PSMA-targeted linear conjugates containing polyethyleneimine and polyethylene glycol and polyplexes containing the same

Abstract

The present invention relates to a polyplex comprising a linear conjugate of LPEI and PEG. The LPEI and PEG fragments of the linear conjugate are preferably linked by a [3+2] cycloaddition between azide and alkene or alkyne to generate 1,2,3 triazole or 4,5-dihydro-1H-[1,2,3]triazole. The linear conjugate is further conjugated to a targeting fragment that can bind to prostate-specific membrane antigen (PSMA) to enable selective interaction with specific cell types. The conjugate can form a polyplex with a therapeutic agent, such as a nucleic acid, to deliver the therapeutic agent to cells.

Description

[Background technology]

[0001] Cancer remains a leading cause of death worldwide. For most solid tumors after surgical removal, chemotherapy is an important treatment option for managing remaining cancer cells. The main reason for chemotherapy failure is inefficient targeting and uptake of chemotherapeutic agents by tumors (Vasir & Labhasetwar Technology in Cancer Research & Treatment 4(4), 363-374(2005)). Insufficient tumor accessibility necessitates higher doses, and due to the nature of chemotherapeutic agents, this leads to nonspecific uptake and toxicity in healthy cells. Targeted drug delivery strategies, in which therapeutic agents reversibly bind to targeting ligands and are selectively delivered to cells for treatment, are currently applied to many chemotherapeutic agents in clinical use. This strategy shows promise for maximizing the safety and efficacy of a given chemotherapeutic agent, as their selective delivery to target cells avoids nonspecific uptake and associated toxicity in healthy cells, which can result in a higher maximum tolerated dose (Srinivasarao & Low, Chem. Rev., 117, 12133-12164, (2017)).

[0002] Cationic polymers are known to form polyplexes with negatively charged nucleic acids in solution. For example, linear polyethyleneimine (LPEI) is protonated at physiological pH and therefore has a net positive charge. When LPEI is incubated with a nucleic acid, which has a net negative charge at physiological pH, the LPEI and nucleic acid can form polyplexes held together by electrostatic interactions. These polyplexes can be internalized by cells in vivo and deliver nucleic acid sequences into cells. Therefore, polyplexes containing cationic polymers and nucleic acids can be used as vectors for therapy. Despite their promise, technical challenges have arisen regarding the formation of homogeneous, well-characterized cationic polymers. Polyplexes containing only LPEI are prone to aggregation and interactions with serum proteins, potentially limiting their potential as nucleic acid delivery agents. To overcome these challenges, the polymer LPEI can be conjugated to polyethylene glycol (PEG). PEG fragments can help shield LPEI from the surrounding matrix, improving the biocompatibility and blood circulation of the resulting polyplexes.

[0003] However, coupling of PEG to LPEI occurs via the formation of a covalent bond between electrophilic PEG fragments and secondary amines embedded within the LPEI backbone fragment, resulting in branched, heterogeneous conjugates and vectors that incorporate PEG fragments randomly and undefinedly, characterized based on the average PEG inclusion density. In such conjugates, multiple PEG fragments are typically orthogonally attached to the LPEI fragment without site specificity. Such random synthesis and inaccurate characterization of LPEI-PEG conjugates can make it difficult to establish a clear structure-activity relationship (SAR) between the conjugate structure and the activity of the resulting polyplex. International Publication No. 2015 / 173824 discloses polyplexes of polymer conjugates consisting of double-stranded RNA, such as poly(IC), and LPEI-PEG conjugates bearing orthogonally attached PEG fragments, each of which is linked to a targeting moiety capable of binding to a cancer antigen. As an example, a polymer conjugate and vector targeting prostate-specific membrane antigen (PSMA) are described.

[0004] Prostate-specific membrane antigen (PSMA) is a multifunctional transmembrane protein that exhibits dual enzymatic functions as a glutamate carboxypeptidase and a folate hydrolase, and also exhibits rapid ligand-induced internalization and recycling (Ghosh A et al., J Cell Biochem 2004, 91:528-539; Liu H et al., 1998, Cancer Res 58:4055-4060). PSMA is a type II membrane protein originally characterized by the mouse monoclonal antibody (mAb) 7E11-C5.3. The PSMA protein has a unique tripartite structure: a 19-amino acid internal segment, a 24-amino acid transmembrane segment, and a 707-amino acid external segment (Chang SS, Rev Urol. 2004, 6(suppl 10):S13-S18). PSMA is also known by the additional names glutamate carboxypeptidase II (GCPII), N-acetyl-α-linked acidic dipeptidase, and folate hydrolase (FOLH1) (Jeitner™ et al., Translational Oncology 2022, 22:101450). PSMA is primarily expressed in four tissues of the body, including the prostate epithelium, the proximal tubules of the kidney, the jejunal brush border of the small intestine, and the ganglia of the nervous system (Mhawech-Fauceglia et al., Histopathology 2007, 50:472-483). Because PSMA expression is approximately 1,000-fold higher in prostate tumors than in healthy tissue, PSMA is particularly considered a target for the diagnosis and treatment of prostate cancer (Kularatne SA et al., Molecular Pharmaceutics 2009,6(3):780-789; Rowe SP et al., Prostate Cancer Prostatic Dis. 2016,19(3):223-230; Wang H et al., Small Struct. 2022,3:220003620;9; Juzeniene A et al, Cancers 2021,13(4):779).Furthermore, upregulation of PSMA may provide a growth advantage to prostate cancer cells, implicating PSMA in the metabolism of polyglutamylated folates and subsequent folate uptake (Yao et al., Prostate 2006, 66:867-875; Yao et al., Prostate 2010, 70:305-316). However, PSMA targeting may also be applicable to other PSMA-expressing tumors besides prostate cancer, particularly because PSMA is not expressed on normal vasculature but is expressed on the neovasculature of many solid tumors, e.g., breast, lung, gastric, colorectal, pancreatic, renal cell, and bladder cancers, allowing targeting to occur in the intravascular compartment (Chang SS et al., Cancer Res. 1999, 59(13):3192-3198; Wernicke et al., APMIS 2014, 122(6):482-489; Samplaski MK et al., Mod Pathol. 2011, 24(11):1521-1529; Haffner MC et al., Hum Pathol. 2009, 40(12):1754-1761; Morgenroth A et al., Breast Cancer Research 2019,21:116; Jian D et al., Clinical and Translational Gastroenterology 2019;10:e-00041; Jeitner TM et al., Translational Oncology 2022,22:101450, and references cited therein).

[0005] Overexpression of PSMA in the neovasculature of prostate cancer tissue and most solid tumors makes it a target for the delivery of cancer therapeutics (Barrett JA et al., J Nucl Med 2013, 54:380-387 and references cited therein). When the ligand is recognized by a specific receptor on the membrane of cancer cells, an internalization signal is often generated, followed by cellular uptake via receptor-mediated endocytosis. Therefore, targeting of PSMA has primarily relied on PSMA-targeting antibodies such as J591 or 7E11 (Viola-Villegas NT et al., Mol Pharm 2014,11:3965-3973 and references cited therein), PSMA aptamers (Baek SE et al., J Control Release 2014,196:234-242 and references cited therein), and small ligands such as glutamic acid urea (Roy J et al., Journal of Medicinal Chemistry 2015,58(7):3094-3103; Shallal HM et al., Bioconjug Chem 2014,25:393-405; Lutje S et al., Theranostics 2015,5:1388; Langut Y et al., PNAS 2017,114(52):13655-13660; and references cited therein), and as reported in several studies, is promoted by folic acid (Patil Y et al., Nanomedicine 2018,14(4):1407-1416; Flores O et al., Theranostics 2017,7(9):2477-2494; and references cited therein). Summary of the Invention

[0006] Instead of random and uncontrolled attachment of electrophilic PEG fragments to multiple nucleophiles on the LPEI backbone fragment, the present invention provides PSMA-targeting conjugates comprising LPEI and specifically defined, distinct molecular weight PEG fragments connected by distinct linkages formed by defined chemoselective reactions. Thus, the present invention provides more uniform PSMA-targeting conjugates with defined chemical structures. The distinct, specifically defined components and linkages not only ensure a consistent and predictable ratio of all components of the conjugates of the present invention, including a consistent and predictable ratio of LPEI to PEG fragments, but also ensure a defined, linear conjugate instead of a randomly branched conjugate. Thus, the LPEI fragment is attached in a linear, end-to-end manner to a single, specifically defined, distinct PEG fragment with a defined, distinct molecular weight, which is further connected to a targeting fragment capable of binding to PSMA. The chemoselective attachment of the LPEI fragment to the specifically defined, distinct PEG fragment can be achieved using any suitable chemical precursor capable of forming a chemoselective bond. In a preferred embodiment, the chemoselective attachment of the LPEI fragment to a specifically defined, distinct PEG fragment occurs via a [3+2] cycloaddition between an azide and an alkyne or alkene, resulting in a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole.

[0007] In the conjugates of the present invention, the PEG fragment is further selectively linked to a targeting fragment capable of binding to prostate-specific membrane antigen (PSMA) to target and promote uptake of the compositions, conjugates, and / or polyplexes of the present invention, particularly in PSMA-targeted cell types. Thus, preferred embodiments and conjugates include one or more, typically and preferably one, targeting fragment such as folic acid specifically linked to a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) or an LPEI-PEG diconjugate, forming an LPEI-PEG-targeting fragment triconjugate capable of targeting PSMA in a specific cell type, typically a cancer cell type. In the polyplexes of the present invention, such triconjugates are combined with a polyanion, e.g., a nucleic acid, and thereby preferably with polyinosinic:polycytidylic acid (poly(IC)), which can serve as a cytotoxic and / or immunostimulatory payload to be delivered to and internalized within cells.

[0008] Even more surprisingly and advantageously, the present inventors have found that preferred conjugates and polyplexes obtained according to the present invention, which exhibit significantly reduced heterogeneity and, therefore, significantly reduced numbers of potentially biologically active conjugates and polyplexes due to the defined chemoselective attachment of LPEI fragments to specifically defined, distinct PEG fragments, not only form polyplexes of appropriate size, but also maintain or even increase their overall biological activity, such as potency and selectivity for reducing survival and inducing cell death in targeted cancer cells. Furthermore, compositions and polyplexes of the present invention comprising nucleic acids encoding peptides or proteins of interest, particularly pharmaceutically active peptides or proteins such as cytokines, interferons, or toxins, not only selectively deliver the pharmaceutically active nucleic acids encoding the pharmaceutically active peptides or proteins to target cells, particularly cancer cells, but also result in high expression and efficient protein translation and secretion of the encoded pharmaceutically active proteins.

[0009] Accordingly, in one aspect, the invention provides a composition comprising a conjugate, the conjugate comprising: a linear polyethyleneimine fragment comprising an alpha end and an omega end; a polyethylene glycol fragment comprising a first end and a second end, the polyethylene glycol fragment comprising, and preferably consisting of, a discrete number m of repeating -(O-CH-CH)- units, wherein said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably 25 to 60, and preferably said discrete number m is a discrete number of consecutive repeating -(O-CH-CH)- units, wherein said discrete number of consecutive repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably 25 to 60; the alpha end of the polyethyleneimine fragment is an initiating residue; and the omega end of the polyethyleneimine fragment is a divalent covalent linking group -ZX 1 -(in the formula, -ZX 1- is not a single bond and -Z- is not an amide); the second end of the polyethylene glycol fragment is connected to a divalent covalent linking moiety X 2 wherein the targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), and preferably the targeting fragment is capable of binding to cells expressing PSMA.

[0010] In another aspect, the present invention provides a composition comprising a conjugate, wherein said conjugate is of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); During the ceremony, n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90% of which is H; X 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, ZX 1 is not a single bond and Z is not NHC(O)-; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA; Preferably, a composition comprising the conjugate is provided, which consists of the conjugate.

[0011] In another aspect, the present invention provides a composition comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA.

[0012] Although the N-N=N fragment of the bicyclic ring in Formula I is typically depicted herein using one single bond and one double bond for simplicity, those skilled in the art will recognize that Formula I and related conjugate structures depicted herein can alternatively be depicted as shown below. Such depictions and descriptions of Formula I are used interchangeably herein: [ka] Here, the fragment [ka] is the fragment R 1 (NR 2 CH2CH2) n Two different regioisomeric bonds, i.e., [ka] and [ka] where the wavy line represents a chemical bond to ring A. Thus, Formula I as depicted herein represents two regioisomeric embodiments, namely, fragment R 1 (NR 2 CH2CH2) n is attached to the top nitrogen atom of the above structure or the bottom nitrogen atom of the above structure, but not to the middle nitrogen atom. One of skill in the art will know that the same applies to other formulas herein, including Formula IA, Formula IB, Formula IC, Formula ID, Formula IE, Formula IH, Formula IJ, Formula IK, etc.

[0013] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more RA1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA.

[0014] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n - the R in the part 2 at least 80%, preferably 90% of which is H; Ring A may have one or more R A1 is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with; R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl or heteroaryl is selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 optionally substituted with R 11 , R 12 and R 13 is independently at each occurrence H or C1-C6 alkyl; R 14 is a linking moiety for (each occurrence is independently H, C1-C6 alkyl, or oxo); X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 optionally substituted with R 21 , R 22 and R 23 are each independently at each occurrence -H, -COH, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, C6-C 10 optionally substituted with aryl, or 5-8 membered heteroaryl; R 24 is a linking moiety for (each occurrence is independently -H, -COH, C-C alkyl, or oxo); L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA. Preferably, a composition is provided that consists of the conjugate.

[0015] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n - the R in the part 2 at least 80%, preferably 90% of which is H; Ring A may have one or more R A1 is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2optionally substituted with; R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl or heteroaryl is selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 optionally substituted with R 11 , R 12 and R 13 is independently at each occurrence H or C1-C6 alkyl; R 14 is a linking moiety for (each occurrence is independently H, C1-C6 alkyl, or oxo); X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 optionally substituted with R 21 , R 22 and R 23are each independently at each occurrence -H, -COH, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, C6-C 10 optionally substituted with aryl, or 5-8 membered heteroaryl; R 24 is a linking moiety for (each occurrence is independently -H, -COH, C-C alkyl, or oxo); L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA.

[0016] In a further aspect, the present invention provides methods for synthesizing a composition comprising a conjugate of Formula I, preferably a plurality of conjugates, comprising reacting an azide-containing LPEI fragment with an alkene- or alkyne-containing PEG fragment at a pH below about 5, preferably below about 4. In some preferred embodiments, the LPEI fragment comprises an azide at the omega terminus and the PEG fragment comprises an alkene or alkyne at a first terminus.

[0017] In a further aspect, the present invention provides a polyplex comprising a composition described herein and a polyanion, preferably wherein said polyanion is a nucleic acid, more preferably wherein said nucleic acid is RNA, and again more preferably wherein said polyanion is polyinosinic:polycytidylic acid (poly(IC)).

[0018] In a further aspect, the present invention provides a polyplex comprising a composition described herein and a nucleic acid. In a further aspect, the present invention provides a polyplex comprising a composition described herein and a nucleic acid, wherein the nucleic acid is RNA. In a further aspect, the present invention provides a polyplex comprising a composition described herein and polyinosinic:polycytidylic acid (poly(IC)).

[0019] In another aspect, the present invention provides a polyplex comprising a triconjugate as described herein, preferably said conjugate of Formula I* or Formula I, and a polyanion, e.g., a nucleic acid, preferably polyinosinic:polycytidylic acid (poly(IC)).

[0020] In a further aspect, the present invention provides a polyplex comprising a composition described herein and a nucleic acid, wherein the nucleic acid is mRNA. In a further aspect, the present invention provides a polyplex comprising a composition described herein and a nucleic acid, wherein the nucleic acid is DNA, preferably plasmid DNA.

[0021] In one aspect, the present invention provides pharmaceutical compositions comprising a triconjugate described herein, preferably said conjugate of Formula I* or Formula I, and / or a polyplex, and pharmaceutically acceptable salts thereof.

[0022] In one aspect, the present invention provides a polyplex as described herein, or a pharmaceutical composition comprising a polyplex as described herein, for use in the treatment of a disease or disorder, preferably cancer, more preferably prostate cancer.

[0023] In one aspect, the present invention provides the use of a polyplex as described herein for use in the manufacture of a medicament for the treatment of a disease or disorder, such as cancer, more preferably prostate cancer.

[0024] In another aspect, the present invention provides a method of treating a disease or disorder, e.g., cancer, preferably prostate cancer, in a subject in need thereof, comprising administering to the subject an effective amount of a polyplex described herein.

[0025] The compositions and polyplexes of the invention, including the linear, non-random LPEI-PEG diconjugates and triconjugates described herein, not only ensure a consistent and predictable ratio of LPEI fragments to PEG fragments, but also typically and preferably ensure a structurally defined, linear conjugate of LPEI fragments to PEG fragments. Thus, they offer greater batch-to-batch consistency, ease of manufacturing, and more predictable SAR compared to the branched LPEI-PEG diconjugates currently prepared using the random, uncontrolled synthetic strategies described above.

[0026] Even more advantageously and surprisingly, when the linear, non-random conjugates of the present invention described herein are combined with polyanions and nucleic acids, such as poly(IC), to form polyplexes and administered to cells, the polyplexes surprisingly maintain and even exhibit superior antitumor activity relative to polyplexes made using random, branched conjugates. Thus, despite the significant reduction in variability and number of conjugate structures used and, therefore, in the number of possible (biological) activities, including targeting and presentation of their target fragments to the surface of targeted cells and subsequent uptake, there is no loss in the efficacy of the linear LPEI-l-PEG:nucleic acid polyplexes described herein. In contrast, the conjugates and compositions of the present invention maintain or even increase their overall biological activity. Further features and advantages of this technology will be apparent to those skilled in the art upon reading the following detailed description of the invention, and further aspects and embodiments of the present invention will become apparent as this description continues. [Brief explanation of the drawings]

[0027] [Figure 1]Triplicate DLS backscattering plots of Me-LPEI-l-[N3:BCN]-PEG36-DUPA:poly(IC) polyplexes measuring size distribution and zeta potential in 20 mM HEPES, 5% glucose (pH 7.2, 0.1875 mg / mL, 1.0 mL volume, N / P ratio 4). The z-average diameter was 130 nm and the polydispersity index (PDI) was 0.134. The zeta potential was 26.6 mV. [Figure 2] Triplicate DLS backscattering plots of Me-LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) polyplexes measuring size distribution and zeta potential in 20 mM HEPES, 5% glucose (pH 7.2, 0.1875 mg / mL, 1.0 mL volume, N / P ratio 4). The z-average diameter was 140 nm and the polydispersity index (PDI) was 0.132. The zeta potential was 28.2 mV. [Figure 3]

[0023] Figure 1 depicts differential PSMA expression as determined in vitro by flow cytometry for an array of human prostate cancer cell lines (LNCaP, VCaP, PC-3, DU145). Staggered histograms of fluorescence intensity are shown, with mean fluorescence intensity (MFI) indicated. [Figure 4A] Flow cytometry analysis of MHC I expression on the cell surface of prostate cancer cell lines with high PSMA expression (LNCaP) or no treatment (untreated control) as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu) polyplexes at payloads of 0.0125 and 0.125 μg / mL. Isotype and unstained controls show background fluorescence. Staggered histograms of fluorescence intensity are shown, and mean fluorescence intensity (MFI) is indicated. [Figure 4B]Flow cytometry analysis of MHC I expression on the cell surface of prostate cancer cell lines with low PSMA expression (DU145) or no treatment (untreated control) as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu) polyplexes at payloads of 0.0125 and 0.125 μg / mL. Isotype and unstained controls show background fluorescence. Staggered histograms of fluorescence intensity are shown, and mean fluorescence intensity (MFI) is indicated. [Figure 5A] 1 is a plot of cell viability in LNCaP cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG24-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG24-DUPA:poly(Glu). The x-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 5B] 1 is a plot of cell viability in PC-3 cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG24-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG24-DUPA:poly(Glu). The x-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 5C] 1 is a plot of cell viability in DU145 cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG24-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG24-DUPA:poly(Glu). The x-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 5D] 1 is a plot of cell viability in LNCaP cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG24-folate:poly(IC) and LPEI-l-[N3:DBCO]-PEG24-folate:poly(Glu). The x-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 5E]1 is a plot of cell viability in DU145 cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG24-folate:poly(IC) and LPEI-l-[N3:DBCO]-PEG24-folate:poly(Glu). The x-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 6A] 1 is a plot of cell viability in LNCaP cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu). The X-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 6B] 1 is a plot of cell viability in PC-3 cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu). The x-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 6C] 1 is a plot of cell viability in DU145 cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu). The X-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 7] 1 is a plot of cell viability in LNCaP cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC), LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu), Me-LPEI[N3:DBCO]PEG36-[MAL-S]-DUPA:poly(IC), and Me-LPEI[N3:DBCO]PEG36-[MAL-S]-DUPA:poly(Glu). The X-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 8]1 is a plot of cell viability in LNCaP cells as a function of treatment with LPEI-1-[N3:BCN]-PEG36-[MAL-S]-DUPA:poly(IC), LPEI-1-[N3:BCN]-PEG36-[MAL-S]-DUPA:poly(Glu), Me-LPEI[N3:BCN]PEG36-[MAL-S]-DUPA:poly(IC), and Me-LPEI[N3:BCN]PEG36-[MAL-S]-DUPA:poly(Glu). The X-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 9] 1 is a plot of cell viability in DU145 prostate cancer cells with low PSMA expression as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC), LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu), Me-LPEI[N3:DBCO]PEG36-[MAL-S]-DUPA:poly(IC), and Me-LPEI[N3:DBCO]PEG36-[MAL-S]-DUPA:poly(Glu). The X-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 10] 1 is a plot of cell viability in DU145 prostate cancer cells with low PSMA expression as a function of treatment with LPEI-l-[N3:BCN]-PEG36-DUPA:poly(IC), LPEI-l-[N3:BCN]-PEG36-DUPA:poly(Glu), Me-LPEI[N3:BCN]PEG36-[MAL-S]-DUPA:poly(IC), and Me-LPEI[N3:BCN]PEG36-[MAL-S]-DUPA:poly(Glu). The X-axis shows the logarithm of the concentration of delivered poly(IC) or poly(Glu). [Figure 11]1 is a plot of cell viability in LNCaP cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC); LPEI-l-[N3:DBCO]-PEG36-[(NH2)MAL-S]-DUPA:poly(IC); LPEI-l-[N3:BCN]-PEG36-DUPA:poly(IC); LPEI-l-[N3:SCO]-PEG36-[MAL-S]-DUPA:poly(IC); LPEI-l-[N3:DBCO]-PEG36-[CONH]-DUPA:poly(IC); and LPEI-l-[N3:DBCO]-PEG36-[S-MAL]-DUPA:poly(IC) polyplexes. The X-axis represents the logarithm of the concentration of delivered poly(IC). [Figure 12] 1 is a plot of cell viability in VCaP prostate cancer cells with intermediate PSMA cell surface expression as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu). The X-axis indicates the concentration of delivered poly(IC) or poly(Glu). [Figure 13] 1 is a plot of cell viability in DU145 cells as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC); LPEI-l-[N3:DBCO]-PEG36-[(NH2)MAL-S]-DUPA:poly(IC); LPEI-l-[N3:BCN]-PEG36-DUPA:poly(IC); LPEI-l-[N3:SCO]-PEG36-[MAL-S]-DUPA:poly(IC); LPEI-l-[N3:DBCO]-PEG36-[CONH]-DUPA:poly(IC); and LPEI-l-[N3:DBCO]-PEG36-[S-MAL]-DUPA:poly(IC) polyplexes. The X-axis indicates the concentration of delivered poly(IC). [Figure 14A] 1 is a plot of IP-10 secretion as a function of LPEI-1-[N3:DBCO]-PEG24-DUPA:poly(IC) concentration in LNCaP and PC-3 cells. [Figure 14B]1 is a plot of IP-10 secretion as a function of LPEI-1-[N3:DBCO]-PEG36-DUPA:poly(IC) concentration in LNCaP and PC-3 cells. [Figure 14C] 1 is a plot of IP-10 secretion as a function of LPEI-1-[N3:DBCO]-PEG36-DUPA:poly(IC) concentration in LNCaP and DU145 cells. [Figure 15A] 1 is a plot of RANTES secretion as a function of LPEI-1-[N3:DBCO]-PEG24-DUPA:poly(IC) concentration in LNCaP and PC-3 cells. [Figure 15B] 1 is a plot of RANTES secretion as a function of LPEI-1-[N3:DBCO]-PEG36-DUPA:poly(IC) concentration in LNCaP and PC-3 cells. [Figure 15C] 1 is a plot of RANTES secretion as a function of LPEI-1-[N3:DBCO]-PEG36-DUPA:poly(IC) concentration in LNCaP and DU145 cells. [Figure 16A] 1 is a plot of IFNβ secretion as a function of LPEI-1-[N3:DBCO]-PEG24-DUPA:poly(IC) concentration in LNCaP and PC-3 cells. [Figure 16B] 1 is a plot of IFNβ secretion as a function of LPEI-1-[N3:DBCO]-PEG36-DUPA:poly(IC) concentration in LNCaP and PC-3 cells. [Figure 16C] 1 is a plot of IFNβ secretion as a function of LPEI-1-[N3:DBCO]-PEG36-DUPA:poly(IC) concentration in LNCaP and DU145 cells. [Figure 17]Western blot imaging analysis showing the qualitative levels of caspase-3, cleaved caspase-3, PARP, cleaved PARP, RIG-1; MDA5, and ISG15 as a function of treatment with 0, 0.0625, and 0.625 μg / mL LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu) polyplexes. GAPDH served as a protein loading control. [Figure 18] Immunoblot analysis of prostate cancer cells with high PSMA (LNCaP) and low PSMA expression (DU145) as a function of treatment with LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC) and LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(Glu) polyplexes at 0.02 and 0.2 μg / mL payload (poly(IC) and poly(Glu) respectively) for 5 and 24 hours. The analysis shows qualitative levels of IκB, phospho-IκB, IRF3, phospho-IRF3, NFκB, phospho-NFκB, and PD-L1. GAPDH served as a protein loading control. [Figure 19] SEM images of polyplex particles containing compounds 31 and 31b and poly(IC), i.e., LPEI-l-[N3:DBCO]-PEG36-DUPA:poly(IC), formed at a concentration of 0.1875 mg / mL in HEPES buffer, 5% glucose (HBG), pH 7.2, with an N / P ratio of 4 and 20 mM. [Figure 20]Human prostate cell lines with differential PSMA cell surface expression: Luminescence normalized to viability in PSMA-high-expressing LNCaP cells and PSMA-low-expressing DU145 cells after transfection with PSMA-targeted polyplexes containing mRNA encoding luciferase. The X-axis indicates the concentration of mRNA in the polyplex (0.25, 0.5, and 1.0 μg / mL). The Y-axis indicates luminescence normalized to viability in arbitrary units (AU). Selective transfection of PSMA-overexpressing cells with Luc mRNA and selective expression of luciferase were demonstrated. [Figure 21] Figure 1 shows the levels of secreted human IL-2 from two cell lines with differential PSMA expression: PSMA-high expressing LNCaP cells and PSMA-low expressing DU145 cells after transfection with PSMA-targeted polyplexes containing hIL-2 mRNA. Selective expression of human IL-2 from PSMA-overexpressing cells is demonstrated. [Figure 22] Figure 1 shows the levels of secreted human IFNβ from two cell lines with differential PSMA expression: PSMA-high expressing LNCaP cells and PSMA-low expressing DU145 cells after transfection with PSMA-targeted polyplexes containing hIFNβ mRNA. Selective expression of human IFNβ from PSMA-high expressing cells is demonstrated. [Figure 23] Figure 1 shows the inhibition of protein biosynthesis by DT-A protein in two cell lines with differential PSMA expression: high-PSMA-expressing LNCaP cells and low-PSMA-expressing DU145 cells after transfection with the PSMA-targeted polyplex LPEI-l-[N3:DBCO]PEG36-DUPA containing mRNA DT-A. Western blot analysis using anti-puromycin antibody as a probe was used to detect the inhibition of protein biosynthesis. GAPDH was used as a loading control. Selective inhibition of protein biosynthesis in PSMA-overexpressing cells is demonstrated. [Figure 24]Luminescence from human prostate cell lines with differential cell surface expression of PSMA: high-PSMA-expressing LNCaP cells and low-PSMA-expressing DU145 cells. Cells were treated with PSMA-targeted polyplexes containing plasmid DNA encoding luciferase. The X-axis indicates the concentration of pGreenFire-CMV in the polyplex (0.25, 0.5, and 1.0 μg / mL). The Y-axis indicates luminescence in arbitrary units (AU). The mean and standard deviation from triplicate samples are shown. Selective expression of luciferase after transfection of PSMA-overexpressing cells with plasmid DNA encoding luciferase (pGreenFire-CMV) is demonstrated. [Figure 25] Cell lines with differential PSMA expression: Figure 1 shows the levels of secreted human IL-2 normalized to cell viability in high-expressing LNCaP and C4-2 cells and low-expressing DU145 cells after transfection with PSMA-targeted polyplexes containing a plasmid encoding the IL-2 protein. The X-axis shows the concentration of hIL-2 plasmid DNA (0.25, 0.5, and 1.0 μg / mL) in the polyplexes. The Y-axis shows the concentration of secreted IL-2 normalized to cell viability in arbitrary units (AU). Selective expression / secretion of human IL-2 after transfection of PSMA-overexpressing cells with plasmid DNA encoding hIL-2 is demonstrated. DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The embodiments, preferred embodiments, and highly preferred embodiments described and disclosed herein should apply to all aspects and other embodiments, preferred embodiments, and highly preferred embodiments, whether or not explicitly or specifically mentioned again.

[0029] The present invention provides linear conjugates of LPEI and PEG that can form polyplexes with polyanions and nucleic acids, such as poly(IC), as outlined herein and below. The conjugates comprise an LPEI fragment, a PEG fragment, and a targeting fragment, the targeting fragment capable of binding to prostate-specific membrane antigen (PSMA), and the LPEI fragment and the PEG fragment are coupled separately in an end-to-end manner. In some preferred embodiments, the LPEI fragment and the PEG fragment are coupled via a covalent bond of an azide to an alkene or alkyne to form a 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3]triazole.

[0030] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0031] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0032] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0033] As used herein, the term "about" shall mean ±10%. For example, about 50% shall mean 45% to 55%. Preferably, as used herein, the term "about" shall mean ±5%. For example, about 50% shall mean 47.5% to 52.5%.

[0034] As used herein, the phrase "between number X and number Y" includes number X and number Y. For example, the phrase "0.01 μmol to 50 μmol" refers to 0.01 μmol and 50 μmol, as well as values ​​therebetween. The same applies to the phrase "between about number X and about number Y."

[0035] The term "optionally substituted" is understood to mean that a given chemical moiety (e.g., an alkyl group) can, but need not, be attached to other substituents (e.g., heteroatoms). For example, an optionally substituted alkyl group can be a fully saturated alkyl chain (i.e., pure hydrocarbon). Alternatively, the same optionally substituted alkyl group can have substituents other than hydrogen. For example, it can be attached at any point along the chain to a halogen atom, an alkoxy group, or any other substituent described herein. Thus, the term "optionally substituted" means that a given chemical moiety has the potential to contain other functional groups, but does not necessarily have any additional functional groups.

[0036] The term "optionally replaced" is understood to refer to a situation in which a carbon atom of a methylene group (i.e., -CH-) may, but need not, be replaced by a heteroatom (e.g., -NH-, -O-). For example, a C alkylene (i.e., propylene) group in which one of the methylene groups is "optionally replaced" may have the structure --CH-O-CH- or --O-CH-CH-. Those skilled in the art will understand that a methylene group cannot be replaced if such a replacement would result in an unstable chemical moiety. For example, those skilled in the art will understand that four methylene groups cannot be replaced simultaneously by oxygen atoms. Thus, in some preferred embodiments, when one methylene group of an alkylene fragment is replaced with a heteroatom, one or both of the adjacent carbon atoms are not replaced with a heteroatom.

[0037] The term "aryl" refers to cyclic aromatic hydrocarbon groups having one to two aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, or naphthyl. 10 An aryl group contains 6 to 10 carbon atoms. When containing two aromatic rings (such as bicyclic rings), the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl) or fused (e.g., naphthyl). The aryl group may be optionally substituted at any point of attachment with one or more substituents, e.g., 1 to 5 substituents. The substituents themselves may be optionally substituted. Furthermore, when containing two fused rings, the aryl group defined herein may have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these aryl groups include indanyl, indenyl, tetrahydronaphthalenyl, and tetrahydrobenzoannulenyl. In some preferred embodiments, the aryl group is a phenyl group.

[0038] Unless otherwise specifically defined, "heteroaryl" means a monovalent monocyclic or polycyclic aromatic ring 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. A 5-10 membered heteroaryl group contains 5 to 10 atoms. Heteroaryl, as defined herein, also means a bicyclic heteroaromatic group in which the heteroatoms are selected from N, S, P, or O. Aromatic radicals are optionally substituted independently with one or more substituents described herein. Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl, pyrimidinyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinyl, indolyl, thiophen-2-yl, quinolyl, benzopyranyl, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazolyl, benzopyranyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinyl, imidazolyl, benzopyranyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, thiazolyl, thiaz ...diazole, indazole, benzimidazolyl, thiadiazole, thiadiazole, indazole, benzimidazolyl, thiadiazole, thiadiazole, Zo[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, dihydrobenzothiophene Phenyl, dihydrobenzofuranyl, benzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, dihydrobenzoxanyl, 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]thiazolinyl Zolo[4,3-a]pyridinyl, 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]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 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]thiazinyl, benzoxazolyl, benzisoxazolyl, furo[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, furo[3,2-b]pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, 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-dihydro-2H-benzo[d]imidazol-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[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 derivatives thereof. Furthermore, when containing two fused rings, heteroaryl groups as defined herein can have an unsaturated or partially saturated ring fused to a fully saturated ring. Exemplary ring systems of these heteroaryl groups include indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3,4-dihydro-1H-isoquinolinyl, 2,3-dihydrobenzofuran, indolinyl, indolyl, and dihydrobenzoxanyl.

[0039] The term "alkyl" refers to a straight-chain or branched-chain saturated hydrocarbon. A C1-C6 alkyl group contains 1 to 6 carbon atoms. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and neopentyl.

[0040] The term "alkylene" refers to a straight-chain or branched-chain saturated divalent hydrocarbon fragment. A C0-C6 alkyl group contains 0 to 6 carbon atoms. Examples of C0-C6 alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, isopropylene, isobutylene, sec-butylene, tert-butylene, isopentylene, and neopentylene.

[0041] The term "C1-C6-alkoxy" as used herein refers to a substituted hydroxyl of the formula (-OR'), where R' is an optionally substituted C1-C6 alkyl as defined herein, and the oxygen moiety is directly attached to the parent molecule; thus, the term "C1-C6 alkoxy" as used herein refers to a straight-chain or branched C1-C6 alkoxy, which can be, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, straight-chain or branched pentoxy, straight-chain or branched hexyloxy. Preferred alkoxy is C1-C4 alkoxy and C1-C3 alkoxy.

[0042] The term "cycloalkyl" means a monocyclic or polycyclic saturated carbocyclic ring containing 3 to 18 carbon atoms. A C3-C8 cycloalkyl group contains 3 to 8 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl. A C3-C8 cycloalkyl is a cycloalkyl group containing 3 to 8 carbon atoms.

[0043] The term "cycloalkenyl" means a monocyclic non-aromatic unsaturated carbocyclic ring containing 5 to 18 carbon atoms. Examples of cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and norborenyl. A C5-C8 cycloalkenyl is a cycloalkenyl group containing 5 to 8 carbon atoms.

[0044] The terms "heterocyclyl" or "heterocycloalkyl" or "heterocycle" refer to monocyclic or polycyclic 3- to 24-membered rings containing heteroatoms derived from carbon and oxygen, nitrogen, or sulfur, and lacking shared delocalized π-electrons (aromaticity) between ring carbons or heteroatoms. 3- to 10-membered heterocycloalkyl groups contain 3-10 atoms. Heterocyclyl rings include, but are not limited to, oxetanyl, azetazinyl, tetrahydrofuranyl, pyrrolidinyl, oxazolinyl, oxazolidinyl, thiazolinyl, thiazolidinyl, pyranyl, thiopyranyl, tetrahydropyranyl, dioxalinyl, piperidinyl, morpholinyl, thiomorpholinyl, thiomorpholinyl S-oxide, thiomorpholinyl S-dioxide, piperazinyl, azepinyl, oxepinyl, diazepinyl, tropanyl, and homotropanyl.

[0045] The term "heterocycloalkenyl" refers to a monocyclic or polycyclic 3- to 24-membered ring containing carbon and heteroatoms derived from oxygen, nitrogen, or sulfur, where there are no delocalized π-electrons (aromaticity) shared between ring carbons or heteroatoms, but at least one unsaturated element is present within the ring. 3- to 10-membered heterocycloalkenyl groups contain 3 to 10 atoms.

[0046] As used herein, the term "halo" or "halogen" means fluoro (F), chloro (Cl), bromo (Br), or iodo (I).

[0047] The term "carbonyl" refers to a functional group containing a carbon atom double-bonded to an oxygen atom, which may be abbreviated herein as "oxo," C(O), or C=O.

[0048] The term "overexpression" refers to increased expression of a gene or protein in 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 a cell that overexpresses a cell surface receptor. In one embodiment, the cell that overexpresses a cell surface receptor means that the level of the cell surface receptor expressed in the cell in a particular tissue is elevated compared to the level of the cell surface receptor measured in normal healthy cells of the same tissue type under similar conditions. In one embodiment, the cell that overexpresses a cell surface receptor refers to an increase in the level of the cell surface receptor in a cell compared to the level in the same cell or a closely related non-malignant cell under normal physiological conditions.

[0049] The term "polyanion," as used herein, refers to a polymer, preferably a biopolymer, having two or more negatively charged sites. Typically and preferably, the term "polyanion," as used herein, refers to a polymer, preferably a biopolymer, composed of repeating units that contain residues that may be negatively charged. In a further embodiment, the polyanion is a polymer, preferably a biopolymer, composed of repeating units that contain negatively charged residues. In another preferred embodiment, the polyanion is a nucleic acid, more preferably DNA, RNA, polyglutamic acid, or hyaluronic acid.

[0050] The term "nucleic acid," as used herein, includes deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), or a combination thereof. In preferred embodiments, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA), and as used herein, refers to genomic, viral, and recombinantly prepared and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded and in the form of linear or covalently closed circular molecules, may include chemical derivatization of the nucleic acid on the nucleotide base, sugar, or phosphate, and may contain non-natural nucleotides and nucleotide analogs.

[0051] The term "dispersity" (abbreviated as D), as used herein, refers to the distribution of molar masses in a given polymer sample, such as the polymer fragments used herein for the conjugates and polyplexes of the invention. This is defined herein as D = (M w / M n ) where D is the dispersity; M w is the weight average molecular weight of the polymer sample or polymer fragment; M n is the number average molecular weight of the polymer sample or polymer fragment.

[0052] The term "weight average molecular weight," as used herein, refers to the sum of the products of the weight fraction of a given molecule in a mixture and the molecular mass of each molecule in the mixture, and is typically and preferably represented by the symbol Mw.

[0053] The term "number average molecular weight," as used herein, refers to the total weight of a mixture divided by the number of molecules in the mixture, and is typically and preferably represented by the symbol Mn.

[0054] The term "polydispersity index" (abbreviated as PDI) as used herein refers to the polydispersity index in dynamic light scattering measurements of polyplex nanoparticles, such as the polyplexes of the present invention. This index is a number calculated from a simple two-parameter fit to correlation data (cumulant analysis). The polydispersity index is dimensionless and is scaled so that values ​​below 0.05 are rarely observed except in highly monodisperse standards. Values ​​above 0.7 indicate a sample has a very broad size distribution and are likely unsuitable for dynamic light scattering (DLS) techniques. Various size distribution algorithms work with data that fall between these two extremes. The zeta-average diameter (z-average diameter) and polydispersity index of the polyplexes of the present invention are determined by dynamic light scattering (DLS) based on the assumption that the polyplexes are isotropic and spherical. The calculation of these parameters is defined and determined in accordance with the ISO standard document ISO 22412:2017.

[0055] The term "amino acid residue" refers to a divalent residue derived from an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH), typically and preferably with a side chain specific to each amino acid. In a preferred embodiment of the present invention, the amino acid residue is a divalent residue derived from an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH), the divalency being provided by the amine and carboxylic acid functional groups, and thus the -NH- and -CO- moieties. In an alternative preferred embodiment of the present invention, the amino acid residue is a divalent residue derived from an organic compound containing the functional groups amine (-NH) and carboxylic acid (-COOH), the divalency being provided by the amine or carboxylic acid functional group and also by functional groups present in the amino acid residue. As a preferred example and embodiment, an amino acid residue according to the present invention derived from cysteine ​​comprises the divalent structure -S-(CH)-CH(COOH)-NH-, where the divalency is provided by the amino functionality and the contained thiol functionality. The term "amino acid residue," as used herein, typically and preferably includes amino acid residues derived from naturally occurring or non-naturally occurring amino acids. Furthermore, the term "amino acid residue," as used herein, typically and preferably also includes amino acid residues derived from chemically synthesized non-natural amino acids, including alpha- (α-), beta- (β-), gamma- (γ-), or delta- (δ-) amino acids, as well as mixtures thereof in any ratio. Furthermore, the term "amino acid residue," as used herein, typically and preferably also includes amino acid residues derived from alpha amino acids, including any isomeric forms thereof, particularly their D- and L-stereoisomers (alternatively addressed by the (R) and (S) nomenclature), as well as mixtures thereof in any ratio, preferably a 1:1 racemic ratio. The terms "D-stereoisomer," "L-stereoisomer," "D-amino acid," or "L-amino acid" refer to the chiral alpha carbon of an amino acid. Thus, in a preferred embodiment, the amino acid residue is a divalent group of the structure -NH-CHR-C(O)-, where R is an amino acid side chain.Two or more consecutive amino acid residues preferably form a peptide (i.e., amide) bond between both the amine and carboxylic acid moieties of each amino acid residue. Di-, tri-, or polypeptide amino acid residues are typically (AA). a When described herein as a tripeptide, the sequences provided are presented from left to right in the N-terminal direction. Thus, by way of example, the depiction of Trp-Trp-Gly should refer to the amino acid residues, where Trp corresponds to the N-terminus of the tripeptide with an -NH- valence and Gly corresponds to the C-terminus of the tripeptide with an -CO- valence.

[0056] The terms "peptide," "polypeptide," and "protein," as used herein, refer to a substance comprising about two or more consecutive amino acid residues linked together via peptide bonds. The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues of any length. In one embodiment, the term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, while in one embodiment, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 8 or more, or about 20 or more amino acids in length, and up to about 50, about 100, or about 150 amino acids in length. The term "epitope," as used herein, refers to an antigenic determinant in a molecule, such as an antigen. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acids in length, for example the epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0057] The term "antibody" refers to any immunoglobulin, whether natural or wholly or partially synthetically produced, as well as derivatives and characteristic portions thereof. Antibodies can be monoclonal or polyclonal. Antibodies can be members of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. As used herein, an antibody fragment (i.e., a characteristic portion of an antibody) refers to any derivative of an antibody that is less than full-length. Generally, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, single-chain and double-chain fragments, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragments can be produced by any means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively, or additionally, antibody fragments can be wholly or partially synthetically produced. 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 include multimolecular complexes. Functional antibody fragments typically comprise 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, antibodies may have reduced effector function and / or bispecific molecules. In some embodiments, antibodies may include fragments produced by Fab expression libraries. Single-chain Fvs (scFvs) are recombinant antibody fragments consisting only of a variable light chain (VL) and a variable heavy chain (VH) covalently connected to each other by a polypeptide linker. Either the VL or VH may comprise an NH2-terminal domain. The polypeptide linker may be of variable length and composition, so long as the two variable domains are bridged without significant steric hindrance.Typically, the linker contains primarily a stretch of glycine and serine residues, with some glutamic acid or lysine residues interspersed for solubility. Diabodies are dimeric scFvs. Diabodies typically have shorter peptide linkers than most scFvs and often preferentially associate as dimers. Fv fragments are antibody fragments consisting of one VH and one VL domain held together by noncovalent interactions. The term "dsFv" as used herein refers to an Fv with an engineered intermolecular disulfide bond to stabilize the VH-VL pair. F(ab')2 fragments are antibody fragments essentially equivalent to those obtained from immunoglobulins by digestion with the enzyme pepsin at pH 4.0-4.5. Fragments can be produced recombinantly. Fab' fragments are antibody fragments essentially equivalent to those obtained by reduction of one or more disulfide bridges joining the two heavy chains in the F(ab')2 fragment. Fab' fragments can be produced recombinantly. 1. Fab fragments are antibody fragments essentially equivalent to those obtained by digesting immunoglobulins with enzymes (e.g., papain). Fab fragments can be produced recombinantly. The heavy chain segment of a Fab fragment is an Fd subfragment.

[0058] The term "alpha end of a linear polyethyleneimine fragment" (α-end of an LPEI fragment), as used herein, refers to the end of an LPEI fragment at which initiation of polymerization occurs using an electrophilic initiator, as further described below for the term "initiating residue."

[0059] The term "omega end of a linear polyethyleneimine fragment" (ω end of an LPEI fragment), as used herein, refers to the end of an LPEI fragment at which polymerization termination occurs using a nucleophile, such as an azide, thiol, and other nucleophiles described herein.

[0060] The term "organic residue" refers to any suitable organic group that can be bonded to a nitrogen atom embedded within an LPEI fragment. In a preferred embodiment, the organic residue is connected to the nitrogen atom via a carbonyl group, forming an amide linkage. Without wishing to be bound by theory, the organic residue is incorporated into the nitrogen atom of a poly(2-oxazoline) during ring-opening polymerization of the 2-oxazoline (see, e.g., 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). Typically and preferably, the organic residue is cleaved from the poly(2-oxazoline) (i.e., the amide is typically cleaved), yielding the -(NH-CH-CH)- moiety embedded within the LPEI and LPEI fragment, and thus the conjugate of the present invention. However, if the cleavage reaction is not complete, a fraction of the organic residue will not be cleaved. Thus, in a preferred embodiment of the present invention, the R 1 -(NR 2 -CH2-CH2) n -R in part 2 at least 80%, preferably 90%, of the R of the conjugates of the invention, including those of formula I* or I, are H; 1 -(NR 2 -CH2-CH2) n -R in part 2 Preferably at least 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%, and most preferably 99% of is H.

[0061] The term "initial residue" refers to the LPEI fragment and R 1 -(NR 2 -CH2-CH2) n- refers to a residue present in the moiety, which is derived from any initiator, typically and preferably any electrophilic initiator, capable of initiating the polymerization of poly(2-oxazoline) from 2-oxazoline. As described in 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, "Different initiator systems can be used, including toluenesulfonic acid (TsOH), or alkylsulfonates such as methyl p-toluenesulfonate (MeOT), which are most frequently found in the literature, p-nitrobenzenesulfonate (nosylate) and trifluoromethanesulfonate (triflate), alkyl halides, benzyl halides, and acetyl halides, oxazolinium salts, and Lewis acids." Thus, in a preferred embodiment, R 1 is -H or -CH3, but those skilled in the art will recognize that R 1 is any other suitable residue, e.g., C where n is greater than 1. n Alkyl groups, typically C 1~6 It will be understood that these groups may also include, but are not limited to, alkyl groups, benzyl groups, or acetyl groups.

[0062] Accordingly, in one aspect, the invention provides a composition comprising a conjugate, the conjugate comprising: a linear polyethyleneimine fragment comprising an alpha end and an omega end; a polyethylene glycol fragment comprising a first end and a second end, the polyethylene glycol fragment comprising, and preferably consisting of, a discrete number m of repeating -(O-CH-CH)- units, wherein said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably 25 to 60, and preferably said discrete number m is a discrete number of consecutive repeating -(O-CH-CH)- units, wherein said discrete number of consecutive repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably 25 to 60; the alpha end of the polyethyleneimine fragment is an initiating residue; and the omega end of the polyethyleneimine fragment is a divalent covalent linking group -ZX 1 -(in the formula, -ZX 1 - is not a single bond and -Z- is not an amide); the second end of the polyethylene glycol fragment is connected to a divalent covalent linking moiety X 2 wherein the targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), and preferably the targeting fragment is capable of binding to cells expressing PSMA.

[0063] In a further aspect, the invention provides a composition comprising a conjugate, the conjugate comprising: a linear polyethyleneimine fragment comprising an alpha end and an omega end; a polyethylene glycol fragment comprising a first end and a second end, the polyethylene glycol fragment comprising, and preferably consisting of, a discrete number m of repeating -(O-CH-CH)- units, wherein said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, preferably said discrete number m is a discrete number of consecutive repeating -(O-CH-CH)- units, wherein said discrete number of consecutive repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60; the alpha end of the polyethyleneimine fragment is an initiating residue; and the omega end of the polyethyleneimine fragment is a divalent covalent linking group, -ZX 1 - (wherein -Z- is not a single bond and -Z- is not an amide) connected to a first end of a polyethylene glycol fragment; -X 1 - is a divalent covalent linking moiety; the second end of the polyethylene glycol fragment is linked to a divalent covalent linking moiety X 2 wherein the targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), and preferably the targeting fragment is capable of binding to cells expressing PSMA.

[0064] In another aspect, the present invention provides a composition comprising a conjugate, wherein said conjugate is of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); During the ceremony, n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90% of which is H; X 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, ZX 1 is not a single bond and Z is not NHC(O)-; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA; Preferably, a composition comprising the conjugate is provided, which consists of the conjugate.

[0065] In yet another aspect, the present invention provides a composition comprising a conjugate, said conjugate being of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); [In the formula, n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90% of which is H; X 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, Z is not a single bond, and Z is not NHC(O)-; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA. Preferably, a composition comprising the conjugate is provided, which comprises a plurality of said conjugates. In a preferred embodiment, the composition of the invention comprises a plurality of said conjugates, and more preferably the composition of the invention consists of said conjugates.

[0066] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof, wherein R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2-L (Formula I*); wherein n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, wherein Z is not -NHC(O)-; and L is a targeting fragment, wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA], more preferably consisting of said conjugate.

[0067] In another aspect, the present invention provides a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); wherein n is any integer from 1 to 1500; m is any integer from 1 to 200, preferably m is a discrete number of repeating -(O-CH-CH)- units, wherein said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, more preferably said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2-CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, wherein Z is not NHC(O)-; L is a targeting fragment, wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably wherein said targeting fragment is capable of binding to cells expressing PSMA].

[0068] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof, wherein R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); wherein n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units being 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, Z is not a single bond, Z is not NHC(O)-; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA, preferably a composition comprising said conjugate. The term "said cell surface receptor is PSMA" is intended to mean that the cell surface receptor is derived from PSMA, is typically and preferably provided on the cell surface, and more typically and preferably is such a portion and / or part of PSMA that corresponds to the extracellular domain of PSMA and / or a cell surface-exposed portion of PSMA.

[0069] In another aspect, the present invention provides a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); wherein n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units being 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, Z is not a single bond, and Z is not NHC(O)-; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell surface receptor, and said cell surface receptor is PSMA.

[0070] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single bond or a double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, wherein said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n - the R in the part 2 at least 80%, preferably 90%, of are H; ring A may contain one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H, or -OSO3H; X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl or heteroaryl is selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 optionally substituted with R 11 , R 12 and R 13 is independently at each occurrence H or C1-C6 alkyl; R 14 is a linking moiety for (each occurrence is independently H, C1-C6 alkyl, or oxo); X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is independently selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 optionally substituted with R 21 , R 22 and R 23are each independently at each occurrence -H, -COH, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, C6-C 10 optionally substituted with aryl, or 5-8 membered heteroaryl; R 24 is a linking moiety of the formula (III), wherein each occurrence is independently -H, -COH, C1-C6 alkyl, or oxo; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA), preferably consisting of said conjugate.

[0071] As referred to herein, the depiction of Formula I above refers to the fragment R 1 (NR 2 CH2CH2) n Two different regioisomeric bonds, i.e., [ka] and [ka] where the wavy line represents a chemical bond to ring A. Thus, Formula I as depicted herein represents two regioisomeric embodiments, namely, fragment R 1 (NR 2 CH2CH2) n is attached to the upper nitrogen atom of the above structure or to the lower nitrogen atom of the above structure, but not to the middle nitrogen atom. Formula I, as depicted above, includes an equivalent depiction of Formula I including the following fragment N—N=N, i.e., [ka] are used interchangeably herein.

[0072] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single bond or a double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, wherein said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably, said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n - the R in the part 2 at least 80%, preferably 90%, of are H; ring A may contain one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, -SO3H, or -OSO3H; X 1 is the formula -(Y 1) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl or heteroaryl is selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 optionally substituted with R 11 , R 12 and R 13 is independently at each occurrence H or C1-C6 alkyl; R 14 is a linking moiety for (each occurrence is independently H, C1-C6 alkyl, or oxo); X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is independently selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 optionally substituted with R 21 , R 22 and R 23 are each independently at each occurrence -H, -COH, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, C6-C 10 optionally substituted with aryl, or 5-8 membered heteroaryl; R 24is a linking moiety of (each occurrence is independently -H, -COH, C1-C6 alkyl, or oxo); L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA). 1 is -H. In a preferred embodiment, 1 is -CH3.

[0073] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0074] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0075] In some preferred embodiments, the divalent covalent linking moiety Z comprises a triazole.

[0076] 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 are connected to the PEG fragments by a single covalent linking moiety, which creates a linear end-to-end linkage between the LPEI and PEG fragments, preferably as determined by UV spectroscopy or mass spectroscopy. 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 included in the conjugate are connected to the PEG fragments by a single covalent linking moiety, which creates a linear end-to-end linkage between the LPEI and PEG fragments, preferably as determined by UV spectroscopy or mass spectroscopy. In some embodiments, at least 60%, at least 70%, or at least 80%, at least 90%, at least 95%, or at least 99% of the LPEI in the composition are included in the conjugate, preferably as determined by UV spectroscopy or mass spectroscopy. In some embodiments, the composition consists essentially of the conjugate. In some embodiments, the composition consists of the conjugate.

[0077] In some embodiments, at least 60% of the LPEI fragments in the composition are connected to a single PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 60% of the LPEI fragments in the composition are connected to a PEG fragment by a single triazole linker, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 70% of the LPEI fragments in the composition are connected to a PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 70% of the LPEI fragments in the composition are included in the conjugate, preferably, as determined by UV spectroscopy or mass spectrometry. In some embodiments, at least 80% of the LPEI fragments in the composition are connected to a PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 80% of the LPEI fragments in the composition are comprised in the conjugate, preferably as determined by UV spectroscopy or mass spectroscopy. In some embodiments, at least 90% of the LPEI fragments in the composition are connected to the PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 90% of the LPEI fragments in the composition are comprised in the conjugate, preferably as determined by UV spectroscopy or mass spectroscopy. In some embodiments, at least 95% of the LPEI fragments in the composition are connected to the PEG fragment by a single covalent linking moiety Z, preferably, the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 95% of the LPEI fragments in the composition are comprised in the conjugate, preferably, as determined by UV spectroscopy or mass spectroscopy.In some embodiments, at least 99% of the LPEI fragments in the composition are connected to the PEG fragment by a single covalent linking moiety Z, preferably the covalent linking moiety Z creates a linear end-to-end linkage between the LPEI fragment and the PEG fragment. In some embodiments, at least 99% of the LPEI fragments in the composition are comprised in the conjugate, preferably as determined by UV spectroscopy or mass spectrometry. In some embodiments, the composition consists essentially of the conjugate. In some embodiments, the LPEI fragment does not include substitutions beyond its first and second ends.

[0078] In some embodiments, Formula I* has the structure: R 1 -(NH-CH2-CH2) n -NHC(O)-(CH2-CH2-O) m -X 2 In some embodiments, formula I* does not include the structure R 1 -(NR 2 -CH2-CH2) n -NHC(O)-X 1 -(O-CH2-CH2) m -X 2 In some embodiments, the composition does not include the structure R 1 -(NH-CH2-CH2) n -NHC(O)-X 1 -(O-CH2-CH2) m -X 2 In some embodiments, the composition does not include a conjugate of structure R 1 -(NR 2 -CH2-CH2) n -NHC(O)-(CH2-CH2-O) m -X 2 -L conjugates are not included.

[0079] In some embodiments, R 1 is -H.

[0080] In some embodiments, R in the composition 2 At least 80% of the R in the composition are —H. 2 At least 85%, preferably 90%, preferably 95%, more preferably 99% of the R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -the R in the moiety 2 At least 85%, preferably 90% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -the R in the moiety 2 At least 90% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -the R in the moiety 2 At least 90% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -the R in the moiety 2 At least 91%, preferably at least 92%, more preferably at least 93% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -the R in the moiety 2 At least 94%, preferably at least 95%, more preferably 96% of R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -the R in the moiety 2 At least 95%, preferably at least 97%, even more preferably at least 98%, and even more preferably 99% of the total is H.

[0081] In some embodiments, ring A is an 8-membered cycloalkenyl, a 5-membered heterocycloalkyl, or a 7-8-membered heterocycloalkenyl, and each cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl may have one or more R A1 is optionally substituted with

[0082] In some embodiments, ring A is cyclooctene, maleimide, or a 7-8 membered heterocycloalkenyl, where the heterocycloalkenyl contains no heteroatoms other than N, O, and S, and each cyclooctene or heterocycloalkenyl may contain one or more R A1 is optionally substituted with

[0083] In some embodiments, ring A is cyclooctene, maleimide, or a 7-8 membered heterocycloalkenyl, where the heterocycloalkenyl contains one or more heteroatoms, preferably 1 or 2 heteroatoms selected from N, O, and S, and each cyclooctene or heterocycloalkenyl contains one or more R A1 is optionally substituted with

[0084] In some embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, where the heterocycloalkene contains exactly one heteroatom selected from N, O, and S, and each cyclooctene or heterocycloalkene contains one or more R A1 is optionally substituted with

[0085] 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 --OSO3H or --SO3H.

[0086] In some embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, where the heterocycloalkene contains exactly one heteroatom selected from N, O, and S, and each cyclooctene or heterocycloalkene contains one or more R A1 and optionally substituted with R A1 is oxo or fluorine, or two R A1 are combined to form one or more fused phenyl rings, preferably one or two fused phenyl rings.

[0087] In some embodiments, ring A is cyclooctene, maleimide, or an 8-membered heterocycloalkene, where the heterocycloalkene contains exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one or two R A1 is optionally substituted with

[0088] In some embodiments, R A1 is -H, oxo or fluorine, or two R A1 are combined to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each phenyl ring containing one or more R A2 is optionally substituted with

[0089] In some embodiments, ring A is cyclooctene, maleimide, or an 8-membered heterocycloalkene, wherein the heterocycloalkene contains exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one or two R A1 and optionally substituted with 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 -OSO3H or -SO3H.

[0090] In some preferred embodiments, ring A is a cyclooctene, maleimide, or an 8-membered heterocycloalkene, wherein the heterocycloalkene contains exactly one heteroatom selected from N, and each cyclooctene or heterocycloalkene contains one or two R A1 and optionally substituted with R A1 is -H 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 -OSO3H or -SO3H.

[0091] Preparation of linear conjugates The conjugates of the present invention can be prepared by several methods well known to those skilled in the art of polymer synthesis. For 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 recognized by those skilled in the art. The methods include, but are not limited to, the methods described below. The conjugates of the present invention can be synthesized according to the steps outlined in General Schemes 1, 2, 3, 4, 5, 6, 7, and 8, or can be prepared using an alternating sequence of assembling intermediates without departing from the present invention. The conjugates of the present invention can also be synthesized using slight variations of the steps outlined below. For example, while Scheme 3 shows the use of a tetrafluorophenyl ester as an electrophilic functional group to couple with a PSMA targeting fragment containing a nucleophilic amine group to form an amide functional group, those skilled in the art will recognize other suitable electrophilic functional groups other than tetrafluorophenyl ester that can be used for the same purpose.

[0092] In some preferred embodiments, the LPEI fragment and the PEG fragment are coupled via a [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole. In some preferred embodiments, the LPEI fragment contains an azide functional group and the PEG fragment contains an alkene or alkyne functional group.

[0093] LPEI fragment The conjugates of the present invention can comprise an LPEI fragment and a PEG fragment. Linear polyethyleneimine (LPEI) has the chemical formula -[NH-CH-CH]-. LPEI can be synthesized according to several methods known in the art, including, inter alia, polymerization of 2-oxazoline followed by hydrolysis of the pendant amide bond (see, e.g., Brissault et al., Bioconjugate Chem., 2003, 14, 581-587). As noted above, polymerization of poly(2-oxazoline) from 2-oxazoline (i.e., a suitable precursor for LPEI) can be initiated with any suitable initiator. In some embodiments, the initiator leaves an initiator residue at the alpha-terminus of the poly(2-oxazoline). In preferred embodiments, the initiator residue (i.e., Formula I* or R of Formula I) is used. 1 ) is a hydrogen atom or C 1~ C6 alkyl, preferably hydrogen or C1-C4 alkyl, more preferably hydrogen or methyl group; most preferably hydrogen atom. In a preferred embodiment, the initial residue R of formula I 1 is a hydrogen atom or C 1~ C6 alkyl, preferably hydrogen or C1-C4 alkyl, more preferably hydrogen or methyl group; most preferably hydrogen atom. In a preferred embodiment, the initial residue (i.e., R of Formula I* or Formula I) 1 ) is -H or -CH3, most preferably -H. In a preferred embodiment, said initial residue R of formula I* 1 is —H. In a preferred embodiment, the initial residue R of formula I 1 is -H. In a preferred embodiment, said initial residue R of formula I*1 is —CH3. In a preferred embodiment, the initial residue R of formula I 1 is —CH3. However, one skilled in the art will understand that the initiating residue can be the residue remaining from any suitable initiator capable of initiating the polymerization of 2-oxazoline to poly(2-oxazoline).

[0094] In some embodiments, the LPEI fragment can be coupled to a PEG fragment via a [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole, and the LPEI fragment contains an azide (-N3) 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 contains the repeating formula -[NH-CH2-CH2]- and is substituted at the omega-terminus with an azide group that can be coupled to an alkyne or alkene substituent on the PEG fragment. In some preferred embodiments, the alpha-terminus of the LPEI fragment is substituted with a hydrogen atom or a C 1~ It may be substituted with a C6 alkyl, preferably hydrogen or a C1-C4 alkyl, more preferably hydrogen or a methyl group; most preferably a hydrogen atom.

[0095] For example, in some preferred embodiments, the LPEI fragment contains a hydrogen atom or a C at the alpha terminus. 1~ The LPEI fragment may be substituted with a C6 alkyl, preferably a hydrogen atom or a C1-C4 alkyl, more preferably a hydrogen atom or a methyl group, and at the omega terminus with an azide group; in some preferred embodiments, there are no additional substitutions on the LPEI fragment. For example, the conjugates of the present invention may have the following formula: [ka] (In the formula, R 1 is any suitable starting residue, preferably hydrogen or C 1~C6 alkyl, preferably hydrogen or C1-C4 alkyl, more preferably hydrogen or methyl, most preferably hydrogen) It can be prepared from the LPEI fragment of

[0096] In some embodiments, the LPEI fragment can be terminated with a thiol group, and thus in some embodiments, the omega-terminus of the LPEI fragment comprises, and preferably is, a thiol group, which can be coupled to a reactive alkene group on a PEG fragment via a thiol-ene reaction. Thus, in some embodiments, the conjugates of the present invention have the following formula: [ka] (In the formula, R 1 may be any suitable starting residue, preferably hydrogen or methyl, preferably hydrogen). It can be prepared from the LPEI fragment of

[0097] In some embodiments, the LPEI fragment can be terminated with an alkene group, and thus in some embodiments, the omega end of the LPEI fragment comprises, and preferably is, an alkene group, which can be coupled to a reactive thiol group on a PEG fragment via a thiol-ene reaction. Thus, in some embodiments, the conjugates of the present invention have the following formula: [ka] (In the formula, R 1 may be any suitable starting residue, preferably hydrogen or methyl, preferably hydrogen). It can be prepared from the LPEI fragment of

[0098] LPEI fragments can include a range of lengths (i.e., repeat units, as represented above by the variable "n"). For example, LPEI fragments can include 1 to 1000 repeat units (i.e., -NH-CH2-CH2-). In some embodiments, LPEI fragments can be present as dispersed polymer moieties and do not include a discrete number of -NH-CH2-CH2- repeat units. For example, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 5 to 50 KDa and a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 10 to 40 KDa and a dispersity of about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 12 to 30 KDa and a dispersity of about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 15-27 KDa and a dispersity of about 2 or less, preferably about 1.5 or less. In some embodiments, LPEI fragments can be present as dispersed polymer moieties having a molecular weight of about 17-25 KDa and a dispersity of about 1.2 or less.

[0099] For example, LPEI fragments may be present as dispersed polymer moieties containing about 115-1150 repeating units and preferably having a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments may be present as dispersed polymer moieties containing about 230-930 repeating units and having a dispersity of about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments may be present as dispersed polymer moieties containing about 280-700 repeating units and having a dispersity of about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In some embodiments, LPEI fragments may be present as dispersed polymer moieties containing about 350-630 repeating units and having a dispersity of about 2 or less, preferably about 1.5 or less. In some embodiments, LPEI fragments may be present as dispersed polymer moieties containing about 400-580 repeating units and having a dispersity of about 1.2 or less.

[0100] In some embodiments, R 1 -(NR 2 -CH2-CH2) n - moiety is a dispersed polymer moiety having 115 to 1150 repeating units n and a dispersity of about 5 or less, and preferably 1 -(NR 2 -CH2-CH2) n The R - moiety is a dispersed polymer moiety having 280 to 700 repeating units n and a dispersity of about 3 or less, and more preferably 1 -(NR 2 -CH2-CH2) n - moiety is a dispersed polymer moiety having 350 to 630 repeat units n and a dispersity of about 2 or less, and again more preferably 1 -(NR 2 -CH2-CH2) n The - moiety is a dispersed polymer moiety having 400 to 580 repeating units n and a dispersity of about 1.2 or less.

[0101] In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 115 to about 1150 repeating units and a dispersity of about 5 or less, preferably about 230 to about 930 repeating units and a dispersity of about 4 or less; more preferably about 280 to about 700 repeating units and a dispersity of about 3 or less; again more preferably about 350 to about 630 repeating units and a dispersity of about 2 or less; even more preferably about 400 to about 580 repeating units and a dispersity of about 1.2 or less.

[0102] In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 115 to about 1150 repeating units and a dispersity of about 5 or less, preferably about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 230 to about 930 repeating units and a dispersity of about 4 or less, preferably about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having 280 to about 700 repeating units and a dispersity of about 3 or less, preferably about 2 or less, and preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 350 to about 630 repeating units and a dispersity of about 2 or less, preferably about 1.5 or less. In a preferred embodiment, the polyethyleneimine fragment is a dispersed polymer moiety having about 400 to about 580 repeating units and a dispersity of about 1.2 or less.

[0103] As discussed above, those skilled in the art will understand that in some embodiments, LPEI fragments can include organic residues (i.e., pendant amide groups) connected by nitrogen atoms embedded within the LPEI chain. Those skilled in the art will understand that such organic residues (i.e., amide groups) can be formed during the ring-opening polymerization of 2-oxazolines to form poly(2-oxazolines). Without wishing to be bound by theory, LPEIs can be formed from poly(2-oxazolines) by cleavage of amide groups (e.g., using an acid such as HCl). However, in some cases, not all amide linkages may be cleaved under these conditions. Thus, in some embodiments, no more than about 5% of the nitrogen atoms in LPEI fragments can be connected to organic residues to form amides. In some embodiments, no more than about 4%, no more than about 3%, no more than about 2%, no more than about 1%, no more than about 0.5%, no more than about 0.4%, no more than about 0.3%, no more than about 0.2%, or no more than about 0.1% of the nitrogen atoms in LPEI fragments can be connected to organic residues to form amides. Those skilled in the art will understand that the molecular weight of an LPEI fragment includes the percentage of the LPEI fragment that is attached to an organic residue as an amide. Furthermore, those skilled in the art will understand that, although the chemical structures depicted herein show repeating -NH-CH2-CH2- fragments, trace amounts of residual organic residues, such as pendant amide groups (e.g., as defined above), may still be present in the resulting triconjugates or polyplexes of the present disclosure. The term "triconjugate," as occasionally used herein, refers to the conjugates of the present invention. The prefix "tri-" refers to the three components included in the conjugates of the present invention: the LPEI fragment, the PEG fragment, and the targeting fragment.

[0104] PEG fragment Polyethylene glycol (PEG) has the chemical formula -[O-CH2-CH2]-.

[0105] The PEG fragments contained in the conjugates and compositions of the invention comprise, and preferably consist of, a discrete number m of repeating -(O-CH-CH)- units and are not defined in terms of average chain length. Thus, the PEG fragments contained in the conjugates and compositions of the invention comprise, and preferably consist of, a discrete number m of repeating -(O-CH-CH)- units and are not defined in terms of average chain length, but have specifically defined, distinct molecular weights associated with the discrete number m of repeating -(O-CH-CH)- units. In a preferred embodiment, the PEG fragments comprise, and preferably consist of, a discrete number m of repeating -(O-CH-CH)- units, typically and preferably, the discrete number (m) is between 25 and 100, more preferably between 25 and 60. In a preferred embodiment, the PEG fragment comprises, preferably consists of, a discrete number m of consecutive repeating -(O-CH-CH) units, typically and preferably, said discrete number (m) is between 25 and 100, more preferably between 25 and 60.

[0106] The phrases "polyethylene glycol fragment comprising a discrete number (m) of repeating -(O-CH2-CH2)- units" or "PEG fragment comprising a discrete number (m) of repeating -(O-CH2-CH2)- units" refer to a fragment comprising, preferably consisting of, a discrete number, typically referred to herein as a discrete number m, of repeating -(O-CH2-CH2)- units, wherein said discrete number (m) is discrete, i.e., a specific single, defined integer number (m) between 25 and 100, preferably between 25 and 60. Thus, the phrases "polyethylene glycol fragments comprising a discrete number (m) of repeating -(O-CH-CH)- units" or "PEG fragments comprising a discrete number (m) of repeating -(O-CH-CH)- units" refer to fragments comprising, preferably consisting of, a discrete number m of repeating -(O-CH-CH)- units, where m is a discrete, specific, single, defined integer number (m) between 25 and 100, preferably between 25 and 60. Thus, the defined PEG fragments comprise, preferably consist of, a discrete number m of repeating -(O-CH-CH)- units, each having a specifically defined, discrete molecular weight, although not defined in terms of average chain length. Reference herein to a discrete number between 25 and 100 refers to any integer between 25 and 100, i.e., any integer between 25 and 100, including the integers and discrete numbers mentioned herein as boundaries, such as 25 and 100. As a further example, a PEG fragment comprising a discrete number (m) of repeating -(O-CH-CH)- units, where m is 36, refers to a PEG fragment comprising a chain of -(O-CH-CH)- units containing exactly 36 -(O-CH-CH)- units. Such a chain of exactly 36 -(O-CH-CH)- units is a PEG fragment. 36These PEG fragments are typically contrasted with "polymeric PEG fragments," "polydisperse PEG fragments," or "disperse PEG fragments," which refer to a heterogeneous mixture of sizes and molecular weights resulting from a polymer reaction, typically in a Poisson distribution (J. Herzberger et al.; Chem Rev, 2016, 116:2170-2243). The PEG fragments of the present invention, which contain a discrete number (m) of repeating -(O-CH-CH)- units, are not synthesized by a polymerization process. The PEG fragments of the present invention are single molecular fragments that contain a discrete number (m) of repeating -(O-CH-CH)- units and have a discrete, i.e., defined and specified, chain length. Thus, the PEG fragments of the present invention, which contain a discrete number (m) of repeating -(O-CH-CH)- units, are single molecular fragments that have a discrete, i.e., defined and specified, chain length. The PEG fragments of the present invention are not a mixture of molecular entities (such as those resulting from a random polymerization reaction). The discrete nature of the discrete PEG fragments of the present invention distinguishes them from polydisperse techniques.

[0107] The PEG fragments of the present invention can comprise, and preferably consist of, homogeneous discrete PEG fragments or heterogeneous discrete PEG fragments, typically and preferably homogeneous discrete PEG fragments. The term "homogeneous discrete PEG fragments," as used herein, refers to a discrete PEG structure whose entire chemical backbone is composed solely of a specific, discrete number of continuous, consecutive ethylene oxide units. In other words, no other functionality is present within the homogeneous discrete PEG fragments. However, the term "homogeneous discrete PEG fragments" refers to a discrete PEG structure in which the basic ethylene oxide backbone, comprising a discrete number of ethylene oxide units, can, and typically does, have a functional group for conjugation with PEI fragments and target fragments. The term "heterogeneous discrete PEG fragments," as used herein, refers to a discrete PEG structure in which the basic ethylene oxide backbone, comprising a discrete number of ethylene oxide units, is divided by the inclusion of, or substituted with, other functional groups or units within the structure, such as amide or ester bonds or other functional units. In a preferred embodiment of the present invention, the PEG fragments are homogeneous discrete PEG fragments.

[0108] In some preferred embodiments, PEG fragments can be coupled to LPEI fragments via a [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole, and each reactive precursor molecule comprising a PEG fragment further comprises an alkene or alkyne functional group. For example, in some preferred embodiments, the reactive precursor molecule comprising a PEG fragment comprises the repeating formula -[O-CH-CH]-, with the alkene or alkyne group (e.g., a linking moiety "X" as discussed herein) being capable of coupling to the azide group of each reactive precursor molecule comprising the corresponding LPEI fragment. 1 ") at the first end (i.e., terminal). In some preferred embodiments, the alkene or alkyne group is an activated alkene or alkyne group that can spontaneously react with an azide (e.g., without the addition of a catalyst such as a copper catalyst). For example, an activated alkyne group can be incorporated into a 7- or 8-membered ring, resulting in a strained species that spontaneously reacts with the azide group of an LPEI fragment. The activated alkene can include a maleimide moiety, where the alkene is activated by conjugation to an adjacent carbonyl group. In some preferred embodiments, the second end (i.e., terminal) of the PEG fragment is substituted with a targeting fragment (e.g., DUPA) (e.g., a linking moiety "X" discussed herein). 2 "), wherein the targeting segment is capable of binding to prostate-specific membrane antigen (PSMA), and preferably the targeting segment is capable of binding to cells expressing PSMA.

[0109] The PEG fragments included in the conjugates and compositions of the invention, as in the case of polymeric PEG fragments, comprise, and preferably consist of, a discrete number m of repeating -O-CH-CH- units, and are not defined in terms of average chain length. In a preferred embodiment, the -(O-CH-CH) mIn a preferred embodiment, the -(O-CH-CH) - unit comprises, and preferably consists of, a discrete number of repeating units m. m -unit comprises, preferably consists of, a discrete number of consecutive repeating units.

[0110] In a preferred embodiment, the PEG fragment comprises, preferably consists of, 25 to 100 discrete number of repeating units m, preferably 25 to 60 discrete number of repeating units m. In a preferred embodiment, the PEG fragment comprises, preferably consists of, 25 to 60 discrete number of repeating units m, preferably 30 to 50 discrete number of repeating units m. In a preferred embodiment, the PEG fragment comprises, preferably consists of, 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, 52, 53, 54, 55, 56, 57, 58, 59, or 60 discrete number of repeating units m. A discrete number of repeating -(O-CH2-CH2) m The synthesis of the PEG fragments comprising or consisting of discrete PEG units is described in WO 2004 / 073620 and WO 2013 / 033476. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, 28, 32, 36, 40, 44, 48, 52, 56, or 60 discrete numbers of repeating units m. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, 28 discrete numbers of repeating units m. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, 32 discrete numbers of repeating units m. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, 36 discrete numbers of repeating units m. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, 40 discrete numbers of repeating units m. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, 44 discrete numbers of repeating units m. In a preferred embodiment, the PEG fragment comprises, and preferably consists of, 48 discrete numbers of repeating units m.

[0111] In a preferred embodiment, the PEG fragment comprises, preferably consists of, 25 to 100 discrete numbers of consecutive repeating units m, preferably 25 to 60 discrete numbers of consecutive repeating units m. In a preferred embodiment, the PEG fragment comprises, preferably consists of, 25 to 60 discrete numbers of consecutive repeating units m, preferably 30 to 50 discrete numbers of consecutive repeating units m. In a preferred embodiment, the PEG fragment comprises, preferably consists of, 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, 52, 53, 54, 55, 56, 57, 58, 59 or 60 discrete numbers of consecutive repeating units m. In preferred embodiments, the PEG fragment comprises, preferably consists of, 28, 32, 36, 40, 44, 48, 52, 56, or 60 discrete numbers of consecutive repeating units m. In preferred embodiments, the PEG fragment comprises, preferably consists of, 28 discrete numbers of consecutive repeating units m. In preferred embodiments, the PEG fragment comprises, preferably consists of, 32 discrete numbers of consecutive repeating units m. In preferred embodiments, the PEG fragment comprises, preferably consists of, 36 discrete numbers of consecutive repeating units m. In preferred embodiments, the PEG fragment comprises, preferably consists of, 40 discrete numbers of consecutive repeating units m. In preferred embodiments, the PEG fragment comprises, preferably consists of, 44 discrete numbers of consecutive repeating units m. In preferred embodiments, the PEG fragment comprises, preferably consists of, 48 discrete numbers of consecutive repeating units m.

[0112] In a preferred embodiment, the —(O—CH—CH) of formula I* or formula I m The - moiety consists of a discrete number of repeating units m between 25 and 100, preferably a discrete number of repeating units m between 25 and 60. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety consists of 25 to 60 discrete repeating units m, preferably 30 to 50 discrete repeating units m. In a preferred embodiment, the -(O-CH2-CH2) mThe - moiety is comprised of a discrete number of repeating units m of 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, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of a discrete number of repeating units m of 28, 32, 36, 40, 44, 48, 52, 56, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 28 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 32 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 36 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 40 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 44 discrete repeating units m. In a preferred embodiment, the -(O-CH-CH) m -The part consists of 48 discrete repeating units m.

[0113] In a preferred embodiment, the —(O—CH—CH) of formula I* or formula I m The - moiety consists of a discrete number of consecutive repeating units m between 25 and 100, preferably a discrete number of consecutive repeating units m between 25 and 60. In a preferred embodiment, the -(O-CH2-CH2) m The - moiety consists of 25 to 60 discrete consecutive repeating units m, preferably 30 to 50 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH2-CH2) mThe - moiety consists of a discrete number of consecutive repeating units m of 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, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of a discrete number of consecutive repeating units m of 28, 32, 36, 40, 44, 48, 52, 56, or 60. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 28 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 32 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 36 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 40 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m The - moiety consists of 44 discrete consecutive repeating units m. In a preferred embodiment, the -(O-CH-CH) m - The part consists of 48 discrete consecutive repeating units m.

[0114] In another aspect, the present invention provides a composition comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0115] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably said discrete number m of repeating -(O-CH-CH)- units is 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0116] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0117] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA.1 is -H. In a preferred embodiment, 1 is -CH3.

[0118] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number m of consecutive repeating -(O-CH-CH)- units, which is 25 to 100, preferably a discrete number m of consecutive repeating -(O-CH-CH)- units, which is 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0119] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number m of consecutive repeating -(O-CH-CH)- units, which is 25 to 100, preferably a discrete number m of consecutive repeating -(O-CH-CH)- units, which is 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0120] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is the discrete number m of consecutive repeating -(O-CH2-CH2)- units 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0121] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is the discrete number m of consecutive repeating -(O-CH2-CH2)- units 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to a cell expressing PSMA, more preferably said targeting fragment is capable of binding to a cell surface receptor, said cell surface receptor being PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3.

[0122] In some preferred embodiments, the conjugates of the present invention comprise LPEI fragments present as dispersed polymer moieties, where n is from about 280 to about 700 with a dispersity of about 3 or less, preferably from about 350 to about 630 with a dispersity of about 2 or less, and more preferably from about 400 to 580 with a dispersity of about 1.2 or less, and the conjugates of the present invention further comprise PEG fragments present as a discrete number of repeating -(O-CH-CH)- units m, where m is any discrete number between 25 and 100, preferably between 25 and 60, and preferably m is a discrete number of consecutive repeating -(O-CH-CH)- units, and m is any discrete number between 25 and 100, preferably between 25 and 60.

[0123] In some embodiments, conjugates of the invention comprise LPEI fragments present as dispersed polymer moieties of about 17-25 KDa with a dispersity of about 1.2 or less, and PEG fragments comprising, preferably consisting of, a discrete number of repeating -(O-CH2-CH2)- units, m, where m is any discrete number between 25 and 60. In some preferred embodiments, conjugates of the invention comprise LPEI fragments present as dispersed polymer moieties of about 17-25 KDa with a dispersity of about 1.2 or less, and PEG fragments comprising, preferably consisting of, a discrete number of repeating -(O-CH2-CH2)- units, m, where m is 36.

[0124] target fragment The conjugates of the present invention comprise a targeting moiety that enables the conjugates, compositions, and polyplexes of the present invention to be directed to a specific target cell type, collection of cells, organ, or tissue. Typically and preferably, the targeting moiety is capable of binding to a target cell, preferably its cell receptor or cell surface receptor.

[0125] As used herein, the term "cell surface receptor" refers to a protein, such as a glycoprotein or lipoprotein, that is present on the surface of a cell and that is typically and preferably a characteristic marker for cell recognition. Typically and preferably, said cell surface receptor can bind to ligands, including hormones, neurotransmitters, cytokines, growth factors, cell adhesion molecules, or nutrients, in the form of peptides, small molecules, saccharides and oligosaccharides, lipids, amino acids, and other binding moieties, such as antibodies, aptamers, affibodies, antibody fragments, and the like.

[0126] Conjugates and polyplexes of the invention that include targeting fragments are intended to mimic such ligand-receptor interactions. Thus, in preferred embodiments, the targeting fragments are capable of binding to cell surface receptors.

[0127] In a preferred embodiment, said cell surface receptor is a peripheral membrane protein or a transmembrane protein, preferably a type II transmembrane protein.

[0128] In a preferred embodiment, the cell surface receptor is prostate-specific membrane antigen (PSMA). The term "the cell surface receptor is PSMA" is intended to mean that the cell surface receptor is derived from PSMA, is typically and preferably provided on the cell surface, and is more typically and preferably such a portion and / or part of PSMA that corresponds to the extracellular domain of PSMA and / or the cell surface-exposed portion of PSMA.

[0129] The targeting fragment according to the present invention is intended to position and deliver, particularly selectively, the polyplexes and payloads, e.g., nucleic acids, of the present invention to a desired target, particularly a desired target cell. Furthermore, the conjugates of the present invention comprising said targeting fragment not only enable the selective delivery of the conjugates and polyplexes to a target, e.g., a target cell, but also enable internalization and promote selective cellular uptake of the polyanionic payload by the target, particularly by the target cell. Thus, the targeting fragment according to the present invention represents a portion of the conjugates and polyplexes of the present invention capable of specific binding to a selected target, preferably a selected target cell, and more preferably a cellular receptor.

[0130] In a preferred embodiment, the targeting fragment is capable of binding to a target cell that expresses PSMA. In a preferred embodiment, the targeting fragment is capable of binding to a selected target cell type that expresses PSMA. In a preferred embodiment, the targeting fragment is capable of binding to a target cell receptor, and the target cell receptor is PSMA. In a preferred embodiment, the targeting fragment is capable of binding to a target cell surface receptor, and the target cell surface receptor is PSMA.

[0131] In a preferred embodiment, the targeting fragment functions to bind to a target cell that expresses PSMA. In a preferred embodiment, the targeting fragment functions to bind to a selected target cell type that expresses PSMA. In a preferred embodiment, the targeting fragment functions to bind to a target cell receptor, and the target cell receptor is PSMA. In a preferred embodiment, the targeting fragment functions to bind to a target cell surface receptor, and the target cell surface receptor is PSMA.

[0132] In a preferred embodiment, the targeting fragment is capable of specifically binding to a target cell receptor that expresses PSMA. In a preferred embodiment, the targeting fragment is capable of specifically binding to a selected target cell type that expresses PSMA. In a preferred embodiment, the targeting fragment is capable of specifically binding to a target cell receptor, and the target cell receptor is PSMA. In a preferred embodiment, the targeting fragment is capable of specifically binding to a target cell surface receptor, and the target cell surface receptor is PSMA.

[0133] In one embodiment, specifically binding to a target cell, target cell receptor, or target cell surface receptor means that the targeting fragment and the conjugate of the invention and / or the polyplex of the invention bind to said target cell, said target cell receptor, said target cell surface receptor, respectively, at least 2-fold, preferably at least 3-fold, more preferably at least 4-fold, and again more preferably at least 5-fold stronger than they bind to other non-targeted cells, cell receptors, cell surface receptors, typically and preferably as measured by a dissociation constant (KD). Preferably, the targeting fragment binds to said target cell, said target cell receptor, said target cell surface receptor, respectively, at least 2-fold, preferably at least 3-fold, more preferably at least 4-fold, and again more preferably at least 5-fold stronger than they bind to other non-targeted cells, cell receptors, cell surface receptors, typically and preferably as measured by a dissociation constant (KD). -5 Less than M, preferably 10 -6 Less than M, more preferably 10 -7 less than M, and even more preferably 10 -8 Binds to selected cell surface receptors with a KD less than M.

[0134] In one embodiment, the specific binding to a target cell, target cell receptor, or target cell surface receptor means that the targeting fragment and the conjugate of the present invention and / or the polyplex of the present invention, respectively, bind to the target cell, the target cell receptor, or the target cell surface receptor at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, again more preferably at least 10-fold, and even more preferably at least 100-fold stronger than a corresponding conjugate and / or polyplex identical to the conjugate of the present invention and / or the polyplex of the present invention, but comprising a non-specific fragment such as a hydroxyl group or an -OMe moiety, preferably an -OMe moiety, instead of the targeting fragment. Binding to a target cell, target cell receptor, or target cell surface receptor is typically and preferably measured by a dissociation constant (KD). Preferably, the targeting fragment binds ... that is identical to the conjugate of the present invention and / or the polyplex of the present invention, but comprising a non-specific fragment such as a hydroxyl -5 Less than M, preferably 10 -6 Less than M, more preferably 10 -7 less than M, and even more preferably 10 -8 It binds to a selected target cell surface receptor with a KD of less than M. In a preferred embodiment, said binding or said specific binding, and thus the binding level of the conjugates of the invention and the polyplexes of the invention, respectively, can be determined by binding or displacement assays, or by FRET or other measures demonstrating the interaction between the target fragment and the cell receptor, preferably the cell surface receptor.

[0135] The term "binding", as used herein with respect to the binding of a targeting moiety to a cell, cell receptor or cell surface receptor, preferably refers to interactions via non-covalent bonds, such as electrostatic interactions, van der Waals interactions, hydrogen bonds, hydrophobic interactions, ionic bonds, charge interactions, affinity interactions, and / or dipole-dipole interactions.

[0136] In another embodiment, said specific binding to a target cell, target cell receptor or target cell surface receptor results in a biological effect caused by said specific binding of a targeting fragment and a conjugate and / or polyplex of the invention, respectively, and / or caused by a delivered conjugate and / or polyplex of the invention and a polyanion payload, which biological effect is at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, even more preferably at least 10-fold, and even more preferably at least 25-fold, at least 50-fold, or at least 100-fold greater than said biological effect of a non-targeted cell, non-targeted cell receptor, or non-targeted cell surface receptor.

[0137] In another embodiment, said specific binding to a target cell, target cell receptor, or target cell surface receptor results in a biological effect caused by said specific binding of a targeting fragment to a conjugate and / or polyplex of the invention, respectively, and / or caused by the delivered conjugate and / or polyplex of the invention with a polyanionic payload, which biological effect is at least 2-fold, preferably at least 3-fold, more preferably at least 5-fold, again more preferably at least 10-fold, and again more preferably at least 25-fold, at least 50-fold, or at least 100-fold greater than a corresponding conjugate and / or polyplex of the invention that is identical to a conjugate and / or polyplex of the invention but that comprises a non-specific fragment such as a hydroxyl group or an -OMe moiety, preferably an -OMe moiety, instead of the targeting fragment.

[0138] Binding and specific binding can also be determined by measuring activation of protein signaling, and thus can be measured by measuring protein phosphorylation or protein expression, mRNA expression in cells or tissues (using Western blot analysis, real-time PCR, RNAseq, IHC, etc.). The level of delivery of the polyplexes of the present invention to a particular tissue can be measured by comparing the amount of protein produced in overexpressing cells compared to normal and low-expressing cells, by measuring protein secretion by Western blot analysis, luminescence / fluorescence assay, flow cytometry assay, ELISA, ECLIA, etc., by comparing the amount of expression or secretion of downstream protein (from the delivered nucleic acid, such as poly(IC)) in cells / tissues overexpressing the target receptor compared to normal or low-expressing cells / tissues, by measuring protein secretion by Western blot analysis, luminescence / fluorescence assay, flow cytometry assay, ELISA, ECLIA, etc. Delivery levels can also be measured by cytotoxicity using cell viability or cell death assays, including (MTT, methylene blue assay, CellTiter-Glo assay, propidium iodide assay): by comparing the amount of protein produced in a tissue to the weight of the tissue, comparing the therapeutic and / or prophylactic amount in a tissue to the weight of the tissue, comparing the amount of protein produced in a tissue to the total amount of protein in the tissue, or comparing the therapeutic and / or prophylactic amount in a tissue to the total therapeutic and / or prophylactic amount in the tissue. It will be understood that delivery of the polyplexes of the invention to target cells or target tissues need not be determined in the subject being treated, but can be determined in surrogates, such as animal or cell models.

[0139] Thus, in preferred embodiments, the biological effect is selected from (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 delivered poly(IC), in cells / tissues in which the target cell surface receptor is overexpressed compared to normal cells / tissues or cells / tissues with low expression, and (v) cytotoxicity.

[0140] In one embodiment, the target cells include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, and vascular smooth muscle cells. Thus, in one embodiment, the target cells are cells in the liver. In one embodiment, the target cells are epithelial cells. 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 cells are endothelial cells. In one embodiment, the target cells are tumor cells or cells in the tumor microenvironment. In one embodiment, the target cells are blood cells. In one embodiment, the target cells are cells in lymph nodes. In one embodiment, the target cells are cells in the lungs. 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 cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In one embodiment, the target cells are dendritic cells in the spleen. In one embodiment, the target cells are T cells. In one embodiment, the target cell is a B cell. In one embodiment, the target cell is a NK cell. In one embodiment, the target cell is a monocyte.

[0141] In some embodiments, the targeting moiety selectively or preferentially interacts with a particular cell type. The targeting moiety not only serves to selectively target the conjugates and polyplexes of the present invention to a particular cell, but also typically promotes selective uptake of the conjugates and corresponding polyplexes of the present invention within a particular cell type. In some embodiments, the targeting moiety selectively or preferentially interacts with a particular cell surface receptor. When the targeting moiety of a conjugate and / or polyplex selectively or preferentially interacts with a cell surface receptor, the conjugate and / or polyplex can be selectively or preferentially taken up by cells containing the cell surface receptor.

[0142] In a preferred embodiment, the targeting moiety is a peptide, a protein, a small molecule ligand, a saccharide, an oligosaccharide, a lipid, an amino acid, wherein the peptide, the protein, the small molecule ligand, the saccharide, the oligosaccharide, the lipid, the amino acid is selected from a hormone, a neurotransmitter, a cytokine, a growth factor, a cell adhesion molecule, or a nutrient, and the targeting moiety is an antibody, an antibody fragment, an aptamer, or an affibody.

[0143] The term "small molecule ligand," as used herein, particularly with respect to the targeting fragments of the present invention, relates to a chemical moiety having a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, and more preferably at least 200 g / mol, and preferably having a molecular weight of less than about 2000 g / mol. In some embodiments, the small molecule has a molecular weight of less than about 1500 g / mol, more preferably less than about 1000 g / mol. In further preferred embodiments, the small molecule has a molecular weight of less than about 800 g / mol, again more preferably less than about 500 g / mol. The term "small molecule ligand," as used herein, particularly with respect to the targeting fragments of the present invention, more preferably relates to such a ligand that binds, preferably specifically binds, to a target cell, a target cell receptor, or preferably a target cell surface receptor. In a preferred embodiment, the small molecule ligand has a molecular weight of at least 75 g / mol, preferably at least 100 g / mol, and more preferably at least 200 g / mol, and preferably has a molecular weight of less than about 2000 g / mol, and preferably less than about 1500 g / mol. In preferred embodiments, 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 less than about 2000 g / mol, preferably less than about 1500 g / mol, and the small molecule ligand is capable of binding, preferably specifically binding, to a target cell surface receptor.

[0144] In some embodiments, the targeting fragment is a native, natural, or modified ligand or a paralog thereof, or a non-natural ligand such as an antibody, a single-chain variable fragment (scFv), or an antibody mimic such as an affibody. In preferred embodiments, the targeting fragment is a native, natural, or modified cell surface antigen ligand or a paralog thereof, or a non-natural cell surface antigen ligand, such as an antibody, a single-chain variable fragment (scFv), or an antibody mimic such as an affibody. In preferred embodiments, the targeting fragment is a native, natural, or modified cell surface receptor ligand or a paralog thereof, or a non-natural cell surface receptor ligand, such as an antibody, a single-chain variable fragment (scFv), or an antibody mimic such as an affibody. In preferred embodiments, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a native, natural, or modified ligand, and / or a paralog thereof. In preferred embodiments, the targeting moiety is a small molecule ligand, peptide, protein, aptamer, native, natural or modified cell surface antigen ligand and / or paralog thereof, wherein said 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, and preferably has a molecular weight of less than about 2000 g / mol, preferably less than about 1500 g / mol. In preferred embodiments, the targeting moiety is a small molecule ligand, peptide, protein, aptamer, native, natural or modified cell surface receptor ligand and / or paralog thereof, wherein said 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, and preferably has a molecular weight of less than about 2000 g / mol, preferably less than about 1500 g / mol. In preferred embodiments, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a native, naturally occurring or modified ligand and / or a paralog thereof, an antibody, a single chain variable fragment (scFv), or an antibody mimetic such as an affibody.

[0145] In preferred embodiments, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a native, natural, or modified cell surface receptor ligand, and / or a paralog thereof. In preferred embodiments, the targeting fragment is a small molecule ligand, a peptide, a protein, an aptamer, a native, natural, or modified ligand, and / or a paralog thereof, wherein the small molecule ligand, the peptide, the protein, the aptamer, the native, natural, or modified ligand, and / or the paralog thereof is capable of binding, preferably selectively, to a cell surface receptor. In preferred embodiments, the targeting fragment is a small molecule ligand. In preferred embodiments, the targeting fragment is a small molecule ligand, wherein the small molecule ligand is capable of binding, preferably selectively, to a cell surface receptor. In preferred embodiments, the targeting fragment is a peptide. In preferred embodiments, the targeting fragment is a peptide, wherein the peptide is capable of binding, preferably selectively, to a cell surface receptor. In preferred embodiments, the targeting fragment is a protein. In preferred embodiments, the targeting fragment is a protein, wherein the protein is capable of binding, preferably selectively, to a cell surface receptor. In a preferred embodiment, the targeting fragment is an aptamer. In a preferred embodiment, the targeting fragment is an aptamer, and the aptamer is capable of binding, preferably selectively binding, to a cell surface receptor. In a preferred embodiment, the targeting fragment is a native, natural, or modified ligand and / or a paralog thereof, preferably a native, natural, or modified cell surface receptor ligand and / or a paralog thereof. In a preferred embodiment, the targeting fragment is a native, natural, or modified ligand and / or a paralog thereof, wherein the native, natural, or modified ligand and / or a paralog thereof is capable of binding, preferably selectively binding, to a cell surface receptor. In a preferred embodiment, the targeting fragment is an antibody, a single-chain variable fragment (scFv), or an antibody mimetic, such as an affibody.In a preferred embodiment, the targeting fragment is an antibody, a single chain variable fragment (scFv), or an antibody mimetic, such as an affibody, which is capable of binding, preferably selectively binding, to a cell surface receptor.

[0146] In a preferred embodiment, the targeting moiety is selected from a small molecule ligand, a peptide, a protein, an aptamer, an antibody, an antibody fragment, preferably a single chain variable fragment (scFv), an antibody mimetic, preferably an affibody, a nanobody, a diabody, a designed ankyrin repeat protein (DARPin), a cytokine or a functional fragment thereof, an integrin, an interleukin or a functional fragment thereof, an enzyme, a nucleic acid, a fatty acid, a carbohydrate, a monosaccharide, an oligosaccharide or a polysaccharide, a peptidoglycan, a glycopeptide, asialoorosomucoid, mannose-6-phosphate, mannose, Sialyl-Lewis x , N-acetyllactosamine, galactose, a lysosomotropic agent, and / or a nuclear localization agent, preferably T antigen, tumor low pH inserting 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.

[0147] In some embodiments, the targeting moiety is a non-natural ligand such as an antibody or antibody fragment (e.g., a single-chain variable fragment (scFv), an antibody mimetic, e.g., an affibody, nanobody, diabody, designed ankyrin repeat protein (DARPin), or other antibody variant). In some embodiments, the targeting moiety is a hormone or a fragment thereof, preferably a functional fragment (e.g., insulin), asialoorosomucoid, mannose-6-phosphate, mannose, sialyl Lewis x, N-acetyllactosamine, galactose, lysosomotropic agents, and / or nuclear localization agents (e.g., T antigen), tumor low pH insert peptide (PHLIP), p32 targeting peptides, such as LyP-1 tumor homing peptide, insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor, and / or fibroblast growth factor. Further non-limiting examples of targeting moieties include enzymes, nucleic acids, fatty acids, carbohydrates, mono-, oligo- or polysaccharides, peptidoglycans, glycopeptides.

[0148] In a preferred embodiment, the targeting moiety is selected from a small molecule ligand, a peptide, a protein, an aptamer, an antibody, an antibody fragment, preferably a Fab, Fab', F(ab')2 or scFv fragment, an antibody mimetic, preferably an affibody, a nanobody, a diabody, a designed ankyrin repeat protein (DARPin), a growth factor or a functional fragment thereof, a hormone or a functional fragment thereof, preferably insulin, a cytokine or a functional fragment thereof, an interleukin or a functional fragment thereof, an enzyme, a nucleic acid, a fatty acid, a carbohydrate, a monosaccharide, an oligosaccharide or a polysaccharide, a peptidoglycan, a glycopeptide, asialoorosomucoid, mannose-6-phosphate, mannose, Sialyl-Lewis x , N-acetyllactosamine, galactose, a lysosomotropic agent, and / or a nuclear localization agent, preferably T antigen, tumor low pH insert 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.

[0149] In another aspect, the present invention provides a composition comprising a conjugate, preferably a plurality of conjugates, of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating units m between 25 and 100, preferably a discrete number of repeating units m between 25 and 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA.

[0150] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number m of consecutive repeat units, which is 25 to 100, preferably 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA.

[0151] The targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), and is also referred to herein as a PSMA-targeting fragment.

[0152] PSMA is primarily expressed in four tissues of the body, including the prostate epithelium, the proximal tubules of the kidney, the jejunal brush border of the small intestine, and the ganglia of the nervous system (Mhawech-Fauceglia et al., Histopathology 2007, 50:472-483). PSMA is overexpressed in neoplastic tissue and malignant prostate, particularly in prostate adenocarcinoma compared to normal tissue, and the level of PSMA expression is further upregulated as the disease progresses to the metastatic stage (Silver et al., 1997, Clin. Cancer Res., 3:81). Because PSMA expression is approximately 1,000-fold higher in prostate tumors, PSMA is particularly considered a target for the diagnosis and treatment of prostate cancer (Kularatne SA et al., Molecular Pharmaceutics 2009, 6(3):780-789; Rowe SP et al., Prostate Cancer Prostatic Dis. 2016, 19(3):223-230; Wang H et al., Small Struct. 2022, 3:220003620;9). Furthermore, upregulation of PSMA may provide a growth advantage to prostate cancer cells, implicating PSMA in the metabolism of polyglutamylated folates and subsequent folate uptake (Yao et al., Prostate 2006, 66:867-875; Yao et al., Prostate 2010, 70:305-316).However, PSMA targeting may also be applicable to other PSMA-expressing tumors besides prostate cancer, particularly because PSMA is not expressed on normal vasculature but is expressed on the neovasculature of many solid tumors, e.g., breast, lung, gastric, colorectal, pancreatic, renal cell, and bladder cancers, allowing targeting to occur in the intravascular compartment (Chang SS et al., Cancer Res. 1999, 59(13):3192-3198; Wernicke et al., APMIS 2014, 122(6):482-489; Samplaski MK et al., Mod Pathol. 2011, 24(11):1521-1529; Haffner MC et al., Hum Pathol. 2009, 40(12):1754-1761; Morgenroth A et al., Breast Cancer Research 2019,21:116; Jian D et al., Clinical and Translational Gastroenterology 2019;10:e-00041; Jeitner TM et al., Translational Oncology 2022,22:101450, and references cited therein).

[0153] In a preferred embodiment, the targeting fragment is capable of binding to cells that express PSMA. In a preferred embodiment, the targeting fragment is capable of binding to cells that overexpress PSMA. In one embodiment, the overexpression of PSMA means that the level of PSMA expressed in the cells of a particular tissue is elevated compared to the level of PSMA measured in normal, healthy cells of the same type of tissue under similar conditions. In one embodiment, the overexpression of PSMA refers to an increase in the level of PSMA in a 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 that overexpress PSMA have at least 10-fold higher expression of PSMA compared to normal cells or normal tissue. In one embodiment, the cells that overexpress PSMA have expression of PSMA with a cutoff of 5% or more PSMA-positive cells, as described, for example, in Mhawech-Fauceglia et al., 2007, which can be used to define PSMA expression in different types of tissues or cells. Therefore, cells or tissues with less than 5% positive cells were considered negative, as described by Hupe et al., 2018 (Hupe MC et al, Frontiers in Oncology 2018, 8(623):1-7), or PSMA expression was classified according to its intensity and scored as 0 (no expression), 1 (low expression), 2 (moderate expression), and 3 (high expression).

[0154] In a preferred embodiment, the targeting fragment is capable of binding to cells that express or overexpress PSMA. PSMA-expressing cells typically include tumor cells such as prostate, bladder, pancreatic, lung, breast, kidney, and colon tumor cells, melanoma, and sarcoma. In a preferred embodiment, the targeting fragment is capable of binding to cells that express or overexpress PSMA, and the cells are tumor cells, preferably selected from prostate cancer, bladder cancer, pancreatic cancer, lung cancer, breast cancer, kidney and colon tumor cells, melanoma, and sarcoma. In a preferred embodiment, the targeting fragment is capable of binding to cells that express or overexpress PSMA, and the cells are tumor cells, and the tumor cells are prostate tumor cells.

[0155] In preferred embodiments, the targeting fragment is capable of specifically binding to PSMA, and typically and preferably, the affinity or specific binding is determined by a dissociation constant (K D ) and the affinity or specific binding is measured by -3 Less than M, preferably 10 -4 less than M, more preferably 10 -5 less than M, more preferably 10 -6 Less than M, more preferably 10 -7 M or less, and even more preferably 10 -8 M, and again more preferably less than 10 -9 M, and again more preferably less than 10 -10 K less than M D In preferred embodiments, the targeting fragment is capable of specifically binding to PSMA, and typically and preferably, the affinity or specific binding is determined by a dissociation constant (K D ) and the affinity or specific binding is measured by -3 Under M, 10 -4 Under M, 10 -5 Under M, 10 -6 Under M, 10 -7 Under M, 10 -8 Less than M and 10 -9 K less than M DPreferably, the binding results in the formation of a complex between the target fragment and PSMA, wherein the binding or complex can be detected, typically and preferably, using a Biacore 3000 instrument (Biacore Inc., Piscataway NJ) or a cell-based binding assay or Flow Induced Dispersion Analysis (FIDA), typically and preferably as described in Kularatne et al., Mol Pharm. 2009;6(3):790-800.

[0156] In a preferred embodiment, the targeting fragment is capable of binding to the extracellular domain of PSMA or a portion thereof, hi a preferred embodiment, the targeting fragment is capable of binding to an epitope on the extracellular domain of PSMA.

[0157] In preferred embodiments, the targeting moiety is a PSMA antibody, a PSMA aptamer, or a small molecule PSMA targeting moiety.

[0158] In preferred embodiments, the targeting fragment is a PSMA antibody, a PSMA aptamer, or a small molecule PSMA targeting fragment. In preferred embodiments, the PSMA targeting fragment is a PSMA antibody, a PSMA aptamer, or a small molecule PSMA targeting fragment. The term "small molecule PSMA targeting fragment," as used herein, refers to a chemical moiety having a molecular weight of less than about 2000 g / mol, typically and preferably capable of binding to PSMA. In some embodiments, the small molecule PSMA targeting fragment has a molecular weight of less than about 1800 g / mol. In some embodiments, the small molecule PSMA targeting fragment has a molecular weight of less than about 1500 g / mol, more preferably less than about 1000 g / mol. In further preferred embodiments, the small molecule has a molecular weight of less than about 800 g / mol, again more preferably less than about 500 g / mol.

[0159] In some embodiments, the PSMA-targeting fragment is a PSMA antibody, which is an antibody capable of binding to PSMA, and is therefore also known by those skilled in the art as an anti-PSMA antibody. In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, and / or an antibody fragment, preferably a functional fragment thereof, a chimeric antibody, a recombinant antibody, and / or a bispecific or multispecific antibody.Such 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; Smith-Jones et al. al., 2000, Cancer Res., 60:5237; Vallabhajosula et al., 2004, Prostate, 58:145; Bander et al., 2003, J. Urol., 170:1717; Patri et al., 2004, Bioconj. Chem., 15:1174; Viola-Villegas NT et al., Mol Pharm 2014, 11:3965-3973; and U.S. Patent 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 U.S. Patent No. 5,162,504); and Chang et al. 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, Int. J. Cancer, 92:871. Those skilled in the art will understand that any antibody that recognizes and / or specifically binds to PSMA can be used in accordance with the present invention. All of the foregoing documents and disclosures are incorporated herein by reference in their entirety.

[0160] In some embodiments, the targeting fragment that can bind to PSMA is an aptamer. PSMA targeting aptamers include, but are not limited to, A10 aptamer or A9 aptamer, their derivatives, and / or their functional fragments (Lupold et al., 2002, Cancer Res., 62:4029; and Chu et al., Nucleic Acids Res 2006, 34(10):e73; Baek SE, et al., J Control Release 2014, 196:234-242). In some embodiments, in the aptamer derivative, 30, 25, 20, 15, 10, 5, 4, 3, 2, or less than 1 nucleic acid is substituted for the aptamer. In some embodiments, the sequence of the aptamer derivative is at least 80%, preferably 85%, more preferably 90%, again more preferably 95%, and most preferably 99% identical.

[0161] In a preferred embodiment, the targeting moiety is a small molecule PSMA targeting moiety. In a preferred embodiment, the PSMA targeting moiety is a small molecule PSMA targeting moiety, preferably a small molecule PSMA targeted peptidase inhibitor. In a preferred embodiment, the small molecule PSMA peptidase inhibitors include 2-PMPA, GPI5232, VA-033, phenylalkylphosphonamidates (Jackson et al., 2001, Curr. Med. Chem., 8:949; Bennett et al., 1998, J. Am. Chem. Soc., 120:12139; Jackson et al., 2001, J Med. Chem., 44:4170; Tsukamoto et al., 2002, Bioorg. Med. Chem. Lett., 12:2189; Tang et al., 2003, Biochem. Biophys. Res. Commun., 307:8; Oliver et al., 2003, Bioorg. Med. Chem., 11:4455; and Maung et al., 2003, Bioorg. Med. Chem., 11:4455). al., 2004, Bioorg. Med. Chem., 12:4969), and / or analogs and derivatives thereof. All of the foregoing documents (scientific and other publications, patents, and patent applications) are incorporated herein by reference in their entirety. In some embodiments, the small molecule PSMA targeting fragment is a protein, peptide, amino acid, or derivative thereof. In preferred embodiments, the small molecule PSMA targeting fragment comprises 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., 4611989; and U.S. Patent Application Publication No. 2005 / 0080128). In some embodiments, the small molecule PSMA targeting fragment comprises a hydroxamate derivative (Stoermer et al., 2003, Bioorg. Med. Chem. Lett., 1312097).In a preferred embodiment, the small molecule PSMA peptidase inhibitor is an androgen receptor targeted agent (ARTA), such as those described in U.S. Pat. Nos. 7,026,500; 7,022,870; 6,998,500; 6,995,284; 6,838,484; 6,569,896; 6,492,554; and U.S. Pat. Publication Nos. 2006 / 0287547; 2006 / 0276540; 2006 / 0258628; 2006 / 0241180; 2006 / 0183931; 2006 / 0035966; 2006 / 0009529; 2006 / 0004042; 2005 / 0033074; 2004 / 0260108; 2004 / 0260092; 2004 / 0167103; 2004 / 0147550; 2004 / 0147489; 2 004 / 0087810; 2004 / 0067979; 2004 / 0052727; 2004 / 0029913; 2004 / 0014975; These include those described in US Patent Application Publication Nos. 2003 / 0232792; 2003 / 0232013; 2003 / 0225040; 2003 / 0162761; 2004 / 0087810; 2003 / 0022868; 2002 / 0173495; 2002 / 0099096; and 2002 / 0099036. In some embodiments, the small molecule PSMA targeting fragment comprises polyamines such as putrescine, spermine, and spermidine (US Patent Application Publication Nos. 2005 / 0233948 and 2003 / 0035804). All of the above documents and disclosures are incorporated herein by reference in their entirety.

[0162] In a preferred embodiment, the small molecule PSMA peptidase inhibitors include PBDA-based and urea-based inhibitors, such as ZJ 43, ZJ, ZJ 17, and 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 their analogs and derivatives. Other agents that bind to PSMA can also be used, such as PSMA-targeting fragments, including those found in Clin. Cancer Res., 2008 14:3036-43, or PSMA-targeting fragments prepared by sequentially adding components to a preformed urea, such as the lysine-urea-glutamic acid compound described in Banerjee et al. (J. Med. Chem., vol. 51, pp. 4504-4517, 2008). In a preferred embodiment, the one or more targeting moieties capable of binding to prostate-specific membrane antigen (PSMA) are small molecule PSMA targeting moieties, more preferably small molecule urea-based inhibitors.

[0163] 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 or a urea-based PSMA inhibitor), more preferably a urea-based inhibitor as described in 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 Publication No. 2017 / 044936, International Publication No. WO2011 / 084518, International Publication No. 2011 / 084521, International Publication No. 2011 / 084513, International Publication No. 2012 / 166923, International Publication No. 2008 / 105773, International Publication No. 2008 / 121949, International Publication No. 2012 / 135592, International Publication No. 2010 / 005740, International Publication No. 2015 / 168379, International Publication No. 03 / 045436, International Publication No. 03 / 045436, International Publication No. 2016 / 183447, International Publication No. 2017 / 005740, International Publication No. 2018 / 005740, International Publication No. 2019 / 005740, International Publication No. 2020 / 005740, International Publication No. 2021 / 005740, International Publication No. 2022 / 005740, International Publication No. 2023 / 005740, International Publication No. 2024 / 005740, International Publication No. 2025 / 005740, International Publication No. 2026 ... and small molecule urea-based inhibitors such as those disclosed in International Publication Nos. 015 / 258102, WO 2011 / 084513, WO 2017 / 089942, U.S. Patent Application Publication Nos. 2010 / 278927, WO 2012 / 016188, WO 2008 / 124634, WO 2009 / 131435, U.S. Patent Application Publication Nos. 2007 / 225213, WO 2017 / 086467, WO 2009 / 026177, WO 2012005572, WO 2014 / 072357, and WO 2011 / 108930. All of the foregoing documents and disclosures are incorporated herein by reference in their entirety.

[0164] In a preferred embodiment, the targeting moiety is a dipeptide urea PSMA peptidase inhibitor, preferably a small molecule dipeptide urea PSMA peptidase inhibitor. In a preferred embodiment, the PSMA targeting moiety is a dipeptide urea PSMA peptidase inhibitor, preferably a small molecule dipeptide urea PSMA peptidase inhibitor.

[0165] The term "urea-based PSMA peptidase inhibitors" refers to PSMA peptidase inhibitors containing a urea group. The term "dipeptide urea-based PSMA peptidase inhibitors" refers to PSMA peptidase inhibitors containing a urea group and two peptides or amino acids independently attached to the -NH2 group of the urea group, while the term "small molecule dipeptide urea-based PSMA peptidase inhibitors" further refers to dipeptide urea-based PSMA peptidase inhibitors having a molecular weight of less than about 2000 g / mol and typically and preferably capable of binding to PSMA. In some embodiments, small molecule dipeptide urea-based PSMA peptidase inhibitors have a molecular weight of less than about 1800 g / mol, less than about 1500 g / mol, and preferably less than about 1000 g / mol. In further preferred embodiments, small molecule dipeptide urea-based PSMA peptidase inhibitors have a molecular weight of less than about 800 g / mol, and more preferably less than about 500 g / mol. The PSMA peptidase inhibitor can reduce the activity of the PSMA transmembrane zinc(II) metalloenzyme, which catalyzes the cleavage of terminal glutamic acid. More preferably, the small molecule urea-based PSMA peptidase inhibitor has a molecular weight of less than about 500 g / mol. Even more preferably, the small molecule urea-based PSMA peptidase inhibitor is preferably a glutamic acid-urea-based PSMA peptidase inhibitor, such as those mentioned in Kopka et al., J Nuc Med, 58(9), suppl. 2, 2017; Wirtz et al., EJNMMI Research (2018) 8:84 and the references cited therein (all of which are incorporated herein by reference in their entirety).

[0166] In a preferred embodiment, the targeting moiety, preferably the urea based PSMA peptidase inhibitor, is a glutamic acid-urea moiety of Formula 1, preferably of Formula 1*: [ka] and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof; wherein R is preferably substituted or unsubstituted alkyl, substituted or unsubstituted aryl, and any combination thereof; more preferably, R is C substituted one or more times, preferably once, with OH, SH, NH, or COOH. 1~6 alkyl, preferably C2-C4 alkyl, wherein one of the NH2, OH or SH or COOH groups is X 2 In another preferred embodiment, R is a C substituted once with OH, SH, NH, or COOH. 1~6 alkyl, preferably C2-C4 alkyl, wherein the NH2, OH or SH or COOH group is each 2 In a highly preferred embodiment, R is a C alkyl substituted once with COOH, wherein each COOH group is X 2 In a preferred embodiment, the COOH group is X 2 When serving as the point of covalent attachment to the linking moiety, the COOH group is X 2 It condenses with the amine group of the linking moiety to form an amide.

[0167] In a preferred embodiment, the targeting fragment is a glutamic acid-urea moiety of Formula 1: [ka] wherein R is a C substituted one or more times, preferably one time, with OH, SH, NH, or COOH.1~6 alkyl, preferably C2-C4 alkyl, wherein one of the NH2, OH or SH or COOH groups is X 2 In another preferred embodiment, R is a C alkyl group substituted once with OH, SH, NH, or COOH. 1~6 alkyl, preferably C2-C4 alkyl, wherein the NH2, OH, or SH or COOH group is each 2 In a highly preferred embodiment, R is a C alkyl substituted once with COOH, each COOH group being X 2 In a preferred embodiment, the COOH group is X 2 When serving as the point of covalent attachment to the linking moiety, the COOH group is X 2 It condenses with the amine group of the linking moiety to form an amide.

[0168] In another preferred embodiment, the targeting fragment is a glutamic acid-urea moiety of formula 1*: [ka] wherein R is a C substituted one or more times, preferably one time, with OH, SH, NH, or COOH. 1~6 alkyl, preferably C2-C4 alkyl, wherein one of the NH2, OH or SH or COOH groups is X 2 In another preferred embodiment, R is a C alkyl group substituted once with OH, SH, NH, or COOH. 1~6 alkyl, preferably C2-C4 alkyl, wherein the NH2, OH or SH or COOH group is each 2In a highly preferred embodiment, R is a C alkyl substituted once with COOH, each COOH group being X 2 In a preferred embodiment, the COOH group is X 2 When serving as the point of covalent attachment to the linking moiety, the COOH group is X 2 It condenses with the amine group of the linking moiety to form an amide.

[0169] In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0170] In a further preferred embodiment, the PSMA targeting fragment is a folate ligand. In a further preferred embodiment, the PSMA targeting fragment is a small molecule PSMA targeting fragment, and the small molecule PSMA targeting fragment is a folate ligand.

[0171] In a preferred embodiment, the folic acid ligand binds to a cell surface receptor, and the cell surface receptor is PSMA. As recently reported, targeting of PSMA-expressing cells has been achieved by amides of folic acid (Flores O et al., Theranostics 2017,7(9):2477-2494).

[0172] As used herein, the term "folate ligand" is understood as folic acid or methotrexate or a derivative or analog thereof. Preferably, said folic acid or methotrexate derivative or analog is a folic acid ligand having a glutamic acid functional group R-NH-[CH(COOH)-CH-CH-C(O)NH] η-CH(COOH)-CH-CH-COOH, where η is an integer from 0 to 100, and R is a group of formula 2: [ka] (In the formula, R 201 is -OH or -NH2; R 202 is -H or -CH3; and (The wavy line indicates the point of attachment to the glutamic acid functional group.) In a preferred embodiment, η is an integer of 0 to 10, preferably η is an integer of 0 to 5, and more preferably η is 0.

[0173] Those skilled in the art will recognize that R 201 is —OH, in a preferred embodiment, said OH tautomerizes to a carbonyl group (═O), and said R 201 It will be understood that the adjacent nitrogen atom of is protonated.

[0174] Those skilled in the art will recognize that the glutamic acid functional group R—NH—[CH(COOH)—CH—CH—C(O)NH] η It will be further understood that -CH(COOH)-CH-CH-COOH includes at least one alpha carboxylate group and a gamma carboxylate group. Specifically, one or more -COOH groups bonded to the same carbon as one or more -NH- groups are understood herein as an alpha carboxylate group. When η = 0, a -COOH group bonded to the same carbon as an R-NH group is understood herein as an alpha carboxylate group. A -COOH group bonded to a -(CH)- group is understood herein as a gamma carboxylate group. Furthermore, those skilled in the art will understand that the carboxylate groups discussed herein, e.g., alpha and gamma carboxylate groups, can be protonated or deprotonated depending on the pH of the surrounding solution. Thus, those skilled in the art will understand that while carboxylate groups are depicted as neutral species (-COOH) for simplicity and clarity, they may also be deprotonated species, i.e., negatively charged species (-COO) at physiological pH.- ) can be present (e.g., can be present primarily).

[0175] In some embodiments, the alpha carboxylate group of the glutamic acid functional group is X 2 In a preferred embodiment, the alpha carboxylate group of the glutamic acid functional group is X 2 When acting as the point of attachment to a linking moiety, the alpha carboxylate group is X 2 In some embodiments, the alpha carboxylate group of the glutamic acid functional group is condensed with the amine group of the linking moiety to form an amide. 2 When acting as the point of attachment to a linking moiety, the alpha carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0176] In a preferred embodiment, the gamma carboxylate group of the glutamic acid functional group is 2 In a preferred embodiment, the gamma carboxylate group of the glutamic acid functional group is X 2 When acting as the point of attachment to a linking moiety, the gamma carboxylate group is X 2 In some embodiments, the gamma carboxylate group of the glutamic acid functional group is condensed with the amine group of the linking moiety to form an amide. 2 When acting as the point of attachment to a linking moiety, the gamma carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0177] In a preferred embodiment, the folate ligand is folic acid: [ka] wherein either the alpha or gamma carboxylate group of the folic acid is X 2(which acts as a covalent attachment point to the linking moiety).

[0178] In some embodiments, the alpha carboxylate group of the folic acid is X 2 In a preferred embodiment, the alpha carboxylate group of the folic acid is X 2 When acting as the point of attachment to a linking moiety, the alpha carboxylate group is X 2 In some embodiments, the alpha carboxylate group of the folic acid is condensed with the amine group of the linking moiety to form an amide. 2 When acting as the point of attachment to a linking moiety, the alpha carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0179] In a preferred embodiment, the gamma carboxylate group of the folic acid is X 2 In a preferred embodiment, the gamma carboxylate group of the folic acid is X 2 When acting as the point of attachment to a linking moiety, the gamma carboxylate group is X 2 In some embodiments, the gamma carboxylate group of the folic acid is condensed with the amine group of the linking moiety to form an amide. 2 When acting as the point of attachment to a linking moiety, the gamma carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0180] In a preferred embodiment, the folate ligand is methotrexate: [ka] wherein either the alpha or gamma carboxylate group of the methotrexate is X 2 (which acts as a covalent attachment point to the linking moiety).

[0181] In some embodiments, the alpha carboxylate group of the methotrexate is X 2 In a preferred embodiment, the alpha carboxylate group of the methotrexate is X 2 When acting as the point of attachment to a linking moiety, the alpha carboxylate group is X 2 In some embodiments, the alpha carboxylate group of the methotrexate is condensed with the amine group of the linking moiety to form an amide. 2 When acting as the point of attachment to a linking moiety, the alpha carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0182] In a preferred embodiment, the gamma carboxylate group of the methotrexate is X 2 In a preferred embodiment, the gamma carboxylate group of the methotrexate is X 2 When acting as the point of attachment to a linking moiety, the gamma carboxylate group is X 2 In some embodiments, the gamma carboxylate group of the methotrexate is condensed with the amine group of the linking moiety to form an amide. 2 When acting as the point of attachment to a linking moiety, the gamma carboxylate group is X 2 It condenses with the hydroxy group of the linking moiety to form an ester.

[0183] In a further aspect, the present invention provides a composition comprising a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2-L (Formula I*); wherein n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, Z is not —NHC(O)—, preferably Z is a divalent covalent linking moiety, where ZX 1 is not a single bond and Z is not -NHC(O)-; L is a targeting moiety capable of binding to cells overexpressing prostate-specific membrane antigen (PSMA), preferably said L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-)], preferably consisting of said conjugate.

[0184] In a further aspect, the present invention provides a composition comprising a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); wherein n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety, Z is not —NHC(O)—, preferably Z is a divalent covalent linking moiety, where ZX 1 is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to prostate-specific membrane antigen (PSMA), preferably said L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-)], preferably consisting of said conjugate.

[0185] In a further aspect, the present invention provides a composition comprising a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); wherein n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety and Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety and Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, said targeting fragment L is a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-).

[0186] In another aspect, the present invention provides a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); wherein n is any integer from 1 to 1500; m is any integer from 1 to 200, preferably m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety and Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety and Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to cells overexpressing prostate-specific membrane antigen (PSMA), preferably said L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-).

[0187] In another aspect, the present invention provides a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L(formula I*); [wherein n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety and Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety and Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment capable of binding to prostate-specific membrane antigen (PSMA), preferably L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-).

[0188] In another aspect, the present invention provides a conjugate of formula I*, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof: R 1 -(NR 2 -CH2-CH2) n -ZX 1 -(O-CH2-CH2) m -X 2 -L (Formula I*); wherein n is any integer from 1 to 1500; m is any integer from 1 to 200, preferably m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m of repeating -(O-CH-CH)- units being any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably 90%, of X are H; 1 and X 2 are independently a divalent covalent linking moiety; Z is a divalent covalent linking moiety and Z is not -NHC(O)-, preferably Z is a divalent covalent linking moiety and Z is not a single bond and Z is not -NHC(O)-; L is a targeting fragment, said targeting fragment L is a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-).

[0189] In some embodiments, the conjugate is of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number from 25 to 100, preferably from 25 to 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n - the R in the part 2 at least 80%, preferably 90% of which is H; Ring A may have one or more R A1 is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with; R A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with; R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13-, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl or heteroaryl is selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 optionally substituted with R 11 , R 12 and R 13 is independently, at each occurrence, H, —SOH, —NH, —COH, or C-C alkyl; each alkyl is optionally substituted with —COH or —NH; R 14 are independently selected at each occurrence as H, C1-C6 alkyl, or oxo, C6-C 10 aryl, or 5-8 membered heteroaryl); X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 is independently selected from -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 optionally substituted with R 21 , R 22 and R 23 are each independently at each occurrence -H, -SO3H, -NH2, -CO2H, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, -CO2H, -NH2, C6-C6 10 optionally substituted with aryl, or 5-8 membered heteroaryl; R 24 is a linking moiety for (each occurrence is independently -H, -COH, C-C alkyl, or oxo); L is a targeting fragment, preferably said targeting fragment L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-).

[0190] In another aspect, the present invention provides a composition comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating units m between 25 and 100, preferably a discrete number of repeating units m between 25 and 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more RA2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, 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 enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating units m between 25 and 100, preferably a discrete number of repeating units m between 25 and 60; R 1 is the starting residue, preferably R 1 is -H or -CH3; R2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0191] In another aspect, the present invention provides a composition comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is the repeating unit m of 36 discrete numbers; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0192] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is the repeating unit m of 36 discrete numbers; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 RA2 is independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0193] In another aspect, the present invention provides a composition comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number m of consecutive repeat units, which is 25 to 100, preferably 25 to 60, which is a discrete number m of consecutive repeat units; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0194] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number m of consecutive repeat units, which is 25 to 100, preferably 25 to 60, which is a discrete number m of consecutive repeat units; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1is -H. In a preferred embodiment, 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0195] In another aspect, the present invention provides a composition comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is the discrete number m of consecutive repeat units 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0196] In another aspect, the present invention provides a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is the discrete number m of consecutive repeat units 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0197] In another aspect, the present invention provides a composition comprising, preferably consisting of, a conjugate of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is a discrete number m of consecutive repeat units, which is 25 to 100, preferably 25 to 60, which is a discrete number m of consecutive repeat units; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n - the R in the part 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl or heteroaryl is selected from one or more R 13 and each divalent heterocycle is optionally substituted with one or more R 14 optionally substituted with R 11 , R 12 and R 13 is independently at each occurrence H or C1-C6 alkyl; R 14 is a linking moiety for (each occurrence is independently H, C1-C6 alkyl, or oxo); X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 50, and Y 2 Each occurrence of represents independently a chemical bond, -CR 21 R 22 -, NR 23-, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is selected from one or more R 23 and each divalent heterocyclic moiety is optionally substituted with one or more R 24 optionally substituted with R 21 , R 22 and R 23 are each independently at each occurrence -H, -COH, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, C6-C 10 optionally substituted with aryl, or 5-8 membered heteroaryl; R 24 is a linking moiety for (each occurrence is independently -H, -COH, C-C alkyl, or oxo); L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA. 1 is -H. In a preferred embodiment, 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0198] In another aspect, the present invention provides a composition comprising, preferably consisting of, a conjugate of formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: [ka] [In the formula, [ka] is a single or double bond; n is any integer from 1 to 1500; m is the discrete number m of consecutive repeat units 36; R 1 is the starting residue, preferably R 1 is -H or -CH3; R 2 are independently —H or an organic residue, and the —(NR 2 -CH2-CH2) n -R in 2 at least 80%, preferably at least 90% of is H; Ring A may have one or more R A1 R is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with A1 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, oxo, or halogen; or two R A1 together with the atoms to which they are attached to form one or more fused C6-C 10 can form an aryl, C-C heteroaryl, or C-C cycloalkyl ring, and each fused aryl, heteroaryl, or cycloalkyl can be joined by one or more R A2 optionally substituted with R A2 are independently selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, —SO3H, or —OSO3H; X 1 is a divalent covalent linking moiety; X 2 is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being a PSMA targeting fragment, preferably said PSMA targeting fragment being capable of specifically binding to cells that express, preferably overexpress, PSMA.1 is -H. In a preferred embodiment, 1 is -CH3. In a further preferred embodiment, the targeting fragment comprises, or preferably consists of, a DUPA residue (HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). In a further highly preferred embodiment, the targeting fragment consists of a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-) in which both chiral C atoms have the (S)-configuration, as shown in formula 1*.

[0199] In a preferred embodiment, the DUPA residue is a linking moiety X 2 is linked to the PEG targeting fragment by

[0200] Such linking moieties are known to those skilled in the art and are disclosed in U.S. Patent Application Publication Nos. 2020 / 0188523, 2011 / 0288152, and 2010 / 324008, the disclosures of which are incorporated herein by reference in their entireties.

[0201] In a preferred embodiment, the linking moiety X 2 is a peptide linker or C1-C 10 In a preferred embodiment, the linking moiety X 2 is a peptide linker.

[0202] In a preferred embodiment, the linking moiety X 2 is a peptide linker, wherein said peptide linker comprises, preferably consists of, the sequence of SEQ ID NO: 3 (-(NH-(CH2)7-CO)-Phe-Phe-(NH-CH2-CH(NH2)-CO)-Asp-Cys-) or the sequence of SEQ ID NO: 1 (-(NH-(CH2)7-CO)-Phe-Gly-Trp-Trp-Gly-Cys-). In a preferred embodiment, said linking moiety X 2is a peptide linker, wherein said peptide linker comprises, preferably consists of, the sequence of SEQ ID NO: 1 (-(NH-(CH2)7-CO)-Phe-Gly-Trp-Trp-Gly-Cys-). In a further preferred embodiment, said linking moiety X 2 comprises, and preferably consists of, SEQ ID NO: 1 or 3, and the target fragment is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO- (DUPA residue). In a highly preferred embodiment, the linking moiety X 2 comprises, and preferably consists of, SEQ ID NO: 1, wherein the targeting fragment L is HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO- (DUPA residue). In a preferred embodiment, said targeting fragment L is HOOC-(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-, which is capable of binding to cells overexpressing PSMA, and said linking moiety X 2 comprises, and preferably consists of, SEQ ID NO:1.

[0203] In another preferred embodiment, the targeting fragment is 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA), wherein typically and preferably, the coupling to the remainder of the conjugate is via the terminal carboxyl group of the DUPA. Thus, in a further preferred embodiment, the targeting fragment L is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-). DUPA can be selectively taken up by cells with increased (e.g., overexpression of) prostate-specific membrane antigen (PSMA).

[0204] Coupling of PEG fragments to target fragments In some embodiments, the second end of the PEG fragment is a linking group (i.e., X) that links the PEG fragment to a targeting fragment. 2 ) is functionalized with a linking moiety X 2contains a reactive group for coupling to an appropriate, i.e., complementary, reactive group on the targeting fragment. 2 It will be appreciated that various complementary reactive groups may be involved in such coupling reactions between the reactive groups. In some embodiments, the targeting fragment L is unmodified and is a PEG fragment and a linking moiety X, respectively. 2 For example, Scheme 3 shows the nucleophilic addition of hEGF to an electrophilic tetrafluorophenyl ester attached to a PEG fragment. As shown in Scheme 3, the nucleophilic amine of hEGF displaces the tetrafluorophenol of the tetrafluorophenyl ester to form a PEG fragment and a linking moiety, X. 2 In some embodiments, the targeting fragment L is linked to the linking moiety X using a suitable chemical linkage, such as an amide or ester bond. 2 For example, Schemes 4 and 5 show X containing an amide linkage. 2 The DUPA and folate groups are shown attached to the PEG fragment by a linker, respectively. The amide groups are linked to the appropriate carboxylic acid groups of DUPA and folate, and to the PEG-X 2 It is formed by a dehydration synthesis reaction between the appropriate amine on the fragment.

[0205] In some preferred embodiments, the first end (i.e., terminal end) of the PEG fragment is functionalized with an alkene or alkyne group, which in some embodiments can be used to react with an azide-functionalized LPEI; the second end (i.e., terminal end) of the PEG fragment is functionalized with a targeting moiety, which in some embodiments can be used to promote uptake of the conjugate and corresponding polyplex in a specific cell type. Thus, in some preferred embodiments, the resulting conjugates of the invention can have the general structure LPEI-PEG-targeting moiety arranged in a linear end-to-end manner.

[0206] The conjugates of the present invention can be prepared using a variety of different methods and processes. Schemes 1 and 2 below show different strategies for disposing the conjugates of the present invention. As shown in Scheme 1 below, the conjugates of the present invention can be prepared by first coupling a PEG fragment to a targeting fragment, and then coupling the targeting fragment-modified PEG fragment to an LPEI fragment. As shown in Scheme 2 below, the conjugates of the present invention can be prepared by first coupling a PEG fragment to an LPEI fragment, and then coupling the LPEI-modified PEG fragment to a targeting fragment.

[0207] Scheme 1. Exemplary coupling of bifunctional PEG to a targeting moiety and subsequently to an LPEI. [ka] As shown in Scheme 1, a bifunctional PEG (e.g., a PEG containing an alkene or alkyne and an electrophile) can first be reacted with a targeting fragment (e.g., hEGF, DUPA, or folic acid) to generate a PEG fragment covalently linked to the targeting fragment. The alkene or alkyne group of the targeting fragment-modified PEG can then be reacted with the azide group of the LPEI fragment via a [3+2] cycloaddition to generate a linear conjugate of the general structure LPEI-PEG targeting fragment.

[0208] Scheme 2. Exemplary coupling of bifunctional PEG to LPEI and subsequent targeting fragment [ka]

[0209] As shown in Scheme 2, a bifunctional PEG (e.g., a PEG containing an alkene or alkyne and an electrophile) can first react with the azide group of an LPEI fragment via a [3+2] cycloaddition to generate a linear conjugate of LPEI and PEG covalently linked by a 1,2,3 triazole or A4,5-dihydro-1H-[1,2,3]triazole. The linear LPEI-PEG fragment can then be reacted with a targeting fragment (e.g., hEGF, DUPA, or folic acid) to generate a linear conjugate of the general structure LPEI-PEG targeting fragment.

[0210] Schemes 3-5 below illustrate general methods for coupling PEG fragments to various targeting fragments. Those skilled in the art will appreciate that PEG fragments can be coupled to various targeting fragments using any suitable chemistry (e.g., nucleophilic substitution, peptide coupling, etc.). For example, those skilled in the art will appreciate that it is not necessary to use a tetrafluorophenyl ester as the electrophile to couple the PEG fragment to hEGF as shown in Scheme 3, but that other electrophilic groups, such as maleate (as shown in Scheme 4), can also be used. Furthermore, those skilled in the art will appreciate that the reactive group on the bifunctionalized PEG fragment need not necessarily be an electrophilic group, but instead can be a nucleophilic group that reacts with, for example, an electrophilic moiety on a targeting fragment.

[0211] Scheme 3. Exemplary coupling of bifunctional PEG to hEGF [ka] As shown in Scheme 3 above, in some embodiments, PEG can be modified to contain electrophilic groups such as tetrafluorophenyl esters and / or activated alkyne groups such as DBCO. Treatment of a PSMA-targeting fragment containing a nucleophilic group, such as an -NH group, in solution with the tetrafluorophenyl ester-modified PEG results in nucleophilic displacement, producing a PEG fragment conjugated to the PSMA-targeting fragment. The DBCO group can be used in subsequent reactions for coupling to the LPEI fragment. The variable m represents a discrete number of repeating -(O-CH-CH)- positions, with the discrete number m of repeating -(O-CH-CH)- units being any discrete number between 25 and 100, preferably between 25 and 60, and more preferably 36.

[0212] Scheme 4. Exemplary coupling of bifunctional PEG to DUPA [ka] 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 can be coupled to a nucleophilic partner, such as the illustrated DUPA-derived moiety (as shown in the scheme above, containing the peptide spacer Aoc-Phe-Gly-Trp-Trp-Gly-Cys (SEQ ID NO: 1)) N-terminally derivatized with 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA), which contains a nucleophilic group, i.e., a thiol, due to the cysteine-derived amino acid residue. Treatment of the MAL-modified PEG in solution with the thiol-modified DUPA-derived moiety results in nucleophilic 1,4-addition via the nucleophilic thiol of the DUPA-derived moiety, yielding the DUPA-modified PEG. The variable m represents a discrete number of repeating -(O-CH2-CH2)- positions, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36.

[0213] Scheme 5. Exemplary coupling of bifunctional PEG to folic acid [ka] As shown in Scheme 5 above, PEG can be modified to contain an electrophilic maleimide (MAL) group. The maleimide-substituted PEG can be coupled to a nucleophilic partner, such as a folic acid residue, that has itself been modified to contain a nucleophilic group (e.g., a thiol). Treatment of the MAL-modified PEG in solution with folic acid thiol results in nucleophilic 1,4 addition of folic acid via the nucleophilic thiol, yielding the folic acid-modified PEG. The variable m represents a discrete number of repeating -(O-CH2-CH2)- positions, and the discrete number m of repeating -(O-CH2-CH2)- units can be any discrete number between 25 and 100, preferably between 25 and 60, and more preferably, m is 36.

[0214] Coupling of PEG fragments to LPEI fragments The bifunctionalized PEG fragment can be coupled to the LPEI fragment before or after coupling to the targeting fragment. In a preferred embodiment, the bifunctionalized PEG fragment is coupled to the LPEI using cycloaddition chemistry, such as 1,3-dipolar cycloaddition or [3+2] cycloaddition between an azide and an alkene or alkyne to form a 1,2,3 triazole or a 4,5-dihydro-1H-[1,2,3]triazole. In another preferred embodiment, the bifunctionalized PEG fragment is coupled to the LPEI using thiol-ene chemistry between a thiol and an alkene to form a thioether.

[0215] Those skilled in the art will understand that any suitable alkene or alkyne group can be used to react with the azide group to couple the LPEI fragment to the PEG fragment. In some preferred embodiments, the incorporation of an alkene or alkyne group into the ring system introduces strain into the ring system. The strain on the ring system can be released upon reaction of the alkene or alkyne group, preferably without the use of an additional catalyst such as copper, to produce a 1,2,3-triazole or a 4,5-dihydro-1H-[1,2,3]triazole. Thus, in some preferred embodiments, suitable ring systems include a 7-, 8-, or 9-membered ring containing an alkyne group, or an 8-membered ring containing a trans alkene group. For example, suitable alkyne groups, such as cyclooctyne (OCT), monofluorinated cyclooctyne (MOFO), difluorocycloalkyne (DIFO), dibenzocyclooctynol (DIBO), dibenzoazacyclooctyne (DIBAC), bicyclononyne (BCN), biarylazacyclooctynone (BARAC), and tetramethylthiepinium (TMTI), can be used. Additionally, suitable alkene groups, such as transcyclooctene, transcycloheptene, and maleimide, can be used. For example, conjugates of the present invention can be prepared from a PEG fragment and a moiety containing an alkene or alkyne group according to one of the following formulas: [ka] or [ka] (wherein the variable X 1 , X 2 , R A1 , L and m are defined above).

[0216] Without wishing to be bound by theory, azide and alkene or alkyne groups can react spontaneously (i.e., without the addition of a catalyst) to form 1,2,3-triazole or 4,5-dihydro-1H-[1,2,3]triazole. In some embodiments, the azide group reacts with an alkyne to form a 1,2,3-triazole. In some embodiments, the azide group reacts with an alkene to form a 4,5-dihydro-1H-[1,2,3]triazole.

[0217] Those skilled in the art will understand 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 strained alkene or alkyne). Thus, in some embodiments, the LPEI fragment contains an alkene or alkyne group (e.g., a strained alkene or alkyne) and the bifunctionalized PEG fragment contains an azide group. In some preferred embodiments, the bifunctionalized PEG fragment contains an alkene or alkyne group (e.g., a strained alkene or alkyne) and the LPEI fragment contains an azide group.

[0218] Those skilled in the art will also appreciate that the [3+2] cycloaddition between an azide and an alkene or alkyne group can afford adducts with different regiochemistries, as shown below in Schemes 6-8. Those skilled in the art will appreciate that all possible regiochemistries of the [3+2] cycloaddition are contemplated by the present invention.

[0219] In some preferred embodiments, the [3 + 2] azide-alkyne cycloaddition reaction occurs at a pH of 5 or less, preferably 4 or less. As shown below in the comparative example, when a PEG fragment modified with an activated alkyne was treated with a non-azide-containing LPEI fragment at pH 4, no reaction occurred. 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 a strained alkyne or alkene) group of the PEG fragment. However, at higher pH, the comparative example teaches that a by-product characterized by a hydroamination reaction between the nitrogen atom of the LPEI fragment and the alkene or alkyne was formed. Without wishing to be bound by theory, the present invention teaches that an LPEI fragment (e.g., containing a terminal azide) can be chemoselectively attached to a PEG fragment (e.g., containing an activated, preferably a strained alkene or alkyne) at a pH of less than about 5, preferably about 4 or less.

[0220] In another aspect, the present invention provides a method of synthesizing a conjugate of Formula I, comprising reacting a thiol-containing LPEI fragment with an alkene-containing PEG fragment, as shown below in Scheme 9.

[0221] In another aspect, the present invention provides a method of synthesizing a conjugate as described and defined herein, preferably a method of synthesizing a conjugate of Formula I, wherein the method comprises reacting an omega end of a linear polyethyleneimine fragment with a first end of a polyethylene glycol fragment, wherein said reaction occurs at a pH of less than about 5, preferably 4 or less, and preferably said omega end of said linear polyethyleneimine fragment comprises an azide and said first end of said polyethylene glycol fragment comprises an alkene or alkyne, and said reaction is between said azide and said alkene or alkyne.

[0222] Scheme 6. Coupling of LPEI to dibenzocyclooctyne (DBCO)-modified PEG [ka] As shown in Scheme 6 above, in some embodiments, PEG can be modified to include a strained alkyne group, such as DBCO. Treatment of DBCO-modified PEG in solution with azide-modified LPEI results in a [3 + 2] cycloaddition of the azide to the alkyne of DBCO, yielding a 1,2,3 triazole. One of skill in the art will appreciate that the reaction shown above in Scheme 6 can produce triazole adducts with different regiochemistry, as shown above. The variables m and n represent the number of repeating PEG and LPEI units described herein, where m is any discrete number of repeating -(O-CH-CH)- units, and the discrete number m of repeating -(O-CH-CH)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36.

[0223] Scheme 7. Coupling of LPEI to bicyclononyne (BCN)-modified PEG [ka] As shown in Scheme 7 above, in some embodiments, PEG can be modified to include a strained alkyne group, such as bicyclononyne (BCN). Treatment of the BCN-modified PEG in solution with an azide-modified LPEI results in a [3 + 2] cycloaddition of the azide to the BCN alkyne, yielding a 1,2,3 triazole. One of skill in the art will appreciate that the reaction shown above in Scheme 7 can produce triazole adducts with different regiochemistry, as shown above. The variables m and n represent the number of repeating PEG and LPEI units described herein, where m is any discrete number of repeating -(O-CH-CH)- units, and the discrete number m of repeating -(O-CH-CH)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36.

[0224] Scheme 8. Coupling of LPEI to maleimide (MAL)-modified PEG [ka] As shown in Scheme 8 above, in some embodiments, PEG can be modified to include an alkene group, such as maleimide (MAL). Treatment of the MAL-modified PEG in solution with an azide-modified LPEI results in a [3 + 2] cycloaddition of the azide to the alkene of the MAL, yielding 4,5-dihydro-1H-[1,2,3]triazole. The variables m and n represent the number of repeating PEG and LPEI units as described herein, where m is any discrete number of repeating -(O-CH2-CH2)- units, and the discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably 36.

[0225] Scheme 9. Coupling of LPEI to alkene-modified PEG [ka] As shown in Scheme 9 above, in some embodiments, PEG can be modified to contain a terminal alkene group, and LPEI can be modified to contain a terminal thiol group. Treatment of thiol-modified LPEI in solution with alkene-modified PEG can result in a thiol-ene reaction, yielding a thioether. The variables m and n represent the number of repeating PEG and LPEI units described herein, where m is any discrete number of repeating -(O-CH-CH)- units, and the discrete number m of repeating -(O-CH-CH)- units can be any discrete number between 25 and 100, preferably between 25 and 60, and more preferably 36.

[0226] X 1 and X 2 connecting part In some embodiments, the PEG fragment of the conjugates of the invention may be connected to the alkene or alkyne group and / or the targeting fragment by a covalent linking moiety.

[0227] X 1 connecting part In some embodiments, the PEG fragment of the conjugate of the invention is connected to a terminal activated (e.g., cyclic) alkene or alkyne group by a linking moiety. For example, X 1 Linking moieties can be formed as a result of selecting a PEG fragment and an alkene or alkyne group, each containing reactive functional groups that can be combined by well-known chemical reactions. For example, a PEG fragment can be coupled to an activated (e.g., cyclic) alkene or alkyne group by standard means, such as peptide coupling (e.g., to form an amide), nucleophilic addition, or other means known to those skilled in the art.

[0228] In one embodiment, X 1 is the formula -(Y 1 ) p - [wherein p is an integer of 1 to 20, and Y 1 Each occurrence of represents a chemical bond, -CR 11 R 12 -, -C(O)-, -O-, -S-, -NR 13 -, an amino acid residue, a divalent phenyl moiety, a divalent carbocyle moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl or heteroaryl is independently selected from one or more R 11 and each divalent heterocycle is optionally substituted with one or more R 14 optionally substituted with R 11 , R 12 and R 13 is independently at each occurrence -H, -SO3H, -NH2, or C1-C6 alkyl, where each alkyl is optionally substituted with -CO2H or NH2; and R 14 is independently, at each occurrence, -H, C1-C6 alkyl or oxo, C6-C 10 aryl or 5-8 membered heteroaryl).

[0229] In some embodiments, Y 1is an amino acid residue, it can be oriented in any direction, i.e., —C(O)—CHR—NH— or —NH—CHR—C(O)—, where “R” represents the side chain of a naturally occurring amino acid.

[0230] In some embodiments, the divalent heteroaryl moiety is a divalent heteroaryl group containing one or more heteroatoms selected from O, N, S, and P, preferably one or two 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, wherein the divalent heteroaryl is selected from one or more, preferably one or zero, R 14 is optionally substituted with

[0231] X 1 In the following embodiments for , unless otherwise specified, the wavy line indicates a bond of any orientation, i.e., to a PEG fragment or a divalent covalent linking moiety (e.g., "Z" or ring A).

[0232] In some embodiments, the divalent heterocyclic moiety is a divalent heterocyclic group containing one or more heteroatoms selected from O, N, S, and P, preferably one or two atoms selected from O and N. In some embodiments, the divalent heterocyclic moiety is a divalent heterocyclic group containing one or more heteroatoms selected from O, N, S, and P, preferably one or two atoms selected from O and N. In some embodiments, the divalent heterocyclic moiety is a divalent heterocyclic group containing one or more R 14 In some preferred embodiments, the divalent heterocyclic moiety is succinimide. In some preferred embodiments, the two Y 1 Combining these, the formula [ka] can form a linking or partially linking moiety of

[0233] In a further preferred embodiment, two Y 1 Combining these, the formula [ka] where the wavy line next to the sulfur indicates the direction of the connection towards the target fragment.

[0234] In a further preferred embodiment, Y 1 is the expression: [ka] The linking moiety may comprise a linking moiety or partial linking moiety of

[0235] In a further preferred embodiment, Y 1 is the expression: [ka] where the wavy line next to the sulfur indicates the direction of the connection towards the target fragment.

[0236] In some embodiments, X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 8, and Y 1 Each occurrence of represents a chemical bond, -CHR 11 -, -C(O)-, -O-, -S-, -NH-, -C6H4-, [ka] or [ka] is a linking moiety of

[0237] In some embodiments, X 1is the formula -(Y 1 ) p where p is an integer from 1 to 8, and Y 1 Each occurrence of represents a chemical bond: -CH2-, -C(O)-, -O-, -S-, -NH-, -C6H4-, [ka] or [ka] is a linking moiety of

[0238] In some embodiments, X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 8, and Y 1 Each occurrence of represents a chemical bond: -CH2-, -C(O)-, -O-, -S-, -NH-, [ka] or [ka] is a linking moiety of

[0239] In some embodiments, X 1 is the formula -(Y 1 ) p where p is an integer from 1 to 8, and Y 1 Each occurrence of represents a chemical bond: -CH2-, -C(O)-, -O-, -NH-, [ka] or [ka] where Y1 is the linking moiety of (only -NH- when adjacent to a -C(O)- group to form a carbamate or amide).

[0240] In some embodiments, X 1 teeth, [ka] (wherein r is an integer of 1 to 8, preferably 1 to 4, more preferably 1 to 2; R 11 and R 12 are independently —H or C1-C6 alkyl, preferably —H or C1-C2 alkyl, more preferably —H).

[0241] In some embodiments, X 1 teeth, [ka] or [ka] (wherein 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; R 11 and R 12 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0242] In some embodiments, X 1 teeth, [ka] (wherein 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 -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0243] In some embodiments, X 1 teeth, [ka] (wherein r is an integer of 0 to 3, preferably 1 to 3, more preferably 1 to 2; s and t are each independently an integer of 0 to 2, preferably 0 to 1; the sum of r, s, and t is 6 or less; and R 11 and R 12 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "t" is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0244] In some embodiments, X 1 teeth, [ka] or [ka] (wherein 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; R 11 , R 12 and R 13 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0245] In some embodiments, X 1 teeth, [ka] or [ka] (wherein 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; R 11 , R 12 and R 13are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0246] In some embodiments, X 1 teeth, [ka] or [ka] (wherein r and t are each an integer of 0 to 3, and s is an integer of 0 to 3; preferably, r is 0, s is 2 or 3, and t is 2; the sum of r, s, and t is 5 or less; and R 11 , R 12 and R 13 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "t" is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0247] In some embodiments, X 1 teeth, [ka] or [ka] (wherein r and t are each an integer of 0 to 3; s is an integer of 0 to 3; the sum of r, s, and t is 5 or less; R 11 and R 12 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "t" is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0248] In some embodiments, X 1 teeth, [ka] or [ka] (wherein r and s each independently represent an integer of 0 to 3, preferably 0 to 2; the sum of r and s is 5 or less; R 11 , R 12 and R 13 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" is the bond to the PEG fragment -[OCH2-CH2] m-, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0249] In some embodiments, X 1 teeth, [ka] (wherein r is independently an integer of 0 to 4, preferably 0 to 2, more preferably 1 to 2; R 11 and R 12 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A), and the wavy line closest to the carbonyl group is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0250] In some embodiments, X 1 teeth, [ka] (wherein 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; R 11 and R 12 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A), and the wavy line closest to the carbonyl group is the bond to the PEG fragment -[OCH2-CH2] m-, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0251] In some embodiments, X 1 teeth, [ka] (wherein r and s each independently represent 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 C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A), and the wavy line closest to the carbonyl group is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0252] In some preferred embodiments, X 1 teeth, [ka] or [ka] (In the formula, r is independently at each occurrence 0 to 6, preferably 0, 1, 2, or 5; s is independently, at each occurrence, 0 to 6, preferably 0, 2, or 4; t is independently, at each occurrence, 0 to 6, preferably 0, 1, 2, or 4; R 11 and R 12 is independently selected at each occurrence from -H, -C1-C2 alkyl, -SO3H, and -NH2; more preferably -H, -SO3H, and -NH2; even more preferably -H; R 13 is -H). Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" or "t" or the carbonyl group is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0253] In some preferred embodiments, X 1 teeth, [ka] or [ka] (In the formula, r is independently at each occurrence 0 to 6, preferably 0, 1, 2, or 5; s is independently, at each occurrence, 0 to 6, preferably 0, 2, or 4; t is independently, at each occurrence, 0 to 6, preferably 0, 1, 2, or 4; R 11 and R 12 is independently selected at each occurrence from -H, -C1-C2 alkyl, preferably -H; R 13is -H). Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" or "t" or the carbonyl group is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0254] In some preferred embodiments, X 1 teeth, [ka] (In the formula, r is independently, at each occurrence, 0 to 6, preferably 0, 1, 2, or 5; more preferably 0; s is independently, at each occurrence, 0 to 6, preferably 0, 2, 3, or 4; more preferably 2 or 3; t is independently, at each occurrence, 0 to 6, preferably 0, 1, 2, or 4; more preferably 2; R 11 and R 12 is independently selected at each occurrence from -H, -C1-C2 alkyl, preferably -H; and R 13 is -H). Preferably, the wavy line closest to the integer "r" is the bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the wavy line closest to the integer "s" or "t" group is the bond to the PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0255] In some preferred embodiments, X 1 teeth, [ka] (In the formula, X A is -NHC(O)- or -C(O)NH-; and [ka] Preferably, the left wavy line is a bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the right wavy line is a bond to a PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0256] In some preferred embodiments, X 1 teeth, [ka] and [ka] Preferably, the left wavy line is a bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the right wavy line is a bond to a PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0257] In some preferred embodiments, X 1 teeth, [ka] and [ka] Preferably, the left wavy line is a bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the right wavy line is a bond to a PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0258] In some embodiments, X 1 teeth, [ka] and [ka] Preferably, the left wavy line is a bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the right wavy line is a bond to a PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0259] In some preferred embodiments, X 1 teeth, [ka] and [ka] Preferably, the left wavy line is a bond to a divalent covalent linking moiety (e.g., "Z" or ring A) and the right wavy line is a bond to a PEG fragment -[OCH2-CH2] m -, wherein m is a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number from 25 to 100, preferably from 25 to 60, and more preferably m is 36.

[0260] In some preferred embodiments, X 1 is -(CH2) 1~6 -; preferably, X 1 is -(CH2) 2~4 -; more preferably, X 1 is -(CH2)2-.

[0261] X 2 connecting part In some embodiments, the PEG fragment of the conjugate of the invention is connected to a terminal targeting fragment by a linking moiety. For example, X 2 Linking moieties can be formed as a result of selecting PEG and targeting fragments, each containing reactive functional groups that can be combined by well-known chemical reactions. For example, PEG fragments can be coupled to targeting groups by standard means, such as peptide coupling (e.g., to form amides), nucleophilic addition, or other means known to those skilled in the art.

[0262] In one embodiment, X 2 is the formula -(Y 2 ) q - [wherein q is an integer of 1 to 50, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NR 23 -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is independently selected from one or more R 23and each divalent heterocyclic moiety is optionally substituted with one or more R 24 optionally substituted with; R 21 , R 22 and R 23 are each independently at each occurrence -H, -SO3H, -NH2, -CO2H, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, -CO2H, -NH2, C6-C6 10 optionally substituted with aryl or 5-8 membered heteroaryl; R 24 is a linking moiety for (each occurrence is independently -H, -CO2H, C1-C6 alkyl, or oxo).

[0263] In some embodiments, R 21 , R 22 and R 23 is independently at each occurrence -H, -COH, or C-C alkyl. In some embodiments, R 21 , R 22 and R 23 are each independently —H or C1 to C4 alkyl, preferably C1 to C2 alkyl.

[0264] In some embodiments, R 21 , R 22 , R 23 and R 24 is -H.

[0265] In some embodiments, R 24 are independently -H, C1-C6 alkyl, or oxo.

[0266] In some embodiments, the divalent heteroaryl moiety is a divalent heteroaryl group containing one or more heteroatoms selected from O, N, S, and P, preferably one or two 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, wherein the divalent heteroaryl is selected from one or more, preferably one or zero, R 21 is optionally substituted with

[0267] X 2 In the following embodiments for , unless otherwise stated, the wavy line represents any orientation of the PEG fragment (-[OCHCH] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably, m is 36; or to a targeting fragment (i.e., "L"), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably, said targeting fragment is capable of binding to cells expressing PSMA.

[0268] In some embodiments, the divalent heterocyclic moiety is a divalent heterocyclic group containing one or more heteroatoms selected from O, N, S, and P, preferably one or two atoms selected from O and N. In some embodiments, the divalent heterocyclic moiety is a divalent heterocyclic group containing one or more heteroatoms selected from O, N, S, and P, preferably one or two atoms selected from O and N. In some embodiments, the divalent heterocyclic moiety is a divalent heterocyclic group containing one or more R 24 In some preferred embodiments, the divalent heterocyclic moiety is succinimide. In some preferred embodiments, the two Y 2 Combining these, the formula [ka] can form a linking or partially linking moiety of

[0269] In a further preferred embodiment, two Y 2 Combining these, the formula [ka] wherein the wavy line next to the sulfur represents a bond to a targeting fragment (L), wherein said targeting fragment L is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA; the wavy line next to the nitrogen represents a bond to a PEG fragment (-[OCH-CH] m -), wherein the variable m represents a discrete number of repeating -(O-CH-CH)- units, and the discrete number m of repeating -(O-CH-CH)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably, m is 36.

[0270] In a further preferred embodiment, two Y 2 Combining these, the formula [ka] where the wavy line next to the sulfur represents the PEG fragment (-[OCH-CH] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and the discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably, m is 36; and the wavy line adjacent to the nitrogen represents a bond to a targeting fragment (L), wherein said targeting fragment L is capable of binding to prostate-specific membrane antigen (PSMA), preferably wherein said targeting fragment is capable of binding to cells expressing PSMA.

[0271] In a further preferred embodiment, Y 2 is the expression: [ka] The linking moiety may comprise a linking moiety or partial linking moiety of

[0272] In a further preferred embodiment, Y 2 is the expression: [ka] where the wavy line next to the sulfur indicates the direction of the connection towards the target fragment.

[0273] In some embodiments, X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 40, and Y 2 Each occurrence of represents a chemical bond, -CR 21 R 22 -, NH-, -O-, -S-, -C(O)-, amino acid residues, and [ka] are independently selected from; R 21 and R 22 is independently at each occurrence -H, -CO2H, or C1-C6 alkyl, where each C1-C6 alkyl is selected from one or more of -OH, oxo, C6-C 10 aryl, or 5-8 membered heteroaryl) is a linking moiety.

[0274] In some embodiments, X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 40, and Y 2 Each occurrence of represents a chemical bond, -CHR 21 -, NH-, -O-, -S-, -C(O)-, amino acid residues, and [ka] are independently selected from; R 21 is independently, at each occurrence, -H, -CO2H, or C1-C4 alkyl (preferably C1 alkyl), wherein each C1-C4 alkyl is selected from the group consisting of one or more C6-C 10 is a linking moiety for (optionally substituted with aryl or 5-8 membered heteroaryl).

[0275] In some embodiments, X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 40, and Y 2 Each occurrence of represents a chemical bond, -CHR 21 -, NH-, -O-, -S-, -C(O)-, amino acid residues, and [ka] are independently selected from; R 21 is independently, at each occurrence, -H, -CO2H, or C1-C4 alkyl (preferably C1 alkyl), wherein each C1-C4 alkyl is selected from the group consisting of one or more C6-C 10 is a linking moiety for (optionally substituted with aryl or 5-8 membered heteroaryl).

[0276] In some embodiments, X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 40, and Y 2 Each occurrence of represents a chemical bond, -CHR 21 -, NH-, -O-, -S-, -C(O)-, amino acid residues, and [ka] are independently selected from; R 21is independently, at each occurrence, —H, —COH, or C1-C3 alkyl (preferably C1 alkyl), where each C1-C3 alkyl is optionally substituted with one or more phenyl or indole, and is a linking moiety.

[0277] In some embodiments, X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 40, and Y 2 Each occurrence of represents a chemical bond, -CHR 21 -, NH-, -O-, -S-, -C(O)-, amino acid residues, and [ka] are independently selected from; R 21 is independently, at each occurrence, —H, —COH, or C1-C3 alkyl (preferably C1 alkyl), where each C1-C3 alkyl is optionally substituted with one or more phenyl or 3-indole, and is a linking moiety.

[0278] In some embodiments, X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 40, and Y 2 Each occurrence of represents a chemical bond, -CHR 21 -, NH-, -O-, -S-, -C(O)-, amino acid residues, and [ka] are independently selected from; Y 2 is only -NH- when adjacent to a -C(O)- group to form a carbamate or amide; R 21is independently, at each occurrence, —H, —COH, or C1-C3 alkyl (preferably C1 alkyl), where each C1-C3 alkyl is optionally substituted with one or more phenyl or 3-indole, and is a linking moiety.

[0279] In some embodiments, X 2 is the formula -(Y 2 ) q - (wherein q is an integer from 1 to 40, and Y 2 Each occurrence of represents a chemical bond, -CHR 21 -, NH-, -O-, -S-, -C(O)-, amino acid residues, and [ka] are independently selected from; Y 2 is only -NH- when adjacent to a -C(O)- group to form an amide; R 21 is independently, at each occurrence, —H, —COH, or C1-C3 alkyl (preferably C1 alkyl), where each C1-C3 alkyl is optionally substituted with one or more phenyl or 3-indole, and is a linking moiety.

[0280] In some embodiments, Y 2 is an amino acid residue, Y 2 represents a naturally occurring L-amino acid residue. 2 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” represents the side chain of a naturally occurring amino acid.

[0281] In some embodiments, X 2 teeth, [ka] (wherein r is an integer of 1 to 8, preferably 1 to 4, more preferably 1 to 2; R 21and R 22 are independently —H or C1-C6 alkyl, preferably —H or C1-C2 alkyl, more preferably —H).

[0282] In some embodiments, X 2 teeth, [ka] or [ka] (wherein 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; R 21 and R 22 are independently —H or C1-C6 alkyl, preferably —H or C1-C2 alkyl, more preferably —H).

[0283] In some embodiments, X 2 teeth, [ka] (In the formula, s and t each independently represent an integer of 0 to 4, preferably 1 to 3, and more preferably 1 to 2; the sum of r and s is 7 or less; R 21 , R 22 , and R 23 are independently —H or C1-C6 alkyl, preferably —H or C1-C2 alkyl, more preferably —H).

[0284] In some embodiments, X 2 teeth, [ka] (wherein r is an integer of 0 to 3, preferably 1 to 3, more preferably 1 to 2; s and t are each independently an integer of 0 to 2, preferably 0 to 1; the sum of r, s, and t is 6 or less; and R 21 and R 22 are independently —H or C1-C6 alkyl, preferably —H or C1-C2 alkyl, more preferably —H).

[0285] In some embodiments, X 2 teeth, [ka] or [ka] (wherein 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; R 21 , R 22 and R 23 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H). Preferably, the wavy line closest to the integer "r" represents a PEG fragment (-[OCH2-CH2] m -), wherein variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36; and the wavy line closest to the integer "s" is attached to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0286] In some embodiments, X 2 teeth, [ka] or [ka] (wherein 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; R 21 , R 22 , and R 23 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H). Preferably, the wavy line closest to the integer "r" represents a PEG fragment (-[OCH2-CH2] m -), wherein variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36; and the wavy line closest to the integer "s" is attached to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0287] In some embodiments, X 2 teeth, [ka] or [ka] (wherein r and t are each an integer of 0 to 3, and s is an integer of 0 to 3; preferably, r is 0, s is 2 or 3, and t is 2; the sum of r, s, and t is 5 or less; and R 21 , R 22 and R 23 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H). Preferably, the wavy line closest to the integer "r" represents a PEG fragment (-[OCH2-CH2] m-), wherein variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36; and the wavy line closest to the integer "t" is attached to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0288] In some embodiments, X 2 teeth, [ka] or [ka] (wherein r and t are each an integer of 0 to 3; s is an integer of 0 to 3; the sum of r, s, and t is 5 or less; and R 21 and R 22 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H). Preferably, the wavy line closest to the integer "r" represents a PEG fragment (-[OCH2-CH2] m -), wherein variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36; and the wavy line closest to the integer "t" is attached to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0289] In some embodiments, X 2 teeth, [ka] or [ka] (wherein r and s each independently represent 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 C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line closest to the integer "r" represents a PEG fragment (-[OCH2CH2] m -), wherein variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36; and the wavy line closest to the integer "s" is attached to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0290] In some embodiments, X 2 teeth, [ka] (wherein 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; R 21 and R 22 are independently -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H). Preferably, the wavy line closest to the integer "r" represents a PEG fragment (-[OCH2-CH2] m-), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36, and the wavy line closest to the carbonyl group is attached to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0291] In some embodiments, X 2 teeth, [ka] (wherein r and s each independently represent 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 C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H). Preferably, the wavy line closest to the integer "r" represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36, and the wavy line closest to the carbonyl group is attached to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0292] In some embodiments, X 2 teeth, [ka] [ka] or [ka] (In the formula, r, s, t, and u each independently represent an integer of 0 to 6, preferably 0 to 4; v represents an integer of 0 to 10; w represents an integer of 0 to 10; AA is an amino acid residue, preferably a naturally occurring amino acid residue; even more preferably, AA is an amino acid selected from Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, 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 selected from -H, C1-C6 alkyl or (-COOH), preferably -H, C1-C2 alkyl or (-COOH), more preferably -H or (-COOH). Preferably, the wavy line on the left represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0293] 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).

[0294] In some embodiments, X2 teeth, [ka] [ka] or [ka] (In the formula, r, s, t, and u each independently represent an integer of 0 to 6, preferably 0 to 4; v represents an integer of 0 to 10; w represents an integer of 0 to 10; AA is an amino acid residue, preferably a naturally occurring amino acid residue; even more preferably, AA is an amino acid selected from Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, 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 selected from -H, C1-C6 alkyl or (-COOH), preferably -H, C1-C2 alkyl or (-COOH), more preferably -H or (-COOH). Preferably, the wavy line on the left represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0295] In some preferred embodiments, (AA) ais Trp-Trp-Gly-Phe (SEQ ID NO: 2).

[0296] In some embodiments, X 2 teeth, [ka] or [ka] (In the formula, r and s each independently represent an integer of 0 to 4, preferably 0 to 2; w represents an integer of 0 to 10; R 21 , R 22 and R 23 are independently selected from -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line on the left represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0297] In some embodiments, X 2 teeth, [ka] [ka] or [ka] (In the formula, r and s each independently represent an integer of 0 to 4, preferably 0 to 2; w represents an integer of 0 to 10; R 21 , R 22 and R 23 are independently selected from -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line on the left represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0298] In some preferred embodiments, X 2 teeth, [ka] [ka] or [ka] (In the formula, r, s, and t each independently represent an integer of 0 to 4, preferably 0 to 2; w represents an integer of 0 to 10; AA is an amino acid selected from Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, 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 R23 are independently selected from -H or C1-C6 alkyl, preferably -H or C1-C2 alkyl, more preferably -H. Preferably, the wavy line on the left represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0299] In an even more preferred embodiment, (AA) a is Trp-Trp-Gly-Phe (SEQ ID NO: 2).

[0300] In some embodiments, X 2 comprises or is a urea, carbamate, carbonate, or ester. In some preferred embodiments, X 2 teeth, [ka] and [ka] Preferably, the wavy line on the left represents a PEG fragment (-[OCH2-CH2] m-), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0301] In a preferred embodiment, the X 2 teeth [ka] Preferably, the wavy line on the left represents the PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0302] In a further preferred embodiment, the X 2 teeth, [ka] and the L of the triconjugate is a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-). Preferably, the wavy line on the left represents a PEG fragment (-[OCH-CH] m-), where variable m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36, and the wavy line on the right is attached to a DUPA residue.

[0303] In a further preferred embodiment, the X 2 teeth, [ka] wherein L of the triconjugate is a DUPA residue (HOOC(CH2)2-CH(COOH)-NH-CO-NH-CH(COOH)-(CH2)2-CO-), and X 2 The amide group-bearing end of the PEG fragment (-[OCH2-CH2] m -), wherein the variable m is a discrete number of repeating -(O-CH-CH)- units, and said discrete number m of repeating -(O-CH-CH)- units is any discrete number between 25 and 100, preferably 25 and 60, and more preferably m is 36, and the amine-functionalized end is attached to a DUPA residue (HOOC(CH)-CH(COOH)-NH-CO-NH-CH(COOH)-(CH)-CO-).

[0304] In some embodiments, X 2 teeth, [ka] or [ka] [where, X B is -C(O)NH- or -NH-C(O)-, and Y 2 and R 21 is as defined above. Preferably, the wavy line on the left is selected from the PEG fragment (-[OCH2-CH2] m-), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0305] In some embodiments, X 2 teeth, [ka] or [ka] [where, X B is -C(O)NH- or -NH-C(O)-, and Y 2 and R 21 is as defined above. Preferably, the wavy line on the left is selected from the PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0306] In some embodiments, X 2 teeth, [ka] (SEQ ID NO:10, wherein SEQ ID NO:10 is defined as W1-Gly-Trp-Trp-Gly-Phe-W2, wherein W1 is [ka] and W2 is [ka] is), or [ka] (In the formula, Y 2 and R 21 is as defined above). Preferably, the left wavy line represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0307] In some embodiments, X 2 teeth, [ka] or [ka] (SEQ ID NO:14, wherein SEQ ID NO:14 is defined as W9-Gly-Trp-Trp-Gly-Phe-W10, wherein W9 is [ka] and W10 is [ka] and;R 21 is as defined above, preferably R 21 is selected from -H or -CH2-NH2; more preferably -H). Preferably, the wavy line on the left represents a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0308] In some embodiments, X 2 teeth, [ka] (SEQ ID NO:11, wherein SEQ ID NO:11 is defined as W3-Gly-Trp-Trp-Gly-Phe-W4, wherein W3 is [ka] and W4 is [ka] is), or [ka] (SEQ ID NO:14, wherein SEQ ID NO:14 is defined as W9-Gly-Trp-Trp-Gly-Phe-W10, wherein W9 is [ka] and W10 is [ka] Preferably, the wavy line on the left is selected from the group consisting of a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0309] In some embodiments, X 2 teeth, [ka] (SEQ ID NO:11, wherein SEQ ID NO:11 is defined as W3-Gly-Trp-Trp-Gly-Phe-W4, wherein W3 is [ka] and W4 is [ka] (SEQ ID NO:12, wherein SEQ ID NO:12 is defined as W5-Gly-Trp-Trp-Gly-Phe-W6, wherein W5 is [ka] and W6 is [ka] (SEQ ID NO:13, wherein SEQ ID NO:13 is defined as W7-Gly-Trp-Trp-Gly-Phe-W8, wherein W7 is [ka] and W8 is [ka] (SEQ ID NO:14, wherein SEQ ID NO:14 is defined as W9-Gly-Trp-Trp-Gly-Phe-W10, wherein W9 is [ka] and W10 is [ka] or [ka] Preferably, the wavy line on the left is selected from the group consisting of a PEG fragment (-[OCH2-CH2] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0310] In some embodiments, X 2 teeth: [ka] is.

[0311] In some embodiments, X 2 teeth: [ka] (In the formula, X B is -C(O)NH- or -NH-C(O)-). Preferably, the wavy line on the left represents a PEG fragment (-[OCH-CH] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0312] In some embodiments, X 2 teeth: [ka] (In the formula, X B is -C(O)NH- or -NH-C(O)-). Preferably, the wavy line on the left represents a PEG fragment (-[OCH-CH] m -), wherein the variable m represents a discrete number of repeating -(O-CH2-CH2)- units, and said discrete number m of repeating -(O-CH2-CH2)- units is any discrete number between 25 and 100, preferably between 25 and 60, and more preferably m is 36; and the wavy line on the right attaches to a targeting fragment (L), wherein said targeting fragment is capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment is capable of binding to cells expressing PSMA.

[0313] In some embodiments, the composition comprises a conjugate of formula IA: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0314] In some embodiments, the composition comprises a conjugate of formula IA-1: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0315] In some embodiments, the composition comprises a conjugate of formula IA-2: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0316] In some embodiments, the composition comprises a conjugate of formula IA-3: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0317] In some embodiments, the composition comprises a conjugate of formula IA-3a: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0318] In some embodiments, the composition comprises a conjugate of formula IA-3b: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0319] In some embodiments, the composition comprises a conjugate of formula IA-3c: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0320] In some embodiments, the composition comprises a conjugate of formula IA-3d: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0321] In some embodiments, the composition comprises a conjugate of formula IA-4: [ka] (Preferably, in the formula, n is from about 280 to about 700 with a dispersity of about 3 or less, more preferably from about 350 to about 630 with a dispersity of about 2 or less, and again more preferably from about 400 to 580 with a dispersity of about 1.2 or less; and preferably, m is a discrete number of repeating -(O-CH-CH)- units, said discrete number m being any integer between 25 and 100, preferably between 25 and 60, and even more preferably m is 36). Includes.

[0322] In some embodiments, the composition comprises a conjugate of formula IA-4a: [ka] (Pre...

Claims

1. 1. A composition comprising a conjugate, the conjugate comprising: a linear polyethyleneimine fragment comprising an alpha end and an omega end; A polyethylene glycol fragment comprising a first end and a second end, said polyethylene glycol fragment comprising a discrete number m of repeating -(O-CH ２ -CH ２ )- units, preferably consisting of repeating —(O—CH ２ -CH ２ )-units, m being any discrete number between 25 and 100, preferably between 25 and 60; Including; the alpha terminus of the polyethyleneimine fragment is an initiating residue; The omega end of the polyethyleneimine fragment is a divalent covalent linking group -Z-X １ - (in the formula, -Z-X １ - is not a single bond and -Z- is not an amide) to the first end of the polyethylene glycol fragment; the second end of the polyethylene glycol fragment is linked to a divalent covalent linking moiety X ２ connected to the target fragment by A composition wherein said targeting moiety is capable of binding to prostate-specific membrane antigen (PSMA), preferably wherein said targeting moiety is capable of binding to cells expressing PSMA.

2. a composition comprising a conjugate, said conjugate being of formula I* or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or enantiomer thereof; R １ -(NR ２ -CH ２ -CH ２ ) ｎ -Z-X １ -(O-CH ２ -CH ２ ) ｍ -X ２ -L (Formula I*); During the ceremony, n is any integer from 1 to 1500; m is a repeating group -(O-CH ２ -CH ２ )-units, and the repeating -(O-CH ２ -CH ２ said discrete number m of )-units is any discrete number between 25 and 100, preferably between 25 and 60; R １ is the initial residue, preferably R １ is -H or -CH ３ and R ２ are independently —H or an organic residue, and the —(NR ２ -CH ２ -CH ２ ) ｎ The R in - ２ at least 80%, preferably 90%, of is H; X １ and X ２ are independently divalent covalent linking moieties; Z is a divalent covalent linking moiety; Z-X １ is not a single bond, Z is not NHC(O)—; L is a targeting segment, said targeting segment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting segment being capable of binding to cells expressing PSMA.

3. 3. The composition of claim 1 or claim 2, wherein the conjugate is of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof: 【Chemistry 1】 [In the formula, 【Chemistry 2】 is a single or double bond; n is any integer from 1 to 1500; m is a repeating group -(O-CH ２ -CH ２ )-units, and the repeating -(O-CH ２ -CH ２ said discrete number m of )-units is any discrete number between 25 and 100, preferably between 25 and 60; R １ is the initial residue, preferably R １ is -H or -CH ３ and R ２ are independently —H or an organic residue, and the —(NR ２ -CH ２ -CH ２ ) ｎ The R in - ２ at least 80%, preferably at least 90% of is H; Ring A is one or more R Ａ１ is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R Ａ１ is C １ ~C ６ Alkyl, C １ ~C ６ or two R are independently selected from alkoxy, oxo, or halogen; Ａ１ 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 Each fused aryl, heteroaryl, or cycloalkyl can form a cycloalkyl ring, and one or more R Ａ２ optionally substituted with R Ａ２ is C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, halogen, —SO ３ H, or -OSO ３ independently selected from H; X １ is a divalent covalent linking moiety; X ２ is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA.

4. The —(O—CH ２ -CH ２ ) ｍ The part is a repeat of 25 to 60 discrete numbers m -(O-CH ２ -CH ２ )-unit, and preferably the —(O—CH ２ -CH ２ ) ｍ - the part is a repeat of a discrete number m from 25 to 48 - (O-CH ２ -CH ２ )-units, more preferably the repeating —(O—CH ２ -CH ２ 4. The composition of claim 2, wherein the discrete number m of α- and β-hydroxybenzoates is 36.

5. Ring A is an 8-membered cycloalkenyl, a 5-membered heterocycloalkyl, or a 7-8-membered heterocycloalkenyl, wherein each cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl has at any position one or more R Ａ１ 5. The composition of claim 3, wherein the compound is optionally substituted with

6. Ring A is cyclooctene, succinimide, or a 7- to 8-membered heterocycloalkenyl, wherein the heterocycloalkenyl contains one or two heteroatoms selected from N, O, and S, and each cyclooctene or heterocycloalkenyl may contain, at any position, one or more R Ａ１ and is optionally substituted with, preferably, R Ａ１ is oxo or fluorine, or two R Ａ１ are combined to form one or more fused phenyl rings, preferably one or two fused phenyl rings, each phenyl ring containing one or more -SO ３ H or -OSO ３ 6. The composition of claim 3, optionally substituted with H.

7. wherein the conjugate of formula I is 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 and 【Chemistry 11】 The composition of any one of claims 3 to 6, wherein the composition is selected from

8. wherein the conjugate of formula I is 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 and [Chemistry 18] The composition of any one of claims 3 to 7, wherein the composition is selected from

9. wherein the conjugate of formula I is 【Chemistry 19】 and 【Chemistry 20】 9. The composition of claim 3, wherein the composition is selected from

10. wherein the conjugate of formula I is 【Chemistry 21】 9. The composition of claim 3, wherein the composition is selected from

11. wherein the conjugate of formula I is 【Chemistry 22】 and 【Chemistry 23】 9. The composition of claim 3, wherein the composition is selected from

12. X １ but, 【Chemistry 24】 (In the formula, r is independently at each occurrence 0 to 6, preferably 0, 1, 2, or 5; more preferably 0; s is independently, at each occurrence, 0 to 6, preferably 0, 2, 3, or 4; more preferably 2 or 3; t is independently at each occurrence 0 to 6, preferably 0, 1, 2, 4; more preferably 2; R １１ and R １２ is independently, at each occurrence, —H and —C １ ~C ２ alkyl, preferably —H; and R １３ is —H; preferably, the wavy line closest to said integer “r” is a bond to ring A, and the wavy line closest to said integer “s” or “t” is —[OCH ２ -CH ２ ] ｍ - is a bond with 12. The composition of claim 3, wherein the composition is selected from:

13. X １ but, 【Chemistry 25】 (In the formula, X Ａ is —NHC(O)— or —C(O)NH—; and 【Chemistry 26】 Preferably, the wavy line on the left is a bond to ring A and the wavy line on the right is —[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 11, wherein the bond is -.

14. X １ but, 【Chemistry 27】 【Chemistry 28】 【Chemistry 29】 Preferably, the wavy line on the left side is a bond to ring A, and the wavy line on the right side is —[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 11, wherein the bond is -.

15. X ２ but, 【Transformation 30】 and 【Chemistry 31】 wherein X is selected from Ｂ is —C(O)NH— or —NH—C(O)—; Y ２ Each occurrence of -CR ２１ R ２２ -, NR ２３ -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, each divalent phenyl and divalent heteroaryl being independently selected from one or more R ２３ and each divalent heterocyclic moiety is optionally substituted with one or more R ２４ optionally substituted with; R ２１ , R ２２ and R ２３ are each independently, at each occurrence, -H, -SO ３ H, —NH ２ , -CO ２ H or C １ ~C ６ alkyl, where each C １ ~C ６ Alkyl is one or more of -OH, oxo, -CO ２ H, —NH ２ , C ６ ~C １０ optionally substituted with aryl or 5-8 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 —[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 14, wherein the wavy line on the right is a bond to - and the wavy line on the right is a bond to L.

16. X ２ but, 【Chemistry 32】 【Transformation 33】 and 【Transformation 34】 is selected from Y ２ Each occurrence of -CR ２１ R ２２ -, NR ２３ -, -O-, -S-, -C(O)-, an amino acid residue, a divalent phenyl moiety, a divalent carbocyclic moiety, a divalent heterocyclic moiety, and a divalent heteroaryl moiety, wherein each divalent phenyl and divalent heteroaryl is independently selected from one or more R ２３ and each divalent heterocyclic moiety is optionally substituted with one or more R ２４ optionally substituted 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, where each C １ ~C ６ The alkyl group may be one or more of -OH, oxo, -CO ２ H, —NH ２ , C ６ ~C １０ optionally substituted with aryl or 5-8 membered heteroaryl; R ２４ independently, each occurrence represents -H, -CO ２ H, C １ ~C ６ alkyl, or oxo; preferably the wavy line on the left is —[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 14, wherein the wavy line on the right is a bond to - and the wavy line on the right is a bond to L.

17. X ２ but, 【Chemistry 35】 【Transformation 36】 【Chemistry 37】 【Transformation 38】 and 【Chemistry 39】 Preferably, the wavy line on the left side is selected from -[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 14, wherein the wavy line on the right is a bond to - and the wavy line on the right is a bond to L.

18. X ２ but, 【Chemistry 40】 and preferably, the wavy line on the left is —[OCH ２ -CH ２ ] ｍ The composition of any one of claims 3 to 14, wherein the wavy line on the right is a bond to - and the wavy line on the right is a bond to L.

19. X ２ but, 【Chemistry 41】 15. The composition of any one of claims 3 to 14, wherein

20. The composition of any one of claims 1 to 19, wherein the targeting fragment L is capable of binding to a cell surface receptor expressing PSMA, preferably wherein the targeting fragment is capable of specifically binding to a cell surface receptor expressing PSMA.

21. 21. The composition of claim 20, wherein the cell surface receptor is a transmembrane protein, preferably a type II transmembrane protein.

22. 22. The composition of claim 20 or claim 21, wherein the cell surface receptor is prostate-specific membrane antigen (PSMA).

23. 23. The composition of any one of claims 1 to 22, wherein the targeting fragment L is capable of binding to a cell surface receptor expressing PSMA, and the targeting fragment is a peptide, protein, small molecule ligand, sugar, oligosaccharide, oligonucleotide, lipid, amino acid, antibody, antibody fragment, aptamer, or affibody.

24. 24. The composition of any one of claims 1 to 23, wherein the targeting fragment L is selected from a PSMA antibody, a PSMA aptamer, and a small molecule PSMA targeting fragment, preferably wherein the small molecule PSMA targeting fragment is a DUPA residue or a folate ligand.

25. 25. The composition of any one of claims 1 to 24, wherein the targeting fragment L is a small molecule PSMA targeting fragment.

26. 26. The composition of claim 25, wherein the small molecule PSMA targeting fragment is a urea-based PSMA peptidase inhibitor.

27. 26. The composition of claim 25, wherein the small molecule PSMA targeting moiety is a folate ligand.

28. The folate ligand is folate 【Chemistry 42】 and wherein either the alpha or gamma carboxylate group of the folic acid is X ２ Preferably, the gamma carboxylate group of the folic acid serves as the covalent attachment point for the linking moiety. ２ 28. The composition of claim 27, which serves as a covalent attachment point for a linking moiety.

29. The folate ligand is methotrexate 【Chemistry 43】 and wherein either the alpha carboxylate group or the gamma carboxylate group of the folic acid is selected from the group consisting of X ２ Preferably, the gamma carboxylate group of the folic acid serves as the covalent attachment point for the linking moiety. ２ 28. The composition of claim 27, which serves as the covalent attachment point for a linking moiety.

30. the target fragment L is of formula 1, 【Chemistry 44】 In the formula, R is OH, SH, NH ２ or C substituted one or more times, preferably once, with COOH １～６ alkyl, and the NH ２ , OH, SH or COOH group is one of the X ２ 30. The composition of any one of claims 1 to 29, wherein the alkyl group, which serves as the point of covalent attachment to a linking moiety, may be optionally interrupted by N(H), S, or O.

31. The target fragment L contains DUPA residues (HOOC(CH ２ ) ２ -CH(COOH)-NH-CO-NH-CH(COOH)-(CH ２ ) ２ 31. The composition of claim 1, wherein the aryl group is —CO—.

32. 2. The composition of claim 1, wherein the conjugate is selected from Compound 12a, Compound 12b, Compound 19a, Compound 19b, Compound 24, Compound 28a, Compound 28b, Compound 32a, Compound 32b, Compound 37a, Compound 37b, Compound 43, Compound 44a, Compound 44b, Compound 45, Compound 49a, and / or Compound 49b: 【Chemistry 45-1】 【Chemistry 45-2】 【Chemistry 45-3】 【Chemistry 45-4】 【Chemistry 45-5】

33. 33. The composition of any one of claims 1 to 32, further comprising a polyanion, preferably said polyanion being a nucleic acid, said polyanion preferably being non-covalently bound to said conjugate, said polyanion and said conjugate forming a polyplex.

34. 34. The composition of claim 33, wherein the polyanion is a nucleic acid, and the nucleic acid is dsRNA or ssRNA.

35. 35. The composition of claim 34, wherein the nucleic acid is a dsRNA.

36. 36. The composition of claim 35, wherein the dsRNA is polyinosinic:polycytidylic acid (poly(IC)).

37. 35. The composition of claim 34, wherein the nucleic acid is ssRNA.

38. 38. The composition of claim 37, wherein the ssRNA is mRNA.

39. 34. The composition of claim 33, wherein the polyanion is a nucleic acid, and the nucleic acid is DNA.

40. 40. The composition of claim 39, wherein the DNA is plasmid DNA.

41. 41. A polyplex of a conjugate and a polyanion as defined in any one of claims 1 to 40, wherein the polyanion is preferably non-covalently bound to the conjugate, and preferably the polyanion is a nucleic acid.

42. 1. A polyplex comprising a conjugate of Formula I, or a pharmaceutically acceptable salt, solvate, hydrate, tautomer, or enantiomer thereof, and a polyanion, preferably a nucleic acid, wherein said polyanion, preferably said nucleic acid, is preferably non-covalently bound to said conjugate: 【Chemistry 46】 [In the formula, 【Chemistry 47】 is a single or double bond; n is any integer from 1 to 1500; m is a discrete number of repeating units m between 25 and 100, preferably m is a discrete number of repeating units m between 25 and 60; R １ is the initial residue, preferably R １ is -H or -CH ３ and R ２ are independently —H or an organic residue, and the —(NR ２ -CH ２ -CH ２ ) ｎ The R in - ２ at least 80%, preferably at least 90% of is H; Ring A may have one or more R Ａ１ is a 5-10 membered cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl optionally substituted with R Ａ１ is C １ ~C ６ Alkyl, C １ ~C ６ or two R are independently selected from alkoxy, oxo, or halogen; Ａ１ 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 can form a cycloalkyl ring, where each fused aryl, heteroaryl, or cycloalkyl is joined to one or more R Ａ２ optionally substituted with R Ａ２ is C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, halogen, —SO ３ H, or -OSO ３ independently selected from H; X １ is a divalent covalent linking moiety; X ２ is a divalent covalent linking moiety; L is a targeting fragment, said targeting fragment being capable of binding to prostate-specific membrane antigen (PSMA), preferably said targeting fragment being capable of binding to cells expressing PSMA.

43. 43. The polyplex of claim 41 or claim 42, wherein the polyanion is a nucleic acid, and the nucleic acid is RNA.

44. 44. The polyplex of claim 43, wherein the RNA is dsRNA or ssRNA.

45. 44. The polyplex of claim 43, wherein the RNA is a dsRNA.

46. 46. ​​The polyplex of claim 45, wherein the dsRNA is polyinosinic:polycytidylic acid (poly(IC)).

47. 44. The polyplex of claim 43, wherein the RNA is ssRNA.

48. 48. The polyplex of claim 47, wherein the ssRNA is mRNA.

49. 43. The polyplex of claim 41 or claim 42, wherein the polyanion is a nucleic acid, and the nucleic acid is DNA.

50. 50. The polyplex of claim 49, wherein the DNA is plasmid DNA.

51. 51. A pharmaceutical composition comprising the composition of any one of claims 1 to 40 or the polyplex of any one of claims 41 to 50, and optionally one or more pharmaceutically acceptable excipients and / or carriers.

52. 52. A composition according to any one of claims 1 to 40 or a polyplex according to any one of claims 41 to 50 or a pharmaceutical composition according to claim 51 for use in the treatment of cancer, preferably cancer characterized by cells that overexpress prostate-specific membrane antigen (PSMA).

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