover acetate (42 mL), RP-C ₈ -HPLC, copper assay and 280 nm for determination of hEGF content. Characterized by spectrophotometry in . The concentration of the solution was determined to be 2.6 mg/mL total LPEI by copper assay.

Example 18

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of [S-MAL]-hEGF (Compound S 72a and Compound 72b)

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-hEGF was synthesized as a mixture of regioisomers 72a and 72b according to the following reaction scheme. DBCO-PEG ₃₆ -[S-MAL]-hEGF (Compound 74) was prepared by condensing hEGF with MCC-hEGF (Compound 73). The resulting DBCO-PEG ₃₆ -[S-MAL]-hEGF (Compound 74) was reacted with LPEI-N ₃ to obtain Compound 72a and Compound 72b.

Step 1: DBCO-PEG ₃₆ -Synthesis of [S-MAL]-hEGF (Compound 74)

DBCO-PEG ₃₆ -SH (Compound 59) solution (230 μL, 3.0 μmol, 1.0 equiv) was prepared as described in Example 15. Add solution MCC-hEGF (Compound 73; 2.9 μmol, 1.0 eq, 58 mM based on measured peptide content of 7%; CBL Patras, Greece) in 20 mM HEPES pH 7.2 (5.0 mL) , the reaction mixture was incubated at room temperature on a Stuart rotator and monitored by RP-C8-HPLC. After 15 minutes, an additional amount of DBCO-PEG ₃₆ -SH solution (20 μL, 0.3 μmol, 0.1 equiv) was added. After a total of 30 minutes, acetonitrile (0.56 mL) was added and the reaction mixture was purified by RP-C ₁₈ preparative HPLC. DBCO-PEG ₃₆ -[S-MAL]-hEGF (Compound 74) was isolated and the pool of fractions containing Compound 74 was lyophilized. Lyophilisate (15 mg) was analyzed by RP-HPLC-ELSD and RP-HPLC - ESI ⁺ qTOF mass spectrometry (DBCO-PEG ₃₆ -[S-MAL]-hEGF, calculated monoisotopic mass: 8,450.90 Da ; measured value: 8,450.97 Da).

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of [S-MAL]-hEGF (Compound 72a and Compound 72b)

A solution of LPEI-N ₃ (2.5 mL, 2.0 μmol, 1.2 equiv, 0.84 mM) in 50 mM acetate buffer, pH 4.0 was mixed with 4.0 mL of solution DBCO-PEG ₃₆ -[S-MAL]- in 50 mM acetate buffer, pH 4.0. hEGF (Compound 74; 1.7 μmol, 1.0 equiv, 0.43 mM) was added slowly. The mixture was incubated at room temperature on a Stuart rotator and protected from light. After 20 hours, the reaction mixture was supplemented with acetonitrile (0.72 mL) and TFA (73 μL). LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-hEGF was isolated as a mixture of regioisomers 72a and 72b using RP-C ₁₈ preparative HPLC, RP-C ₈ -HPLC, copper Characterized by assay and spectrophotometry at 280 nm for determination of hEGF content. The lyophilisate showed a 25% weight percentage of LPEI and an LPEI to hEGF ratio of 1:1.09.

Step 3: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-hEGF (Compounds 72a and 72b) Preparation of HEPES salts

LPEI- l- [ _N3 :DBCO]-PEG _36- [S-MAL]-hEGF (Compound 72a and Compound 72b) TFA salt (26.4 mg, w _LPEI = 25%, total LPEI 6.6 mg) was dissolved in 0.8 mL of 20 Dissolved in mM HEPES, pH 7.2. Two centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-hEGF solution and 14,000 g Centrifugation was performed once for 30 minutes, and then 400 μL of 20 mM HEPES, pH 7.2, was added three times. Recover approximately 212 μL of LPEI- l- [N ₃ :DBCO]-PEG _36- [S-MAL]-hEGF (Compound 72a and Compound 72b) HEPES salt solution, containing 2.3 mL of 20 mM HEPES, pH 7.2. did. The concentration of the solution was determined to be 2.3 mg/mL total LPEI by copper assay.

Example 19

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of [MAL-S]-CysGE11 (Compound 75a and Compound 75b)

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-GE11 was synthesized as a mixture of regioisomers 75a and 75b in two steps according to the following reaction scheme. In the first step, the human peptide Cys-GE11 (compound 76) is bound to DBCO-PEG ₃₆ -MAL (compound 36) in 20 mM HEPES buffer to form DBCO-PEG ₃₆ -[MAL-S]-CysGE11 (compound 77). produced. In the second step, DBCO-PEG ₃₆ -[MAL-S]-CysGE11 is conjugated to LPEI-N ₃ to produce LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-CysGE11 (Compound 75a and compound 75b) were produced.

Step 1: DBCO-PEG ₃₆ -Synthesis of [MAL-S]-CysGE11

CysGE11 peptide (compound 76; 6.5 μmol, 1.0 eq, 0.93 mM) in solution TCEP (6.5 μmol, 1.0 eq, 85 mM) in 20 mM HEPES, pH 7.4 (76 μL) ) and mixed with. Next, solution DBCO-PEG ₃₆ -MAL (Compound 36; 7.8 μmol, 1.2 equiv, 24 mM) in DMSO (0.32 mL) was added and the reaction mixture was incubated on a Stewart rotator at room temperature. After a total of 30 minutes, acetonitrile (0.8 mL) was added to the reaction mixture, which was purified by RP-C ₁₈ preparative HPLC. Freeze-drying of the fraction pool yielded DBCO-PEG ₃₆ -[MAL-S]-CysGE11 (Compound 77) as a solid (13 mg; calculated monoisotopic mass: 3,725.85 Da; measured: 3,725.90 Da).

Stage 2: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -Synthesis of [MAL-S]-CysGE11

A solution of LPEI-N ₃ (3.0 mL, 2.5 μmol, 1.0 equiv, 0.84 mM) in 50 mM acetate buffer, pH 4.0 was mixed with 4.0 mL of solution DBCO-PEG ₃₆ -[MAL-S]- in 50 mM acetate buffer, pH 4.0. CysGE11 (Compound 77; 4.3 μmol, 1.7 equiv, 1.1 mM) was added slowly. The mixture was incubated at room temperature on a Stuart rotator and protected from light. After 16 hours, acetonitrile (0.78 mL) and TFA (78 μL) were added to the reaction mixture, which was purified using RP-C ₁₈ preparative HPLC. The pool of fractions was lyophilized to obtain LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-CysGE11 (60 mg) as a mixture of regioisomers 75a and 75b, which was purified by RP-C ₈ -HPLC. , copper assay and spectrophotometry at 280 nm for determination of peptide content. The lyophilisate showed a 27% weight percentage of LPEI and an LPEI to CysGE11 ratio of 1:1.1.

Step 3: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ Preparation of -[MAL-S]-CysGE11 (Compound 75a and Compound 75b) HEPES salt

LPEI- l- [ _N3 :DBCO]-PEG _36- [MAL-S]-CysGE11 (Compound 75a and Compound 75b) TFA salt (27.3 mg, w _LPEI = 27%, total LPEI 7.4 mg) was dissolved in 0.8 mL of 20. Dissolved in mM HEPES, pH 7.2. Two centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-CysGE11 solution and 14,000 g Centrifugation was performed once for 30 minutes, and then 400 μL of 20 mM HEPES, pH 7.2, was added three times. Approximately 245 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[MAL-S]-CysGE11-HEPES salt solution was recovered and supplemented with 2.3 mL of 20 mM HEPES, pH 7.2. The concentration of the solution was determined by the copper assay (total LPEI 2.6 mg/mL and LPEI to CysGE11 ratio of 1:1.1).

Example 20

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -(GalNAc) ₃ Synthesis of (Compound 78a and Compound 78b)

Step 1: DBCO-PEG ₃₆ -(GalNAc) ₃ Synthesis of (Compound 80)

Solution (GalNAc) ₃ -PEG ₃ -NH ₂ (Compound 79; 5.6 μmol, 1.0 equiv, 7.2 mM) in 20 mM HEPES, pH 7.4 (0.5 mL) was dissolved in solution DBCO-PEG ₃₆ -TFP ( Compound 52 (7.5 μmol, 1.3 equivalents, 48 mM) was added. The reaction mixture was placed on a Stewart rotator at room temperature and the reaction was monitored by RP-C8-HPLC. After 2 hours, an additional 3.0 mL of 20 mM HEPES buffer, pH 7.4, was added. After a total of 20 hours, MilliQ water (3.4 mL) and acetonitrile (0.78 mL) were added to the reaction mixture, which was purified using RP-C ₁₈ preparative HPLC. A pool of fractions containing purified DBCO-PEG ₃₆ -(GalNAc) ₃ (Compound 80) was lyophilized to obtain a solid (10 mg; ESI ⁺ qTOF mass spectrometry, calculated monoisotopic mass: 3,646.00 Da; determined Value: 3,646.02 Da).

Stage 2: LPEI- l -[N ₃ :DBCO]- PEG ₃₆ -GalNAc) ₃ Synthesis of (Compound 78a and Compound 78b)

Solution LPEI-N ₃ (3.0 mL, 2.5 μmol, 1.0 equiv, 0.84 mM) in 50 mM acetate buffer, pH 4.0 was mixed with 4.0 mL of solution DBCO-PEG _36- (GalNAc) ₃ (compound) in 50 mM acetate buffer, pH 4.0. 80; 3.0 μmol, 1.2 equivalent, 0.76 mM). The mixture was placed on a Stuart rotator and protected from light. After 16 hours, acetonitrile (0.78 mL) and TFA (79 μL) were added to the reaction mixture for preparative chromatography. LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -(GalNAc) ₃ (Compound 78a and Compound 78b) was isolated as a mixture of regioisomers 78a and 78b using RP-C ₁₈ preparative HPLC, and RP-C ₈ -Characterized by HPLC and copper assay. The lyophilisate (63 mg) showed a weight percent LPEI of 27%.

Step 3: LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -GalNAc) ₃ -Preparation of HEPES salts

LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -(GalNac) ₃ (Compound 78a and 78b) TFA salt (42 mg, w _LPEI = 27%, 11.3 mg total LPEI) was dissolved in 1.6 mL of 20 mM HEPES, Dissolved at pH 7.2. Four centrifugal filters (Amicon Ultra - 0.5 mL, 10 kDa MWCO) were each filled with 400 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -(GalNac) ₃ solution and incubated at 14,000 g for 30 min. Centrifugation was performed once, followed by the addition of 400 μL of 20 mM HEPES, pH 7.2, three times. Approximately 418 μL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -(GalNac) ₃ -HEPES salt solution was recovered and supplemented with 4.0 mL of 20 mM HEPES, pH 7.2. The concentration of the solution (4.4 mL) was determined by copper assay (total LPEI 2.2 mg/mL).

Example 21

Multicomplex size determination and zeta potential measurements

General procedure for multicomplex formation.

For the preparation of the desired multicomplexes, the triconjugates were complexed with nucleic acids such as poly(IC) at various N/P ratios in HBG buffer (20 mM HEPES, pH 7.2, 5% glucose, w/v). The nitrogen to phosphorus (N/P) ratio was calculated based on the nitrogen content in the LPEI portion of the triconjugate used and the phosphorus content in the nucleic acid such as poly(IC). Herein, a stock solution of a triconjugate such as LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF (also abbreviated as LPEI -l- PEG ₂₄ -hEGF in the detailed description) is prepared at an appropriate concentration at a selected N/P ratio. It was diluted using HBG. The diluted triconjugate solution was added to an equal volume of nucleic acid solution to a final nucleic acid concentration in the multiplex preparation of 0.1 to 0.2 mg/mL and mixed by vigorous pipetting. The mixture was left at RT for 30 min to allow multicomplex formation. In a similar manner, multicomplexes with polyanions such as poly(Glu) were prepared. Multicomplexes are typically further characterized with respect to size and surface charge.

Figures 1A, 1B and 1C show a comparison of physicochemical characterization for LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplexes as a function of N/P ratio. Figure 1A shows triplicate DLS backscatter plots of z-average diameter and dispersion for a 100 mL sample of LPEI -l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) at an N/P ratio of 2.4. Indicates measurement. Measurements of the same sample were performed in triplicate. The concentration of the multicomplex in HBG is 0.125 mg/mL (pH 7.2). Figure 2b shows triplicate DLS backscatter plots of z-average diameter and dispersion for a 100 mL sample of LPEI -l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) at an N/P ratio of 4.0. Indicates measurement. Measurements of the same sample were performed in triplicate. The concentration of the multicomplex in HBG is 0.125 mg/mL (pH 7.2). Figure 1c shows triplicate DLS backscatter plots of z-average diameter and dispersion for a 100 mL sample of LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) at an N/P ratio of 5.6. Indicates measurement. Measurements of the same sample were performed in triplicate. The concentration of the multicomplex in HBG is 0.125 mg/mL (pH 7.2). As shown in Figures 1A, 1B, and 1C, multicomplexes with N/P ratios of 4 and 5.6 showed average diameters of 116 and 107 nm and PDIs of 0.08 and 0.11, respectively. The multicomplex with an N/P ratio of 2.5 showed an average diameter of 306 nm and a PDI of 0.35.

Similarly, Figures 2 and 3 show LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF, which is non-toxic or non-targeted and used as a control multicomplex in cell probing experiments, respectively, as described herein: Triplicate DLS backscatter measurements of mean diameter and dispersion for poly(Glu) and LPEI- l- [N ₃ :DBCO]-PEG ₂₃ -OMe:poly(IC) multicomplexes are shown. Figure 2 shows the DLS inverse of LPEI -l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu) multicomplex in HBG buffer at pH 7.2, 0.1 mg/mL, 1 mL volume, N/P ratio of 4. indicates scattering. The z-average diameter was 121 nm with a polydispersity index (PDI) of 0.087. The ζ-potential was 28.7 mV. Figure 3 shows LPEI -l- [N ₃ :DBCO]-PEG ₂₃ -OMe:poly(IC) in 50 mM acetate buffer, pH 4.3, 5% glucose, 0.1875 mg/mL, 1 mL volume, N/P ratio of 4. ) shows DLS backscattering of multiple complexes. The z-average diameter was 107 nm with a polydispersity index (PDI) of 0.139. The ζ-potential was 31.4 mV.

The data shown in FIGS. 1A, 1B and 1C are summarized in Table 7 below. For ζ-potential measurements, a larger sample volume of 900 mL is required.

In a similar manner and similarly determined, Figures 4 and 5 show LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) and LPEI- l -[N Physicochemical characterization of both ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) multicomplexes is shown. Figure 4 shows triplicate LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) multicomplexes in 50 mM acetate buffer, pH 4.3, 5% glucose, 0.1875 mg/mL, 1 mL. DLS backscattering is shown to measure size distribution and ζ-potential at volume, N/P ratio of 4. The z-average diameter was 156 nm with a polydispersity index (PDI) of 0.144. The ζ-potential was 38.3 mV. Figure 5 shows the size distribution and ζ- obtained from triplicate LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) multicomplexes at 0.1875 mg/mL, 1 mL volume, N/P 4. Indicates DLS backscattering, which measures the potential. The z-average diameter was 120 nm with a polydispersity index (PDI) of 0.125. The ζ-potential was 31.1 mV.

Physicochemical characterization by DLS of the additional multicomplexes prepared in the above examples are shown in Tables 8 to 10 below.

Example 22

Targeting cells expressing EGFR of the present invention Cytotoxic activity of multiple complexes.

Cell survival experiments investigated the efficacy and selectivity of triconjugated LPEI- l -PEG-targeting fragment:nucleic acid multicomplexes in various cancer cell lines showing differential cell surface expression of receptor proteins. Triconjugated LPEI- l -PEG-targeting fragment:poly(Glu) multicomplexes served as controls to demonstrate that the reduction in survival was primarily mediated by targeted delivery of poly(IC) by the multicomplexes. In addition, a comparison with the multicomplex of the prior art was performed to demonstrate the enhancement of activity of the multicomplex of the present invention.

Cell survival assay of EGFR-targeted multicomplexes in cells showing high and low EGFR expression.

This assay was performed on two cancer cell lines that showed differential EGFR cell surface expression levels, A431 (high EGFR; Phillips et al ., Mol. Cancer. Ther., 2016, 15(4): 661- 669) and MCF7 (low EGFR; see European patent EP3098239B1). The efficacy and selectivity of PEI-PEG-hEGF:poly(IC) multicomplexes were investigated. A431 cells and MCF7 cells were obtained from ATCC. The cell surface density of EGFR for both cell lines is given in Table 11 below.

Figures 6A-9B show similarly performed cell survival experiments in two cancer cell lines, MCF7 (low EGFR) and A431 (high EGFR), that showed the differential EGFR cell surface expression levels. Therefore, cancer cell lines were treated with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC), LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC), LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC), LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC), and their respective control multicomplexes LPEI- l - [N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu), LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu), LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(Glu) and LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu) (Figures 6a to 9b).

LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC), LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC), LPEI- l -[N ₃ : DBCO]-PEG ₂₄ -hEGF:poly(IC), LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu), LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF: Multicomplex samples containing poly(Glu) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu) were incubated at an N/P ratio of 4 in 50 mM acetate containing 5% glucose, pH 4.3. Manufactured in . Cancer cells (3,000 cells/well) showing differential EGFR expression levels were treated with the multicomplex for 72 hours. Cell survival was analyzed using CellTiter-Glo (Promega). The concentration shown as log (multicomplex) in Figures 6A-9B reflects the concentration of poly(Glu) or poly(IC) in the individual multicomplex.

Multicomplex samples containing LPEI- l -PEG ₃₆ -hEGF:poly(IC) or LPEI- l -PEG ₃₆ -hEGF:poly(Glu) were incubated at an N/P ratio of 4 in HBG buffer, 5% glucose, pH 7.2. Formulated. Cancer cell lines (3,000 cells/well) showing differential EGFR expression (MCF7: low EGFR expression; and A431: high EGFR expression) were incubated with LPEI- l -PEG ₃₆ -hEGF:poly(IC) or LPEI- l -PEG _36. -hEGF:treated with poly(Glu) multicomplex for 72 hours. Cell survival was analyzed using CellTiter-Glo (Promega). Concentrations expressed as log (multicomplex) reflect the concentration of poly(Glu) or poly(IC) in the individual multicomplex.

Figure 6A shows LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC) or LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu), i.e. Compound 70a and Compound 70a. Percent survival of MCF7 cells treated with multicomplex containing 70b is shown. As shown in Figure 6a, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu) were 0.625. There was no activity at concentrations as high as μg/mL (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 6B shows LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC) or LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu), i.e. Compound 70a and Compound 70a. Percent survival of A431 cells treated with multicomplex containing 70b is shown. As shown in Figure 6b, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu) were 0.625. There was no activity at concentrations as high as μg/mL (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 7A shows LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) or LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu), Compound 1a and Compound 1a. Percent survival of MCF7 cells treated with multicomplex containing 1b is shown. As shown in Figure 7a, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF:poly(Glu) were 0.625. There was no activity at concentrations as high as μg/mL (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 7b shows LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) or LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu), Compound 1a and Compound 1a. Percent survival of A431 cells treated with multicomplex containing 1b is shown. As shown in Figure 7b, LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu) were 0.625. There was no activity at concentrations as high as μg/mL (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 8A shows LPEI- l- [N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) or LPEI- l- [N ₃ :DBCO]-PEG _12- hEGF:poly(Glu), Compound 4a and Compound 4a. Percent survival of MCF7 cells treated with multicomplex containing 4b is shown. As shown in Figure 8a, LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(Glu) were 0.625. There was no activity at concentrations as high as μg/mL (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 8b shows LPEI- l- [N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) or LPEI- l- [N ₃ :DBCO]-PEG _12- hEGF:poly(Glu), Compound 4a and Compound 4a. Percent survival of A431 cells treated with multicomplex containing 4b is shown. As shown in Figure 8b, LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(Glu) were 0.625. There was no activity at concentrations as high as μg/mL (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 9A shows LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu), Compound 7a and Compound 7a. Percent survival of MCF7 cells treated with multicomplex containing 7b is shown. As shown in Figure 9a, LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu) are 0.625 There was no activity at concentrations as high as μg/mL (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 9B shows LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu), Compound 7a and Compound 7a. Percent survival of A431 cells treated with multicomplex containing 7b is shown. As shown in Figure 9b, LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(IC) showed an IC ₅₀ of 0.002 μg/mL. LPEI- l -[N ₃ :DBCO]-PEG ₄ -hEGF:poly(Glu) showed an IC ₅₀ of 1.026 μg/mL.

Table 12 shows the linear LPEI- l -PEG ₄ -hEGF:poly(IC), LPEI- l -PEG ₁₂ -hEGF:poly described above, measured in MCF7 (low EGFR) cells as well as A431 (high EGFR) cells. (IC), LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) and LPEI- l -PEG ₃₆ -hEGF:poly(IC) provide cell survival as a function of treatment with multicomplexes. . Also provided is cell survival data of randomly branched LPEI- r -PEG _2KDa -hEGF:poly(IC) multicomplexes taught in International Patent Application WO 2015/173824, measured in a similar manner as described above. The data show that the linear multicomplex according to the invention is significantly more potent than the randomly branched LPEI- r -PEG _2KDa -hEGF:poly(IC) multicomplex of the prior art as taught in International Patent Application WO 2015/173824 , demonstrated substantially higher cytotoxic efficacy and selectivity against the EGFR overexpressing cell line A431.

As shown in Figure 7a, the multicomplex [LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu )] treatment had no effect on cell survival of MCF7 cells at concentrations as high as 1 μg/mL, while LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly( IC) of 0.005 μg/mL compared to an IC ₅₀ of 1.432 μg/mL induced by the control polyanion LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(Glu) multicomplex in A431 cells. Cell death was induced with IC ₅₀ . Similarly, as shown in Figure 8a, the multicomplexes [LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly (Glu)] had no effect on cell survival of MCF7 cells at concentrations as high as 1 μg/mL, while LPEI- l- [N ₃ :DBCO]-PEG ₁₂ -hEGF: Poly(IC) was 0.003 μg/mL compared to an IC ₅₀ of 1.020 μg/mL induced by the control polyanion LPEI- l -[N ₃ :DBCO]-PEG ₁₂ -hEGF:poly(Glu) multicomplex in A431 cells. Cell death was induced with an IC ₅₀ of mL. Similar results are also shown in FIGS. 6A and 6B and FIGS. 9A and 9B.

In a preferred embodiment, the multicomplex comprising the poly(IC) of the present invention exhibits high biological efficacy as evidenced by the high cytotoxicity of the triconjugate:nucleic acid multicomplex of the present invention. In a preferred embodiment, the high cytotoxicity of the multicomplex is believed to be caused by poly(IC). Accordingly, in some embodiments the multicomplexes of the invention include poly(IC).

Additionally, the examples herein show that the multicomplex of the present invention is A431 expressing hEGFR at a higher level (i.e., 10 ⁶ molecules/cell) than in cells expressing hEGFR at a low level (i.e., 10 ³ molecules/cell). It was demonstrated that cytotoxicity was significantly high in cells, and thus it showed very high selectivity. Accordingly, in a preferred embodiment, the multicomplex of the invention selectively induces cell death in cells expressing high levels of a specific cell surface receptor, preferably wherein the multicomplex of the invention contains a targeting fragment that targets the cell surface receptor. Includes.

Example 23

Effect of targeting on cytotoxic activity

The triconjugate of LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ (Compound 17a and Compound 17b) was used to prepare multicomplexes containing poly(IC) or poly(Glu). LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ (Compound 17a and Compound 17b) and poly(IC) or poly(Glu) were dissolved in HEPES buffer containing 5% glucose, pH 7.2. A solution containing LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ was added to an equal volume of poly(IC) or poly(Glu) solution to achieve a final nucleic acid concentration of 0.1 mg/mL in the multicomplex preparation. provided. The combined solutions of LPEI- l -]N ₃ :DBCO]-PEG ₂₃ -OCH ₃ and nucleic acid were mixed by vigorous pipetting. Mixture LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(Glu) for 30 minutes at room temperature. This allowed the formation of multiple complexes. The final N/P ratio of the multicomplex was 4.

A431 and MCF7 cells (see Table 11 above) were grown at a density of 3,000 cells/well. Increasing the multicomplex LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC) or LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(Glu) in cells Treated with concentration. Cell survival was analyzed using CellTiter-Glo (Promega). The results are shown in Figures 10A and 10B.

Figure 10A shows MCF7 treated with LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC) or LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(Glu) Indicates the survival percentage of cells. As shown in Figure 10a, LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(Glu) There was no activity at concentrations as high as 0.625 μg/mL (i.e., no significant cell death was observed for the multicomplex at concentrations as high as 0.625 μg/mL).

Figure 10b shows A431 treated with LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC) or LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(Glu) Indicates the survival percentage of cells. As shown in Figure 10b, LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC) showed an IC ₅₀ of 0.313 μg/mL. LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(Glu) had no activity at a concentration as high as 0.625 μg/mL (i.e., significant cell death occurred at a concentration as high as 0.625 μg/mL). not observed at all for multicomplexes).

Figures 10A and 10B show untargeted multicomplex LPEI -l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC) and LPEI- l- measuring cell survival in A431 cells as well as MCF7 cells. [N ₃ :DBCO]-PEG ₂₃ -OCH ₃ : Indicates treatment with poly(Glu). As shown in Figure 10A, treatment with both multicomplexes had no effect on the survival of MCF7 cells, whereas untargeted multicomplex LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OCH ₃ :poly(IC ) induced cell death in A431 cells with an IC ₅₀ of 0.313 μg/mL, and the control polyanion LPEI- l -[N ₃ :DBCO]-PEG ₂₃ -OMe:poly(Glu) multicomplex at 1 μg/mL. Even at high concentrations, there was no effect on the cell survival of these cells (Figure 10b). Accordingly, in a preferred embodiment the cytotoxicity of the triconjugate:nucleic acid multicomplex of the invention is primarily due to the delivery of the selected nucleic acid (e.g., poly (IC)). In a preferred embodiment, the cytotoxicity of the multicomplex of the invention can be increased by adding targeting fragments to the triconjugate of the invention.

Example 24

Selective delivery of the multicomplex of the present invention reduces the survival of cells overexpressing PSMA.

LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC); LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA:poly(IC); LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA:poly(IC); LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA:poly(IC); and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA:poly(IC) multicomplex formulated at an N/P ratio of 4 in 20 mM HEPES, pH 7.2 with 5% glucose. did.

Cancer cell lines showing differential PSMA expression (3,000 cells/well) (PC3: low PSMA expression; DU145 low PSMA expression; and LNCaP: high PSMA expression) were incubated with LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA. :poly(IC); LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC); Alternatively, it was treated with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) multicomplex for 72 hours. Cell survival was analyzed using CellTiter-Glo (Promega). Concentrations expressed as log (multicomplex) reflect the concentration of poly(Glu) or poly(IC) in the individual multicomplex.

Table 13 shows the linear LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) reported above measured in PC-3 and DU145 (low PSMA) cells as well as LNCaP (high EGFR) cells. or provides cell survival as a function of treatment with linear LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) multicomplexes. Additionally, cell survival data of prior art randomly branched LPEI -r -PEG _2KDa -DUPA:poly(IC), measured in a similar manner, are provided. The data confirm that the linear multicomplex according to the present invention is more potent than the randomly branched multicomplexes of the prior art and exhibits higher selectivity (i.e., approximately 10-fold) for cell lines overexpressing PSMA.

Figure 11A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in LNCaP cells. indicates cell survival. While LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) was completely inactive (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL). ), LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) induced a healthy reduction in LNCaP cell survival with an IC ₅₀ value of 0.02 μg/mL.

Figure 11B shows the transformation of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in PC-3 cells. Cell survival is shown as a function of treatment. While LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) was completely inactive (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL). ), LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) inhibited PC-3 cell survival with an IC ₅₀ value of 0.24 μg/mL.

Figure 11C shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) in DU145 cells. indicates cell survival. LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(Glu) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) were not active (i.e., no significant No cell death was observed for both multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 12A shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) in LNCaP cells. represents cell survival. While LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) was completely inactive (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL). ), LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) induced a healthy reduction in LNCaP cell survival with an IC ₅₀ value of 0.02 μg/mL.

Figure 12B shows the transformation of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) in PC-3 cells. Cell survival is shown as a function of treatment. While LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) was completely inactive (i.e., no significant cell death was observed for multicomplexes at concentrations as high as 0.625 μg/mL). ), LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) inhibited PC-3 cell survival with an IC ₅₀ value of 0.22 μg/mL.

Figure 12C shows treatment function with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) in DU145 cells. indicates cell survival. LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) had no activity (i.e., no significant No cell death was observed for both multicomplexes at concentrations as high as 0.625 μg/mL).

Figure 13 shows LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA:poly(IC); LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA:poly(IC); LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA:poly(IC); and LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA:poly(IC) multicomplexes.

Figure 14 shows LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA:poly(IC); LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA:poly(IC); LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA:poly(IC); and LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA:poly(IC) multicomplexes.

Table 14 shows the linear LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) reported above measured in PC-3 and DU145 (low PSMA) cells as well as LNCaP (high EGFR) cells. ; LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[(NH ₂ )MAL-S]-DUPA:poly(IC); LPEI- l -[N ₃ :BCN]-PEG ₃₆ -DUPA:poly(IC); LPEI- l -[N ₃ :SCO]-PEG ₃₆ -DUPA:poly(IC); LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -[CONH]-DUPA:poly(IC); and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -[S-MAL]-DUPA:poly(IC) multicomplex.

While all tested linear conjugates of the invention: poly(IC) multicomplexes induced a similar selective and significant reduction in the survival of PSMA overexpressing cells, there was a much weaker effect on cell survival in low PSMA expressing cells. was observed.

Example 25

Selective delivery of the multicomplex of the present invention reduces the survival of cells overexpressing folate.

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -folate:poly(IC) multicomplex was formulated at an N/P ratio of 4 in 20 mM HEPES, pH 7.2 with 5% glucose.

Cancer cell lines showing differential folate receptor expression (3,000 cells/well) (MCF7: low folate receptor expression; SKOV3: high folate receptor expression) were incubated with LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -pol. Treated with rate:poly(IC) multicomposite for 72 hours. Cell survival was analyzed using CellTiter-Glo (Promega).

As shown in Figure 15, selective delivery of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -folate:poly(IC) reduced survival of folate expressing cells (SKOV3). In contrast, in MCF7 cells, delivery of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -folate:poly(IC) showed no significant effect on cell survival at concentrations as high as 0.625 μg/mL.

.

Example 26

Selective delivery of the multicomplex of the present invention reduces survival of cells overexpressing HER2

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:poly(Glu) multicomplexes. Formulated at an N/P ratio of 4 in 20 mM HEPES with 5% glucose, pH 7.2.

Cancer cell lines showing differential HER2 expression (3,000 cells/well) (MCF7: low HER2 expression; SKBR3 and BT474: high HER2 expression) were incubated with LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody: Treated with poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:poly(Glu) multicomplex for 72 hours. Cell survival was analyzed using CellTiter-Glo (Promega). Selective delivery of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:poly(IC) reduced survival of cells overexpressing HER2, as shown in Figures 16A, 16B and 16C.

Figure 16A shows LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly in MCF7 cells. Cell survival is shown as a function of treatment with (Glu). While LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:Poly(Glu) was completely inactive (i.e., no significant cell death was observed at concentrations as high as 0.625 μg/mL). , LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:poly(IC) inhibited MCF7 cell survival with an IC ₅₀ value of 0.85 μg/mL.

Figure 16B shows LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly in SKBR3 cells. Cell survival is shown as a function of treatment with (Glu). While LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:Poly(Glu) was completely inactive (i.e., no significant cell death was observed at concentrations as high as 0.625 μg/mL). , LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:poly(IC) inhibited SKBR7 cell survival with an IC ₅₀ value of 0.25 μg/mL.

Figure 16C shows LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-apibody:poly in BT474 cells. Cell survival is shown as a function of treatment with (Glu). While LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:Poly(Glu) was completely inactive (i.e., no significant cell death was observed at concentrations as high as 0.625 μg/mL). , LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -HER2-Apibody:poly(IC) inhibited BT474 cell survival with an IC ₅₀ value of 0.34 μg/mL.

Example 27

IP-10 cytokine secretion from EGFR cancer cell lines

IP-10 secretion experiments showed LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) in two cancer cell lines that showed differential EGFR surface expression, A431 (high EGFR) and MCF7 (low EGFR). Selective cytokine release induced by the multicomplex was investigated.

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplex was formulated at an N/P ratio of 4 in 20 mM HEPES, pH 7.2 with 5% glucose. Cancer cells (40,000 cells/well in a 96-well plate) were incubated with various concentrations of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) (extinction coefficient of 22.2 L/(g·cm) (0.125, 0.25, 0.5, 1.0 μg/mL) determined using EM260) for 5 hours. Media from treated cells was collected and analyzed for human IP-10 (CXCL10) using an ELISA assay (Pepprotec) and detection using the microplate reader Synergy H1 (Biotech). Figure 17 shows IP-10 secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) concentration in A431 cells and MCF7 cells.

Example 28

EGFR phosphorylation

The EGFR targeting engagement of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplex was investigated, and the phosphorylation level of EGFR in NIH3T3 cells was analyzed by immunoblot analysis.

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) multicomplex was formulated at an N/P ratio of 4 in 20 mM HEPES, pH 7.2 with 5% glucose. Cells (400,000 cells/well in a 96-well plate) were serum starved and incubated with the carrier LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF (0.04 μg/mL, reflecting total LPEI concentration) and multiplex. Treated with complex LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -hEGF:poly(IC) (0.0615 μg/mL, reflecting poly(IC) concentration) for 30 min. Protein eluates were generated, centrifuged, and subjected to phospho-EGFR immunoblot analysis (total 10 μg protein eluate). Serum starvation conditions served as a negative control, and human EGF treatment served as a positive control for EGFR protein phosphorylation. Tubulin demonstrates equal loading of total protein. Figure 18 _shows phosphorylation of EGFR ₍ It is proven that P-EGFR) was induced in a healthy manner.

Example 29

Cytokine secretion from PSMA cancer cell lines

LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) multicomplexes were grown in 20 mL in the presence of 5% glucose. Formulated at an N/P ratio of 4 in mM HEPES, pH 7.2. Cancer cells showing differential PSMA expression (40,000 cells/well in a 96-well plate) (LNCaP: high PSMA expression; PC-3 and DU145: low PSMA expression) were incubated with various concentrations of LPEI- l- [ _N3 :DBCO. ]-PEG ₂₄ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) multicomplex (0.0625, 0.625 μg/mL) for 6 or 24 hours. did. Media from treated cells was collected and assayed for human IP-10 (CXCL10), RANTES (CCL5) or Interferon beta (IFN-β) was assayed and detected using the microplate reader Synergy H1 (Biotech).

Treatment with the indicated concentrations of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) alc LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) multicomplex Selectively induces IP-10, RANTES and IFN-β cytokine release in PSMA overexpressing cells (LNCaP) compared to low PSMA expressing cells (PC-3 and DU145). The results are shown in Figures 19A to 21C.

Figure 19A shows IP-10 secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) secreted 382 pg/mL and 1245.67 pg/mL of IP-10 at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived. In PC-3 cells, LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) had an IP of 11.33 pg/mL and 37.67 pg/mL at 0.0625 μg/mL and 0.625 μg/mL, respectively. 10 Secretion was induced.

Figure 19B shows IP-10 secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) secreted 582.87 pg/mL and 1524.97 pg/mL of IP-10 at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived. In PC-3 cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) was obtained at 0.0625 μg/mL and 0.625 μg/mL, respectively, at 0 pg/mL and 0 pg/mL. 10 Secretion was induced.

Figure 19C shows IP-10 secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and DUC145 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) secreted 582.87 pg/mL and 1524.97 pg/mL of IP-10 at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived. In DU145 cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) secreted 0 pg/mL and 0 pg/mL of IP-10 at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived.

For Figures 19B and 19C, treatments with multicomplexes were compared side by side in identical experiments in LNCaP, PC3 and DU145 cells. The figure is separated for easy viewing, and the IP-10 secretion values in LNCaP cells are the same.

Figure 20A shows RANTES secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) induced RANTES secretion of 514.33 pg/mL and 1368.33 pg/mL at 0.0625 μg/mL and 0.625 μg/mL, respectively. did. In PC-3 cells, LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) secreted 0 pg/mL and 24 pg/mL of RANTES at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived.

Figure 20B shows RANTES secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) induced RANTES secretion of 209.67 pg/mL and 1057 pg/mL at 0 μg/mL and 0.625 μg/mL, respectively. did. In PC-3 cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) secreted 214.33 pg/mL and 210.33 pg/mL of RANTES at 0 μg/mL and 0.625 μg/mL, respectively. was derived.

Figure 20C shows RANTES secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and DU145 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) induced RANTES secretion of 209.67 pg/mL and 1057 pg/mL at 0 μg/mL and 0.625 μg/mL, respectively. did. In DU145 cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) induced RANTES secretion of 207.67 pg/mL and 167.67 pg/mL at 0 μg/mL and 0.625 μg/mL, respectively. did.

For Figures 20B and 20C, treatments with multicomplexes were compared side by side in identical experiments in LNCaP, PC3, and DU145 cells. The figures are separated for easy viewing, and the RANTES secretion values in LNCaP cells are the same.

Figure 21A shows IFN-β secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) secreted 181.5 pg/mL and 312.3 pg/mL of IFN-β at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived. In PC-3 cells, LPEI -l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) produced 0 pg/mL and 40.47 pg/mL of IFN at 0.0625 μg/mL and 0.625 μg/mL, respectively. -β secretion was induced.

Figure 21B shows IFN-β secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and PC-3 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) secreted 216.27 pg/mL and 606.6 pg/mL of IFN-β at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived. In PC-3 cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) produced 44.17 pg/mL and 86.57 pg/mL of IFN- at 0.0625 μg/mL and 0.625 μg/mL, respectively. β secretion was induced.

Figure 21C shows IFN-β secretion as a function of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) concentration in LNCaP and DU145 cells. In LNCaP cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) secreted 216.27 pg/mL and 606.6 pg/mL of IFN-β at 0.0625 μg/mL and 0.625 μg/mL, respectively. was derived. In PC-3 cells, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) produced 4.37 pg/mL and 5 pg/mL of IFN- at 0.0625 μg/mL and 0.625 μg/mL, respectively. β secretion was induced.

For Figures 21B and 21C, treatments with multicomplexes were compared side by side in identical experiments in LNCaP, PC3, and DU145 cells. The drawings are separated for easy viewing, and the values of IFN-β secretion in LNCaP cells are the same.

Multicomplexes of the invention, LPEI -l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) or LPEI- l- [N ₃ :DBCO] at two concentrations: 0.0625 μg/mL and 0.625 μg/mL. Treatment with -PEG ₂₄ -DUPA:poly(IC) results in (A) IP-10, (B) RANTES, (C) IFN in PSMA overexpressing cells, LNCaP compared to low PSMA expressing cells, PC-3 or DU145. -Selectively induces the release of β cytokines.

Example 30

Signaling in PSMA cancer cell lines

LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) multicomplex were incubated at 20 °C in the presence of 5% glucose. Formulated at an N/P ratio of 4 in mM HEPES, pH 7.2.

Cancer cells that showed differential PSMA expression (400,000 cells/well in a 96-well plate) (LNCaP: high PSMA expression; DU145: low PSMA expression) were incubated with LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA: treated with poly(IC) or LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) multicomplexes. LNCaP cells were incubated with 0.00625 or 0.0625 μg/mL of LPEI- l -[N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) or 0.0625 μg/mL of LPEI- l -[N ₃ :DBCO]-PEG ₃₆ - DUPA: Treated with poly(Glu). DU145 cells were incubated with 0.0625 μg/mL of LPEI- l- [N ₃ :DBCO]-PEG ₂₄ -DUPA:poly(IC) or 0.0625 μg/mL of LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA: Treated with poly(Glu). Next, the cells were eluted, and the protein eluate was loaded onto SDS-PAGE, followed by sequencing of the indicated proteins (Cell Signaling; caspase 3 (9665), cleaved caspase 3 (9664), PARP (9542), cleaved PARP (5625), RIG-1 (3743); MDA5 (Abcam ab126630) and ISG15 (Santa Cruz SC-166755) (total 10 μg protein eluate/lane). GAPDH (Cell Signaling 2118) and beta-actin (Sigma A5441) were used as protein loading controls.

Figure 22 shows LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly at 0, 0.0625 and 0.625 μg/mL. (Glu) Western blot image analysis showing qualitative levels of caspase 3, cleaved caspase 3, PARP, cleaved PARP, RIG-1, MDA5, and ISG15 as a function of treatment with the multicomplex.

Treatment with LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC) is selective in cells overexpressing PSMA (LNCaP), targeting proteins associated with interferon-stimulated gene responses, such as MDA5, RIG-1 and ISG15. Inducing an increase in protein expression and cleavage of apoptosis markers such as PARP and caspase 3 were induced. No effect was observed in low PSMA expressing cells (DU145). The LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(Glu) control multicomplex did not induce this signal.

Example 31

Multicomplex morphology using SEM

Scanning electron microscopy (SEM) was performed on a Thermo-Scientific Teneo SEM instrument with the following parameters: beam energy: 1 keV; Beam current: 25 pA; Image size 1536 × 1024 pixels; It was performed using a dwell time of 30 μsec (500 nsec × 60 linear segments). Samples were “sputter” coated with 5 nm iridium prior to imaging. The multicomplex was prepared by combining Compound 31a and Compound 31b, namely LPEI- l- [N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC), with a N of 4 in HEPES 20 mM buffer, 5% glucose (HBG), pH 7.2. It was formed using a P ratio and a concentration of 0.1875 mg/mL. A drop (20 μL) of the multicomplex mixture on the stub was dried under vacuum and analyzed.

The resulting SEM image (FIG. 23) shows that the multicomposite particles containing Compound 31a and Compound 31b, that is, LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -DUPA:poly(IC), have a constant morphology with small size dispersion. It is confirmed that it is naturally spherical and exhibits a particle size in a range similar to the particle size determined by DLS analysis.

Example 32

Selective delivery of mRNA by multicomplex of the present invention

Materials and Methods

Firefly luciferase (Fluc) mRNA was purchased from Treelink Bitechnology, USA (catalog number L-7602; 1.0 mg/mL in 1 mM sodium citrate, pH 6.4; mRNA length: 1929 nucleotides). Lipofectamine Messenger Max was purchased from Thermo Fisher, and jetPEI was purchased from PolyPlus (catalog number 101000053). Cell culture reagents were purchased from Biology Industries, Israel. All reagents were used at the indicated concentrations according to the manufacturer's instructions.

Multicomplexes containing Fluc mRNA and LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF (i.e., Compound 70a and Compound 70b) were incubated in HEPES-buffered saline (HBS: 20 mM HEPES, 150 mM NaCl, pH 7.2) Triconjugate Fluc mRNA with N/P ratios of 4, 6, 12 (N = nitrogen from LPEI and P = phosphorus from mRNA) LPEI- l -[N ₃ :DBCO]-PEG ₃₆ -hEGF It was created by combining with. For complete formation of the multicomplex particles, LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA], samples were incubated for 30 min at room temperature.

Lenca blasts (mouse renal carcinoma, no human EGFR) and Lenca EGFR M1 H cells (derived from Lenca blasts engineered to overexpress human EGFR) were obtained from ATCC and cultured according to the manufacturer's protocol. Lenca (parent) cells were cultured in RPMI medium supplemented with 10% calf serum (FBS), 104 U/L penicillin, and 10 mg/L streptomycin at 37°C and 5% CO ₂ . 400 μg/mL of G418 was added to the medium of Lenca EGFR M1 H cells. 15,000 cells/well of Renca EGFR M1 H cells and 10,000 cells/well of Renca blast cells were inoculated into 96-well white plates (Greiner) and 96-well transparent plates (Nunc) in triplicate at 90 μL each. Cells were transfected with 0.125 to 1 mg/mL of LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA].

Luciferase activity was measured at the indicated times after treatment using the OneGloX assay (Promega). Luminescence measurements were performed using a Lumiskan Ascent microplate luminometer (Thermo Scientific). Values in arbitrary units (AU) are presented as the mean and standard deviation of luciferase activity from triplicate samples.

Cell survival was measured by a colorimetric assay using the methylene blue assay. Briefly, cells were fixed with 2.5% glutaraldehyde in PBS (pH 7.4), washed with dihydrogen-free water (DDW), and then incubated with 1% (w/v) methylene blue solution in borate buffer for 1 h. dyed. Afterwards, the stain was extracted with 0.1 M HCl and the optical density of the stain solution was read at 630 nm with a microplate reader (Synergy H1, Biotex). Luminescence and cell survival were measured 24 hours after treatment.

Physicochemical characterization of LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] multicomplexes of 3, 4, 5 and 6 in 50 mM acetate buffer, 5% glucose, pH 4.3 using DLS. It was measured by N/P ratio. A summary of the physicochemical measurements is given in Table 16. The z-average diameter ranged from 95 nm to 127 nm with a polydispersity index (PDI) of 134 to 0.209. The ζ-potential range measured by ELS was 29.7 to 45.6 mV.

Figure 24A shows luminescence (AU) of Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to control delivery vehicle Messenger Max. Luminescence was observed after treatment with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and Lipofectamine Messenger Max at N/P ratios of 4, 6, 12 and concentrations of 0.125 to 1.0 mg/mL for 24 hours. Measurements were made over time.

Figure 24B shows luminescence (AU) of Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to the control delivery vehicle jetPEI. Luminescence was measured 24 hours after treatment with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and jetPEI at N/P ratios of 4, 6, and 12 and concentrations of 0.125 to 1.0 mg/mL. .

Figure 24C shows the luminescence (AU) ratio between Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to Messenger Max as a comparative delivery vehicle. . Luminescence was observed after treatment with LPEI- l- [ _N3 :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and Lipofectamine Messenger Max at N/P ratios of 4, 6, and 12 and concentrations of 0.125 to 1.0 mg/mL. Measurements were made over time, and the ratio was calculated by dividing the luminescence signal from Lenca EGFR M1H cells by the luminescence signal from Lenca blast cells.

Figure 24D shows the luminescence (AU) ratio between Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] compared to the control delivery vehicle jetPEI. Luminescence was measured 24 hours after treatment with LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] and jetPEI at N/P ratios of 4, 6, and 12 and concentrations of 0.125 to 1.0 mg/mL. .

Figures 24A-24D show selective mRNA delivery to Lenca EGFR M1H cells over Lenca blast cells achieved using LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 4 to 12. It indicates that In contrast, the non-targeted delivery vehicles Lipofectamine Messenger Max and jetPEI did not show selective mRNA delivery to either cell line. In both cases untargeted delivery systems were demonstrated across all N/P ratios.

Figure 24E shows that LPEI- l -[N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] multicomplexes at N/P ratios of 4 and 6 are not cytotoxic.

Figure 25A shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 4 at 6 hours post-treatment. indicates.

Figure 25B shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 6 at 6 hours post-treatment. indicates.

Figure 25C shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 4 at 22 hours post-treatment. indicates.

Figure 25D shows relative luminescence (AU) in Lenca blasts and Lenca EGFR M1H cells treated with LPEI- l- [N ₃ :DBCO]PEG ₃₆ -hEGF:[Fluc mRNA] at N/P of 6 at 22 hours post-treatment. indicates.

Sequence list electronic file attached

Claims (44)

A composition comprising a conjugate,
The conjugate includes a linear polyethyleneimine fragment comprising an alpha terminus and an omega terminus; and a polyethylene glycol segment comprising a first terminal end and a second terminal end;
The alpha terminus of the polyethyleneimine fragment is the initiation residue;
The omega terminus of the polyethyleneimine fragment is connected to the first terminus of the polyethylene glycol fragment by a covalent bond -ZX ¹ -, where -Z- is not a single bond and -Z- is not an amide, and -X ¹ - is a divalent covalent moiety;
The composition of claim 1, wherein the second terminal end of the polyethylene glycol fragment is capable of binding a targeting fragment. According to clause 1,
The conjugate is a conjugate of formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,
R ¹ -(NR ² -CH ₂ -CH ₂ ) _n -ZX ¹ -(O-CH ₂ -CH ₂ ) _m -X ² -L (Formula I ^* )
here
n is any integer from 1 to 1,500;
m is any integer from 1 to 200;
R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;
R ² is independently -H or an organic moiety, and at least 80%, preferably 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;
X ¹ and X ² are independently divalent covalent moieties;
Z is a divalent covalent moiety, Z is not a single bond and Z is not -NHC(O)-;
L is a targeting fragment, preferably wherein the targeting fragment is capable of binding to a cell. According to claim 1 or 2,
The conjugate is a conjugate of formula I ^* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof,

here
is a single bond or a double bond;
n is any integer from 1 to 1,500;
m is any integer from 1 to 200;
R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;
R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;
Ring A is 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H;
X ¹ is a divalent covalent moiety;
X ² is a divalent covalent moiety;
L is a targeting fragment, preferably wherein the targeting fragment is capable of binding to a cell. According to claim 2 or 3,
The R ¹ -(NR ² -CH ₂ -CH ₂ ) _n - moiety has a repeating unit n of about 115 to about 1,150 and a dispersion of about 280 to about 700 with a dispersion of about 5 or less, preferably about 3 or less. A composition that is a dispersed polymeric moiety having n repeat units, more preferably about 350 to about 630 repeat units with a degree of dispersion of about 2 or less. According to any one of claims 2 to 4,
The -(O-CH ₂ -CH ₂ ) _m - moiety has about 2 to about 80 repeat units m and about 2 to about 70 repeat units m with a dispersity of about 2 or less, preferably about 1.8 or less. , more preferably a dispersed polymeric moiety having a repeating unit m of about 2 to about 50 with a degree of dispersion of about 1.5 or less. According to any one of claims 2 to 4,
The -(O-CH ₂ -CH ₂ ) _m - moiety comprises, preferably consists of, a definite number of repeat units m ranging from about 4 to about 60, and preferably the -(O-CH ₂ -CH ₂ ) _m - a composition wherein the moiety comprises, and preferably consists of, a definite number of contiguous repeat units m, from about 4 to about 60. According to any one of claims 3 to 6,
Ring A is 8-membered cycloalkenyl, 5-membered heterocycloalkyl or 7- to 8-membered heterocycloalkenyl, and each cycloalkenyl, heterocycloalkyl or heterocycloalkenyl may be substituted at any position by one or more A composition, wherein R is optionally substituted with ^A1 . According to any one of claims 3 to 7,
Ring A is cyclooctene, succinimide or 7- to 8-membered heterocycloalkenyl, said heterocycloalkyl or heterocycloalkenyl comprising 1 or 2 heteroatoms selected from N, O and S, Each cyclooctene, heterocycloalkyl or heterocycloalkenyl is optionally substituted at any position with one or more R ^A1 , preferably R ^A1 is oxo or fluorine. Two R ^A1 combine to form one or more fused phenyl rings, preferably 1 or 2 fused phenyl rings, each phenyl ring optionally substituted with one or more -SO ₃ H or -OSO ₃ H , composition. According to any one of claims 3 to 8,
The conjugate of Formula I is

A composition selected from: According to any one of claims 3 to 9,
The conjugate of Formula I is


A composition selected from: According to any one of claims 3 to 10,
The conjugate of Formula I is

A composition selected from: According to any one of claims 3 to 10,
The conjugate of Formula I is

A composition selected from: According to any one of claims 3 to 10,
The conjugate of Formula I is

A composition selected from: According to any one of claims 3 to 13,
X ¹ is

Contains a group selected from;
here
r is, at each occurrence, independently 0 to 6, preferably 0, 1, 2 or 5, more preferably 0;
s at each occurrence is independently 0 to 6, preferably 0, 2, 3 or 4, more preferably 2 or 3;
t is, at each occurrence, independently 0 to 6, preferably 0, 1, 2 or 4, more preferably 2;
R ¹¹ and R ¹² at each occurrence are independently selected from -H and -C ₁ -C ₂ alkyl, and are preferably -H;
R ¹³ is -H;
Preferably, the wavy line closest to the integer "r" is a bond to ring A, and the wavy line closest to the integer "s" or "t" is a bond to -[OCH ₂ -CH ₂ ] _m -, Composition. According to any one of claims 3 to 13,
X ¹ is

is selected from;
where X ^A is -NHC(O)- or -C(O)NH-;
Preferably, the wavy line on the left is a bond to Ring A, and the wavy line on the right is a bond to -[OCH ₂ -CH ₂ ] _m -. According to any one of claims 3 to 13,
X ¹ is

is selected from;
Preferably, the wavy line on the left is a bond to Ring A, and the wavy line on the right is a bond to -[OCH ₂ -CH ₂ ] _m -. According to any one of claims 3 to 16,
X ² is

is selected from;
where X ^B is -C(O)NH- or -NH-C(O)-;
For each Y ² , a chemical bond, -CR ²¹ R ²² -, NR ²³ -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbon ring moiety, is independently selected from a divalent heterocycle moiety and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl are optionally substituted with one or more R ²³ and each divalent heterocycle moiety is optional with one or more R ²⁴ is replaced with;
R ²¹ , R ²² and R ²³ are each independently -H, -SO ₃ H, -NH ₂ , -CO ₂ H or C ₁ -C ₆ alkyl in each case, and each C ₁ -C ₆ alkyl is one or more - optionally substituted with OH, oxo, -CO ₂ H, -NH ₂ , C ₆ -C ₁₀ aryl or 5- to 8-membered heteroaryl;
R ²⁴ is independently -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo at each occurrence, preferably the wavy line on the left is a bond to -[OCH ₂ -CH ₂ ] _m -, The composition wherein the wavy line on the right is a bond to L. According to any one of claims 3 to 16,
X ² is

is selected from;
For each Y ² , a chemical bond, -CR ²¹ R ²² -, NR ²³ -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbon ring moiety, is independently selected from a divalent heterocycle moiety and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl are optionally substituted with one or more R ²³ and each divalent heterocycle moiety is optional with one or more R ²⁴ is replaced with;
R ²¹ , R ²² and R ²³ are each independently at each instance -H, -SO ₃ H, -NH ₂ , -CO ₂ H or C ₁ -C ₆ alkyl, and each C ₁ -C ₆ alkyl is one or more - optionally substituted with OH, oxo, -CO ₂ H, -NH ₂ , C ₆ -C ₁₀ aryl or 5- to 8-membered heteroaryl;
R ²⁴ is independently at each occurrence -H, -CO ₂ H, C ₁ -C ₆ alkyl or oxo, preferably the wavy line on the left is a bond to -[OCH ₂ -CH _{2] m -, and the wavy line on the left is a bond to -[OCH 2 -CH 2} ] _m - The wavy line of is a bond to L, composition. According to any one of claims 3 to 16,
X ² is

is selected from;
Preferably, the wavy line on the left is a bond to -[OCH ₂ -CH ₂ ] _m -, and the wavy line on the right is a bond to L. According to any one of claims 3 to 16,
X ² is

ego;
Preferably, the wavy line on the left is a bond to -[OCH ₂ -CH ₂ ] _m -, and the wavy line on the right is a bond to L. According to any one of claims 3 to 16,
X ² is

Phosphorus, composition. According to any one of claims 3 to 21,
The composition, wherein the targeting fragment L is capable of binding to a cell surface receptor, and preferably the targeting fragment L is capable of specifically binding to a cell surface receptor. According to clause 22,
The cell surface receptors include growth factor receptors, extracellular matrix proteins, cytokine receptors, hormone receptors, glycosylphosphatidylinositol (GPI) anchored membrane proteins, carbohydrate-binding membrane integral proteins, lectins, ion channels, and G-protein coupled receptors. and an enzyme coupled receptor, such as a tyrosine kinase coupled receptor. According to claim 22 or 23,
The cell surface receptors include epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), prostate surface membrane antigen (PSMA), insulin-like growth factor 2 receptor (IGF1R), and vascular endothelial growth factor receptor (VEGFR). , platelet-derived growth factor receptor (PDGFR), asialo glycoprotein receptor (ASGPr), and fibroblast growth factor receptor (FGFR). According to any one of claims 1 to 24,
The targeting fragment L is capable of binding to a cell surface receptor, and the targeting fragment is a peptide, protein, small molecule ligand, saccharide, oligosaccharide, oligonucleotide, lipid, amino acid, antibody, antibody fragment, aptamer or apichain, composition . According to any one of claims 1 to 25,
The targeting fragment L is an EGFR targeting fragment; PSMA targeting fragment, preferably DUPA residues; anti-HER2 peptide, preferably anti-HER2 antibody or apibody; folic acid; A somatostatin receptor targeting fragment, preferably somatostatin and/or octreotide; an integrin targeting fragment, preferably a fragment comprising arginine-glycine-aspartic acid (RGD); low-pH insert peptide; ASGPr targeting fragment, preferably asialoorosomucoid; Insulin receptor targeting fragment, preferably insulin; Mannose-6-phosphate receptor targeting fragment, preferably mannose-6-phosphate; Mannose receptor targeting fragment, preferably mannose; Sialyl Lewis ^X antigen targeting fragment, preferably E-selectin; Sigma-2 receptor agonists, preferably N,N-dimethyltryptamine (DMT), sphingolipid-derived amines and/or steroids, more preferably progesterone; p32 targeting ligand, preferably anti-p32 antibody or p32 binding LyP-1 tumor homing peptide; Trop-2 targeting fragments, preferably anti-Trop-2 antibodies and/or antibody fragments; Insulin-like growth factor 1; vascular endothelial growth factor; platelet-derived growth factor; and fibroblast growth factors. According to any one of claims 1 to 26,
The composition of claim 1, wherein the targeting fragment L is the DUPA moiety (HOOC(CH ₂ ) ₂ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ₂ ) ₂ -CO-). According to any one of claims 1 to 27,
The conjugate is Compound 1a, Compound 1b, Compound 4a, Compound 4b, Compound 7a, Compound 7b, Compound 10a, Compound 10b, Compound 14, Compound 17a, Compound 17b, Compound 18, Compound 19, Compound 22a, Compound 22b, Compound 28a, Compound 28b, Compound 31a, Compound 31b, Compound 38a, Compound 38b, Compound 43, Compound 47a, Compound 47b, Compound 51a, Compound 51b, Compound 56a, Compound 56b, Compound 62a, Compound 62b, Compound 70a, Compound 70b, A composition selected from Compound 72a, Compound 72b, Compound 75a, Compound 75b, Compound 78a, and/or Compound 78b. According to any one of claims 1 to 28,
The composition further comprises a polyanion, preferably the polyanion is a nucleic acid, the polyanion is preferably non-covalently bound to the conjugate, and the polyanion and the conjugate form a polycomplex. Composition. According to clause 29,
The composition, wherein the polyanion is a nucleic acid, and the nucleic acid is dsRNA or ssRNA. According to clause 30,
The composition of claim 1, wherein the nucleic acid is dsRNA. According to clause 31,
The composition of claim 1, wherein the dsRNA is polyinosinic acid:polycytidylic acid (poly(IC)). According to clause 30,
The composition of claim 1, wherein the nucleic acid is ssRNA. According to clause 33,
A composition wherein the ssRNA is mRNA. A polycomplex of a conjugate as defined in any one of claims 1 to 34 and a polyanion, preferably wherein the polyanion is non-covalently bound to the conjugate, and preferably wherein the polyanion is a nucleic acid. complex. A conjugate of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or optical isomer thereof, and a polyanion, preferably a nucleic acid, comprising a polyanion, preferably a nucleic acid. is non-covalently bound to the conjugate;

here
is a single bond or a double bond;
n is any integer from 1 to 1,500;
m is a repeat unit m with a definite number of 2 to 200, preferably a definite number m of repeat units from 4 to 60;
R ¹ is an initiation moiety, preferably R ¹ is -H or -CH ₃ ;
R ² is independently -H or an organic moiety, and at least 80%, preferably at least 90%, of R ² in -(NR ² -CH ₂ -CH ₂ ) _n - is H;
Ring A is a 5- to 10-membered cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalkenyl optionally substituted at any position with one or more R ^A1 ; R ^A1 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, oxo or halogen; Two R ^A1s may combine together with the atoms to which they are attached to form one or more fused C ₆ -C ₁₀ aryl, C ₅ -C ₆ heteroaryl or C ₃ -C ₆ cycloalkyl rings, each fused Aryl, heteroaryl or cycloalkyl is optionally substituted with one or more R ^A2 ; R ^A2 is independently selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, halogen, -SO ₃ H or -OSO ₃ H,
X ¹ is a divalent covalent moiety,
X ² is a divalent covalent moiety,
L is a targeting fragment, preferably a multicomplex wherein the targeting fragment is capable of binding to cells. The method of claim 35 or 36,
The polyanion is a nucleic acid, and the nucleic acid is RNA. According to clause 37,
The RNA is a multicomplex that is dsRNA or ssRNA. According to clause 37,
The RNA is dsRNA. Multiple complex. According to clause 39,
The dsRNA is a polyinosinic acid:polycytidylic acid (poly(IC)) complex. According to clause 37,
The RNA is ssRNA. Multiple complex. According to clause 41,
The ssRNA is mRNA. Multiple complex. Comprising the composition of any one of claims 1 to 34 or the multicomplex of any of claims 35 to 42, and optionally one or more pharmaceutically acceptable excipient(s) and/or carrier(s). A pharmaceutical composition. A composition of any one of claims 1 to 34, a multicomplex of any of claims 35 to 42 or a pharmaceutical composition of claim 43, which is suitable for treating cancer, preferably EGFR; HER2; Prostate-specific membrane antigen; folate receptor; Integrin, preferably RGD integrin; Asialo glycoprotein receptor; insulin receptor; Mannose-6-phosphate receptor; Mannose receptor; Glycoside, preferably sialyl Lewis ^x antigen; Sigma-2 receptor; p32 protein; or a composition, multicomplex or pharmaceutical composition for use in the treatment of cancer characterized by cells overexpressing Trop-2.