Intracellular delivery components

Abstract

A protein conjugate is provided, comprising a protein carrier comprising a plurality of amine groups, a biological payload that interacts with an intracellular target, and a linker connecting them, wherein at least a portion of the amine groups are bound to a protecting group. Pharmaceutical compositions comprising the protein conjugate, as well as methods of using and producing the protein conjugate, are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 275,049, filed November 3, 2021, and U.S. Provisional Patent Application No. 63 / 348,114, filed June 2, 2022, the contents of which are incorporated herein by reference in their entireties. FIELD OF THE INVENTION The present invention is in the field of intracellular delivery of proteins. [Background technology]

[0002] In the search for novel and potent therapeutic targets, particularly in the field of oncology, intracellular targets, especially those deemed "undruggable," are extremely difficult to target. Such targets cannot be targeted by small molecule drugs, primarily because the required therapeutic intervention involves disrupting protein-protein interactions, and small molecule drugs lack efficacy and / or sufficient selectivity. While such targets and biological processes are effectively addressed in traditional ways by biologics, such as monoclonal antibodies, receptors, interleukins, and their derivatives and combinations, such drugs cannot easily penetrate living cells, especially into clinically relevant intracellular compartments such as the cytoplasm, nucleus, and endoplasmic reticulum (ER). This is because the outer cell membrane is impermeable to protein-based molecules.

[0003] When protein-based molecules are internalized into living cells through natural endocytosis-based mechanisms, they enter the endosomal pathway, which leads to lysosomal degradation.Therefore, this pathway is naturally suitable only for the treatment of lysosomal disorders, or antibody-drug conjugates (ADCs).In the former case, therapeutic protein-based drugs usually aim to exert their activity in endosomes or lysosomes, and are designed or naturally adapted to withstand the conditions of endosomes / lysosomes.In the latter case, antibodies are simply used to target / deliver small molecule drugs to specific cells, and lysosomal degradation of the antibody carrier releases small molecule drugs, usually cytotoxins.

[0004] However, if the protein is itself a therapeutic agent and not merely a targeting moiety, the technology of the present invention must also enable so-called "endosomal escape." This critical step liberates the therapeutic biologic from vesicles at different stages of the endosomal pathway, i.e., early or late endosomes and lysosomes, in order to avoid catabolism of the therapeutic agent by this cellular machinery.

[0005] After successful escape from the endosome, the therapeutic agent is released into the cell cytoplasm. However, the cytoplasm is a crowded environment and cannot accommodate most currently employed biological therapeutic agents, such as monoclonal antibodies and their derivatives. Therapeutic biologics must be efficiently distributed throughout the cytoplasm to find and bind to their therapeutic targets or to reach target intracellular organelles, such as the nucleus, ER, or mitochondria.

[0006] One of the traditional and most studied intracellular delivery techniques involves the use of enhanced positive charge. Most classical approaches evolved from an understanding of the intracellular uptake mechanisms employed by viruses. The latter employs positively charged peptides rich in arginine and lysine amino acids, such as the well-known HIV-derived TAT peptide (RKKRRQRRR (SEQ ID NO: 16)). This approach has led to the development of numerous cell-penetrating peptides (CPPs) with different charges, amino acid sequences, additional modifications, and structures (linear, cyclic, etc.), which have been condensed or chemically conjugated to various payloads or used to decorate various nanoparticles. While CPPs demonstrate the ability to internalize into cells, the efficiency of their endosomal escape remains controversial, and their overall efficiency appears insufficient for practical pharmaceutical use. An alternative approach to utilizing charge-based cell penetration is to modify biologics or carriers with highly positively charged polymers. One such polymer is polyethyleneimine (PEI).

[0007] Appropriate PK and biodistribution profiles are important to ensure the efficacy of any drug, especially biologics. However, chemical modification with PEI or similar molecules, which feature a strong positive charge, can have a dramatic interfering effect on PK and biodistribution. Because most circulating proteins and the lining of blood vessels are negatively charged, any positively charged protein introduced into the bloodstream will adhere to these blood components and interfere with its PK and biodistribution. Furthermore, such attachment can also lead to "trapping" of the administered positively charged protein at the injection site. While the use of minimal levels of modification can minimize the impact of positive charge on PK and "trapping" at the injection site, additional solutions are still needed. Thus, there is an unmet need for the development of suitable charge-masking groups that are sufficiently stable in blood or healthy tissues and undergo rapid and selective unmasking / deprotection in target tissues, allowing for cellular penetration and intracellular delivery of biotherapeutics. Summary of the Invention

[0008] The present invention provides a protein conjugate comprising a protein carrier comprising a plurality of amine groups, a biological payload that interacts with an intracellular target, and a linker connecting them, wherein at least a portion of the amine groups are bound to protecting groups. Pharmaceutical compositions comprising the protein conjugate, and methods of using and producing the protein conjugate are also provided.

[0009] In a first aspect, a protein conjugate is provided comprising a biological payload that interacts with an intracellular target, the biological payload being covalently attached to a cell-penetrating moiety comprising a plurality of amine groups, at least a portion of the amine groups being bound to protecting groups, the protecting groups being capable of undergoing cleavage at pH values ​​below 7; and the protein conjugate is characterized by a negative zeta potential.

[0010] In another aspect: a. A protein carrier covalently attached to a cell-penetrating moiety containing multiple amine groups; b. a biological payload that interacts with an intracellular target; and c. a linker between the protein carrier and the biological payload; A protein conjugate is provided, comprising: At least a portion of the amine groups are bound to protecting groups; the protecting group is capable of undergoing cleavage at a pH value of less than 7, and The protein conjugate is characterized by a negative zeta potential. In some embodiments, the protein conjugate is characterized by enhanced stability in blood relative to a similar protein conjugate lacking a protecting group. In some embodiments, the protein conjugate is characterized by increased accumulation in biological tissues having pH values ​​below 7 compared to a similar protein conjugate lacking a protecting group.

[0011] In some embodiments, the plurality of amine groups comprises primary amines, secondary amines, or both, and at least 50% of the plurality of amine groups are bound to protecting groups. In some embodiments, the linker is covalently linked to the carrier, the payload, or both.

[0012] In some embodiments, the protecting group comprises a moiety that is negatively charged at a pH between 6-8.

[0013] In some embodiments, the moiety comprises a carboxy group.

[0014] In some embodiments, the protecting group is represented by Formula 1: [ka] wherein n is an integer ranging from 0 to 5; [ka] represents the point of attachment for the amine group, and [ka] represents a single or double bond; R and R each independently represent a substituent selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl or heteroaryl, and carboxyalkyl, or a combination thereof; or R and R are joined together to form a ring.

[0015] In some embodiments, one of R and R1 is H and the other one of R and R1 comprises alkyl or carboxyalkyl.

[0016] In some embodiments, the protecting group is [ka] and any salt thereof, wherein R and R1 are selected from H and methyl, and R or R1 is methyl.

[0017] In some embodiments, the cell-penetrating moiety comprises an alkylamine, a cationic polymer, or a combination thereof.

[0018] In some embodiments, the cationic polymer is selected from polyamines and polyethyleneimine (PEI).

[0019] In some embodiments, the PEI is a linear or branched PEI having a molecular weight of less than 2000 daltons.

[0020] In some embodiments, the PEI comprises a molecular weight of 100 to 1000 daltons.

[0021] In some embodiments, the biological payload is an antigen-binding molecule that binds an intracellular target.

[0022] In some embodiments, the biological payload lacks a disulfide bond that, upon cleavage, reduces interaction with the intracellular target.

[0023] In some embodiments, the antigen binding molecule is selected from a single chain antibody, a single domain antibody, a variable heavy chain homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a designed ankyrin repeat protein (DARPin), and an antibody mimetic protein.

[0024] In some embodiments, the antigen binding molecule is selected from a VHH and a DARPin.

[0025] In some embodiments, the protein carrier or biological payload comprises multiple PEI molecules, hi some embodiments, the protein carrier comprises between 2 and 30 PEI molecules.

[0026] In some embodiments, the protein carrier is human serum albumin (HSA).

[0027] In some embodiments, the HSA comprises 3 to 10 PEI molecules.

[0028] In some embodiments, the linker comprises a biocompatible polymer, a biodegradable polymer, or both.

[0029] In some embodiments, the biocompatible polymer comprises polyethylene glycol (PEG). In some embodiments, the biodegradable polymer comprises a polyamino acid.

[0030] In some embodiments, the linker further comprises a spacer that is covalently attached to (i) the biocompatible or biodegradable polymer and (ii) the protein carrier. In some embodiments, the covalent attachment is via a click reaction product.

[0031] In some embodiments, the linker comprises a biocleavable bond.

[0032] In some embodiments, the biocleavable bond comprises a disulfide bond.

[0033] In some embodiments, the linker is substantially stable in the blood for at least 24 hours.

[0034] In some embodiments, the linker is a peptide linker.

[0035] In some embodiments, stability includes less than 25% cleavage in the blood after 24 hours.

[0036] In some embodiments, the biocleavable bond is sterically hindered.

[0037] In some embodiments, the HSA comprises the amino acid sequence of SEQ ID NO: 1, or a fragment or homolog thereof that includes cysteine ​​34 (C34).

[0038] In some embodiments, the linker is attached to HSA via a disulfide bond.

[0039] In some embodiments, the linker is attached to C34 of HSA.

[0040] In some embodiments, the disulfide bond is proximal to C34.

[0041] In some embodiments, proximal is a distance from C34 in the range of 5 to 15 angstroms.

[0042] In some embodiments, the protein carrier lacks DNA.

[0043] In some embodiments, the biological payload does not bind a cell surface protein.

[0044] In some embodiments, the protein conjugate is characterized by a negative zeta potential of less than −1 mV.

[0045] In some embodiments, the protein conjugate further comprises a detectable tag, hi some embodiments, the tag is conjugated to a biological payload.

[0046] In some embodiments, the protein conjugate is a cell-penetrating conjugate.

[0047] In some embodiments, the protecting group is citraconic anhydride. In some embodiments, the protecting group is derived from citraconic anhydride.

[0048] In some embodiments, the click reaction product is a succinimide-thioether.

[0049] In some embodiments, the protein conjugate further comprises a targeting moiety that binds to a protein expressed on the surface of a target cell.

[0050] In some embodiments, the targeting moiety is selected from a single chain antibody, a single domain antibody, a variable heavy chain homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a designed ankyrin repeat protein (DARPin), and an antibody mimetic protein.

[0051] In some embodiments, the targeting moiety is conjugated to the protein carrier via a linker.

[0052] In some embodiments, the targeting moiety and the biological payload are comprised in a single polypeptide, hi some embodiments, the targeting moiety and the biological payload are separated by a linker.

[0053] In some embodiments, the targeting moiety is N-terminal to the biological payload, or the biological payload is N-terminal to the targeting moiety.

[0054] In another aspect, a method is provided for producing a charge-masked protein conjugate capable of binding an intracellular target, the method comprising: a. providing a biological payload that binds to an intracellular target, wherein the biological payload is covalently attached to a cell-penetrating moiety comprising a plurality of amine groups; and b. providing the biological payload under conditions sufficient to protect at least a portion of the amine groups with protecting groups capable of undergoing cleavage at a pH value of less than 7, to obtain a charge-masked protein conjugate comprising the protected amine groups; Including, This produces a charge-masked protein conjugate capable of binding an intracellular target.

[0055] In another aspect, a method is provided for producing a charge-masked protein conjugate capable of binding an intracellular target, the method comprising: a. providing a biological payload that binds an intracellular target; b. providing a protein carrier covalently attached to a cell-penetrating moiety comprising a plurality of amine groups; c. providing a biological payload and a protein carrier under conditions sufficient to covalently bond the biological payload to the protein carrier via the linker to produce a protein conjugate; and d. providing the protein conjugate under conditions sufficient to protect at least a portion of the amine groups with protecting groups that are capable of cleavage at a pH value of less than 7, to obtain a charge-masked protein conjugate comprising the protected amine groups; Including, This produces a charge-masked protein conjugate capable of binding an intracellular target.

[0056] In some embodiments, the method further comprises determining the stability of the linker in human blood, plasma or serum and in cytoplasmic conditions; and selecting a charge-masked protein conjugate comprising a linker that is stable in human blood, plasma or serum and unstable in cytoplasmic conditions.

[0057] In some embodiments, the method further comprises determining the stability of the protected amine groups at neutral or basic pH and at acidic pH, and selecting charge-masked protein conjugates comprising protected amine groups that are stable at neutral or basic pH and unstable at acidic pH.

[0058] In some embodiments, providing the protein carrier under conditions sufficient to protect occurs before conjugating the biological payload to the protein carrier.

[0059] In some embodiments, providing the protein carrier under conditions sufficient to protect occurs after conjugating the biological payload to the protein carrier.

[0060] In some embodiments, the determining is performed before formation of the protein conjugate or after formation of the charge-masked protein conjugate.

[0061] In some embodiments, the protein carrier comprises HSA.

[0062] In some embodiments, the cell-penetrating moiety comprises at least one PEI.

[0063] In some embodiments, the charge-masked protein conjugate is characterized by a negative zeta potential.

[0064] In some embodiments, the plurality of amine groups comprises primary amines, secondary amines, or both, and at least 80% of the plurality of amine groups are protected amine groups.

[0065] In some embodiments, the protecting group comprises a moiety that is negatively charged at a pH between 6-8.

[0066] In some embodiments, the moiety comprises a carboxy group.

[0067] In some embodiments, the protein carrier or biological payload is covalently attached to at least two molecules of PEI, hi some embodiments, the protein carrier is covalently attached to at least eight molecules of PEI.

[0068] In some embodiments, the biological payload lacks a disulfide bond that, when cleaved, reduces binding to the intracellular target.

[0069] In some embodiments, the method further comprises contacting the charge-masked protein conjugate with a cell and confirming that the biological payload enters the cytoplasm of the cell.

[0070] In some embodiments, stable includes less than 25% cleavage in blood after 24 hours, and unstable includes at least 50% cleavage in cytoplasmic conditions after 24 hours.

[0071] In some embodiments, the linker comprises a biocompatible polymer.

[0072] In some embodiments, the covalent linking is via a click reaction.

[0073] In some embodiments, the biological payload is covalently attached to a linker comprising a first reactive group, and the protein carrier is covalently attached to a linker comprising a second reactive group reactive to the first reactive group; and the conditions sufficient to covalently attach the biological payload to the protein carrier comprise reacting the first reactive group with the second reactive group, thereby covalently linking the biological agent and the protein carrier.

[0074] In some embodiments, the linker comprises a biocleavable bond.

[0075] In some embodiments, the covalently linking comprises disulfide bond formation.

[0076] In some embodiments, (i) the biological payload is covalently attached to a linker capable of forming a disulfide bond with a cysteine ​​of the protein carrier; or (ii) the protein carrier is covalently attached to a linker capable of forming a disulfide bond with a cysteine ​​of the biological payload. In some embodiments, attachment is via a disulfide bond.

[0077] In some embodiments, the method further comprises selecting a targeting moiety that binds to a protein expressed on the surface of a target cell and conjugating the targeting moiety to a biological payload, protein carrier, or protein conjugate.

[0078] In some embodiments, the targeting moiety is selected from a single chain antibody, a single domain antibody, a variable heavy chain homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a designed ankyrin repeat protein (DARPin), and an antibody mimetic protein.

[0079] In some embodiments, the targeting moiety and the biological payload are comprised in a single polypeptide, hi some embodiments, the targeting moiety and the biological payload are separated by a linker.

[0080] In some embodiments, the targeting moiety is N-terminal to the biological payload, or the biological payload is N-terminal to the targeting moiety. In some embodiments, the charge-masked protein conjugate is a protein conjugate of the present invention.

[0081] In another aspect, there is provided a protein conjugate produced by the method of the invention.

[0082] In another aspect, there is provided a pharmaceutical composition comprising a protein conjugate of the invention and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0083] In some embodiments, the pharmaceutical composition is formulated for systemic administration.

[0084] In another aspect, a method of binding an intracellular target is provided, the method comprising contacting a cell expressing the intracellular target with a protein conjugate of the invention or a pharmaceutical composition of the invention, wherein the biological payload binds the intracellular target, thereby binding the intracellular target.

[0085] In some embodiments, the method is a method for detecting an intracellular target, wherein the protein conjugate comprises a detectable tag, and the method further comprises detecting the detectable tag.

[0086] In some embodiments, the method is a method of modulating an intracellular target and the biological payload is an agonist or antagonist of the intracellular target.

[0087] In some embodiments, the cell is in a subject and the contacting comprises administering to the subject a protein conjugate of the invention or a pharmaceutical composition of the invention.

[0088] In some embodiments, the cells express a target surface protein and the protein conjugate comprises a targeting moiety that binds to the target surface protein.

[0089] In some embodiments, the method is a method of treating a condition in a subject in need thereof, wherein the condition is treatable by modulation of an intracellular target.

[0090] In some embodiments, the condition comprises cancer or inflammation.

[0091] In some embodiments, the condition is cancer, the intracellular target is oncogenic, and the biological payload is an antagonist.

[0092] In some embodiments, the cancer comprises a target surface protein that is a cancer-specific antigen.

[0093] In some embodiments, the contacting is in the absence of an agent other than the carrier protein designed to direct penetration of the protein conjugate into the cell.

[0094] In some embodiments, the method is for delivering a biological payload to a specific tissue in a subject, the specific tissue being characterized by a pH value of less than 7. In some embodiments, the specific tissue is a tumor.

[0095] Further embodiments and full scope of applicability of the present invention will become apparent from the detailed description set forth hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0096] [Figure 1] MALDI-ToF spectra of unmodified and PEI-modified mouse IgG with various modification levels. [Figure 2] Photomicrographs of internalization of PEI-modified or unmodified mouse IgG into A375 cells (blue - nuclei, green - stained mouse IgG). Native IgG was unmodified; all other panels show increasing levels of PEI modification. The numbers in the corners of each panel represent the average number of PEI molecules per IgG molecule. [Figure 3A-3B] (3A) Micrographs of internalization of PEI-modified GFP (average of 6.5 PEI per GFP molecule) into HEK cells (60 μg / mL) and (3B) A375 cells (5 μg / mL). [Figure 4] Bar graph of cellular uptake efficiency of PEI-modified IgG (initial medium concentration 2 μg / mL) at various modification levels, measured by specific ELISA of IgG levels in the medium. [Figure 5]Histogram showing inhibition of caveolin-mediated endocytic internalization of PEI-modified IgG (4.5PEI). NC - unmodified IgG. [Figure 6] Micrographs of HEK293 cells expressing an endosomal marker (top panel) or a lysosomal marker (bottom panel) after incubation (5 h) with 5 μg / mL PEI-modified IgG (3xPEI, left panel; 4.5xPEI, center panel; and 7xPEI, right panel). Endosome and lysosomal markers are stained green, PEI-modified IgG is red, and nuclei are stained blue. [Figure 7] Micrograph of endosomal escape monitoring using live imaging confocal microscopy of PEI-modified IgG (4.5xPEI) labeled with a pH-sensitive dye (5(6)-carboxynaphthofluorescein, red) after overnight incubation of PEI-modified IgG at 10 μg / mL with 2 x 10 HeLa cells. Green - tubulin staining, blue - nuclei staining. [Figure 8] Bar graph of CD3 cell activation reflected by a dose-dependent increase in IFNγ secretion after internalization of anti-CD247, a PEI-modified mAb (4.5xPEI). No change in IFNγ levels was observed with internalized PEI-modified mouse IgG over the concentration range evaluated. [Figure 9] Schematic of the carrier and payload method in which a carrier protein cationized with PEI is chemically linked to a therapeutic agent (an antibody in this example). In this example, the linker is conjugated to the Fc region of the antibody, distal to its CDRs, and contains a disulfide bond that can be reduced and cleaved in the cytoplasm, releasing the therapeutic agent payload from the cationized carrier and allowing free cytosolic transport. [Figure 10]Micrographs showing the intracellular profile of GFP after incubation of A375 cells with GFP conjugated to PEI-modified HSA (7xPEI) via a PEG-based disulfide-containing linker. Cells show dispersed GFP profiles at 24 hours (left) and 48 hours (right) after incubation, as observed by confocal microscopy (green - GFP; blue - nuclei). [Figure 11] Photomicrograph of anti-TNFα antibody conjugated to PEI-modified HSA (11xPEI) via a disulfide bond-containing PEG linker after overnight incubation with A375, detected by confocal microscopy using a fluorescently labeled (AlexaFluor 647) anti-human IgG antibody. [Figure 12] SDS-PAGE Western blot analysis of intact mouse IgG in PBS7.4 (lanes 8 and 9) after 4 hours (lanes 5–7) and overnight (lanes 1–4) in PBS containing 10 mM GSH. The gel was treated with anti-mouse light chain antibody. [Figure 13] Figure 1 shows exemplary results of the production, purification, and characterization of VHH-PEI-modified HSA constructs generated by modifying HSA with PEI followed by modification of HSA with NHS-PEG4-SPDP. The SPDP-modified HSA-PEI was further reacted with VHHs bearing C-terminal free cysteines. The latter was optionally pretreated with a reducing agent (e.g., TCEP) to ensure that all cysteines were free. Lane 1: HSA-8xPEI; Lane 2: HSA-8xPEI-SS-VHH (arrows indicate different reaction products); Lane 3: reaction products after reduction with DTT (suggesting that the bond between HSA and VHH is a labile disulfide bond); Lanes 7-9: fractions of VHH-HSA conjugates after purification with Protein A resin (removal of excess HSA-8xPEI). MS spectra show product profiles for VHH-HSA-0xPEI (top), VHH-HSA-4xPEI (middle), and VHH-HSA-8xPEI (bottom), all indicating that the majority of products contain HSA attached to a single VHH. [Figure 14]Bar graph of medium levels of anti-vimentin VHH conjugated to PEI-modified HSA with an average of 3.5 (blue) or 8 (red) PEIs per HSA in the presence (dark shading) or absence (light shading) of A375 cells. [Figures 15A-15C] Photomicrographs of confocal microscopy imaging of A375 cells stained for VHH (15A), vimentin (15B) and a merge of both (15C) after 24 hours of incubation with anti-vimentin reversibly conjugated to PEI-modified HSA. Nuclei are stained blue. [Figures 16A-16B] Photomicrographs of confocal microscopy imaging of A375 cells stained for VHH (green) after 24 hours of incubation with PEI-modified HSA-reversibly conjugated anti-vimentin VHH (16A) or unconjugated anti-vimentin VHH (16B). Nuclei are stained blue. [Figure 17] Bar graph of HeLa proliferation index. HeLa (HPV+) cells were incubated for 48 hours in medium (black bars), medium supplemented with anti-E7 VHH (gray bars), or medium supplemented with anti-E7 VHH conjugated to PEI-modified HSA at various concentrations (average 3.5 PEI / HSA) (green bars). HeLa cell proliferation after incubation was measured by a standard MTT assay. [Figure 18] Line graph showing the percentage of HPV-positive HeLa FUCCI cells in S, G2, and M cell phases after thymidine cell cycle synchronization. Cells were treated with anti-E7 VHH conjugated to HSA cells modified with an average of 3.5 PEI, and various control treatments. Controls were: no treatment (wo), unmodified VHH anti-E7, anti-E7 VHH conjugated to unmodified HSA, modified HSA conjugated to an irrelevant VHH (anti-vimentin), modified HSA carrier alone, and a cell cycle inhibitor (DP, a CDK4 / 6 inhibitor). [Figure 19]Photomicrographs of the effect of different treatments on HPV-positive HeLa FUCCI cells. Cells treated with an irrelevant VHH (anti-vimentin) conjugated to HSA modified with an average of 3.5 PEI show the same profile as untreated cells (left image), whereas an anti-E7 VHH conjugated to the same HSA carrier resulted in dramatic cell death similar to that observed after treatment with a CDK4 / 6 inhibitor (right image). [Figure 20] Representative micrograph of anti-K-Ras His-tagged DARPin K27 protein conjugated to a PEI-modified HSA carrier internalized in lung adenocarcinoma cells. The DARPin localizes to K-Ras sites (inside the cell membrane) after staining with an anti-His tag antibody. [Figure 21] Figure 1. Apoptosis line graph. The KRAS DARPin K27 conjugated to a PEI-modified HSA carrier was internalized by SU8686 cells, and their apoptotic state was assessed using Annexin V in a continuous Incucyte system. Cells exposed to a DARPin conjugated to a carrier modified with 8 PEI molecules (carrier I) show high levels of apoptosis, while cells exposed to a DARPin conjugated to a carrier with 3.5 PEI molecules (carrier II) also show clear apoptosis, but to a lesser extent. Untreated cells, cells exposed to an unmodified DARPin, and cells exposed to HSA modified with 8 PEI all show baseline apoptosis. [Figures 22A-22B] Line graphs showing the effect of anti-KRAS DARPin K27 conjugated to a PEI-modified HSA carrier on proliferation (22A) and apoptosis (22B) of HeLa cells constitutively expressing GFP in their nuclei. A pan-RAS inhibitor was used as a positive control, and no treatment was used as a negative control. Cells were also treated with unmodified DARPin K27, PEI-modified HSA carrier alone, DARPin K27 conjugated to an unmodified HSA carrier, and anti-vimentin VHH conjugated to a PEI-modified HSA carrier. [Figure 23]Micrograph showing the effect of KRAS DARPin K27 conjugated to a PEI-modified HSA carrier on apoptosis of HeLa cells constitutively expressing GFP in their nuclei. [Figure 24] Mass spectrometry (MALDI-ToF) spectra of HSA modified with various levels of PEI (600 Da), using a constant molar excess of PEI in the reaction and controlling the level of modification by adjusting the level of the carbodiimide coupling agent, EDC. The average level of PEI molecules relative to HSA is shown in black. [Figure 25] Confocal microscopy images of anti-vimentin VHH inside A375 cells after 48 hours of incubation with VHH conjugated to HSA carriers with various levels of PEI modification. The average number of PEI molecules per HSA carrier is shown in each box. Intracellular VHH is visualized using an anti-VHH antibody. [Figure 26] Bar graph of residual levels of VHH carrier conjugates with 8 (orange) or 3.5 (blue) PEI molecules per HSA remaining in the medium, measured by specific ELISA in the medium of A375 cells incubated with the conjugates. [Figures 27A-27B] IVIS images of whole mice (27A) and organs harvested from mice (27B) receiving one of two doses of unmasked or masked carrier. Relative signal intensity was calculated as the radiant efficiency (emitted light [photons / sec / cm² / str] / excitation light [µW / cm²] x 109) per pixel in the region of interest (27A - live imaging of the whole animal; 27B - ex vivo imaging of each organ). Relative signal intensity is presented as a color scale. Colors represent the localization and concentration of the injectate, with yellow indicating increased fluorescence intensity and dark red indicating decreased fluorescence intensity. [Figures 28A-28F]Confocal microscopy images of HeLa GFP cells after 24 hours of incubation with: (28A-28C) anti-E7-HSA-PEIx3.5, or (28D-28E) α-E7-masked-HSA-PEIx3.5. Immunofluorescence staining with anti-VHH antibody—red; nuclear staining (GFP)—green. (28A,D)—nuclear staining; (28B,E)—VHH staining; (28C,F)—merge. Intracellular VHH staining is observed only with the unmasked conjugate. Intracellular VHH staining is observed primarily in the cytoplasm. [Figures 29A-29F] Confocal microscopy images of B16 cells after 24 hours of incubation with: (2A) HSA-PEIx8; (2B) HSA-PEIx8-CA or (2C) HSA-PEIx8-MSA, or (2D) HSA-PEIx8; (2E) HSA-PEIx8-CA or (2F) HSA-PEIx8-MSA, pretreated with acidic conditions for masking removal before incubation with B16 cells. Carrier stain—red; nuclear stain (DAPI)—blue. [Figure 30A-30B] (30A) Percentage of viable MEL-526 cells after treatment with either 1C5-unmasked HSA-PEIx3.5 or 1C5-masked HSA-PEIx3.5 before and after 8 hours of unmasking. Calculations were performed relative to cells without treatment. Cells were exposed to the respective agents at 6 μM for 6 days. (30B) Percentage of viable SK-MEL-28 cells after treatment with either 1C5-masked HSA-PEIx3.5, 1C5-masked HSA-PEIx3.5 after unmasking (1 hour unmasking step), 1C5-HSA-PEIx3.5, or no treatment. Cells were exposed to the respective agents at 10 μM for 6 days. [Figure 31A-31B] (31A) Line graph showing the pharmacokinetics of modified HSA (8 / 3.5 PEI units) with and without masking. The line represents the mean ± SEM of log HSA plasma concentration [μg / mL] (n=3). A two-tailed Student's t-test yields significant differences between masked HSA-PEIx8 versus HSA-PEIx8 and HSA-PEIx3.5. *p<0.01** (31B) Bar graph showing the in vivo plasma exposure of HSA-PEI derivatives. [Figure 32A-32B] (32A) Bar graph of exposure levels in various organs of HSA-PEI derivatives in vivo. (32B) Bar graph of biodistribution of masked HSA-PEIx8 at 1440 min after IV injection. Bars represent mean ± SEM of HSA concentrations [μg / mL] (n=3). One-way ANOVA revealed significant differences in biodistribution between organs. Post-hoc Dunnett's test showed significance of p<0.0001 (****) between tumor HSA concentrations compared to the respective organ assays. Similar results were obtained at other time points. [Figure 33] Plot of HSA-PEI derivatives detected in urine. [Figure 34] Line graph showing the pharmacokinetics of modified IgG (4 PEI units) with and without masking. [Figure 35] Line graph showing the pharmacokinetics of directly modified VHH (PEI 1800) with and without masking. [Figures 36A-36D] (36A) Line graph of the mean tumor volume in mice inoculated with HeLa cells and treated daily for 15 days with 350 nmol / kg of αE7-VHH-S-Mal-PEG11-Mal-S-HSAx3.5 conjugate or αE7-VHH-S-Mal-PEG11-Mal-S-HSA or vehicle (PBS) alone. (36B-36C) Line graphs of tumor volume in mice seeded with HeLa cells and treated (36B) every other day for 5 days with 250 nmol / kg of αE7-VHH-S-Mal-PEG11-Mal-S-HSAx3.5 citraconic anhydride masked conjugate, masked carrier alone, or PBS, followed by 5 daily injections, or (36C) daily for 15 days with 350 nmol / kg of αE7-VHH-S-Mal-PEG11-Mal-S-HSAx3.5 masked conjugate, masked carrier alone, or PBS. (36D) Bar graph of percent tumor inhibition by masked conjugate compared to masked carrier control throughout the experiment. [Figure 37A-37B]Photomicrographs of immunohistochemical detection of (37A) VHH payload and (37B) HSA-modified PEI carrier in tumor sections from mice treated with PBS (left), citraconic anhydride-masked PEI-modified carrier (center), and the masked conjugate of the present invention (right). [Figure 38A-38B] Line graphs of tumor volume in mice inoculated with (38A) B16 cells or (38B) MEL-526 cells and treated with 350 nmol / kg citraconic anhydride-masked 1C5-VHH-S-Mal-PEG11-Mal-S-HSA-PEIx3.5 conjugate (αBRAF(1C5)-masked carrier 3.5) or the masked carrier alone by daily IV infusion for 15 days. *p-value <0.05. [Figure 39] Line graph of the pharmacokinetic profiles of different conjugates of anti-E7 VHH conjugated to masked carriers, differing in the level of PEI and in the reversibility of the payload-carrier bond or the reversibility of the mask. [Figure 40] Bar graph of the biodistribution of different masked conjugates in various organs, including tumor, presented as organ exposure levels (AUC). [Figure 41A-41B] (41A-41B) Confocal microscopy images (X63) of tumor tissue from athymic nude Foxn1 mice bearing a cervical tumor (HeLa-GFP) 6 hours after injection of (41A) Atto542-HSAPEIx8CA and (41B) Atto542-HSAPEIx8MSA. Carrier signal (red); nuclear staining (blue). (41C-41E) Confocal microscopy images (X63) of tumor tissue from C57BL mice bearing a B16 tumor 6 hours after injection of (41C) Atto542-HSAPEIx8CA, (41D) Atto542-HSAPEIx8MSA, and (41E) Atto542-unmasked HSAPEIx8. Carrier signal (red); nuclear staining (blue). [Figures 42A-42D](42A-42B) Confocal microscopy images (x40) of tumor tissue from an athymic nude Foxn1 mouse bearing a cervical tumor (HeLa-GFP) 6 hours after injection of Atto542-HSA-PEIx8 MSA. Carrier staining (red); nuclear staining (blue); nuclear staining (blue); nuclear staining from green fluorescent protein in the tumor (green). (42A) Carrier staining (red) and tumor cell nuclei (green). (42B) Carrier staining (red) and general cell nuclear staining (blue). (42C-42D) Confocal microscopy images (x63) of liver tissue from a C57BL mouse bearing a B16 tumor 6 hours after injection of (42C) Atto542-HSAPEIx8CA and (42D) Atto542-HSAPEIx8 MSA. Carrier signal (red); nuclear staining (blue). [Figure 43A-43B] (43A) Bar graph of BRAF binding by the masked anti-BRAF VHH, 1C5, before and after unmasking. (43B) Bar graph of the percentage of viable MEL-526 cells after treatment with 1C5-Hel-unmasked HSA-PEIx8 or fully masked 1C5-Hel-HSA-PEIx8, before and after 8 hours of unmasking. Calculations were performed relative to untreated cells. Cells were exposed to each drug at 6 μM for 6 days. [Figure 44] Bar graph representing the percent internalization of HSA-PEIx3.5 at different masking levels compared with the level of internalization of HSA-PEIx3.5 without masking. [Figure 45A-45B] Bar graph summarizing FACS data for binding to PSMA-positive and -negative cells of tandem agents containing anti-PSMA targeting moieties without (45A) and with (45B) a masked carrier. [Figure 46] Line graph of binding of agents containing anti-PSMA targeting moieties to BRAF. [Figure 47] Bar graph of the cytotoxic effect of anti-BRAF VHH alone or expressed in tandem with anti-PSMA VHH on cells after conjugation to carrier (3.5PEI) as measured by Cell Titer Glo viability assay. DETAILED DESCRIPTION OF THE INVENTION

[0097] In some embodiments, the present invention provides a protein carrier covalently bound to a cell-penetrating moiety, wherein the cell-penetrating moiety comprises a plurality of amine groups; at least a portion of the amine groups are bound to protecting groups; and the protecting groups are stable at pH values ​​greater than 7 and can be dissociated from a portion of the amine groups at pH values ​​less than 7. In some embodiments, the protected protein carrier is characterized by a negative zeta potential.

[0098] The present invention is based on the discovery of a transient masking technology suitable for targeted intracellular delivery. This technology allows for the masking of the positive charge of a therapeutic agent or carrier for several minutes, preferably several hours, after injection, allowing sufficient time for "injection site escape" and for the carrier and its payload to circulate in the blood and reach the target site. Charge masking is based on covalent masking. This approach is similar to prodrugs, in which a "problematic" group on a drug molecule is replaced with a covalent bond, thereby changing the properties of the original group, i.e., its polarity, solubility, or charge. The substitution is designed to be unstable under general physiological conditions or under specific conditions, such as a specific pH or the presence of a specific enzyme. The unstable substitution is then removed under the target conditions, leaving behind a positively charged molecule that is internalized and the payload that is delivered to the cytoplasm.

[0099] In a first aspect, a protein conjugate is provided, comprising a protein carrier covalently linked to a biological payload that interacts with an intracellular target. In some embodiments, the protein conjugate of the present invention is a charge-masked conjugate.

[0100] In another aspect, a biological payload that interacts with an intracellular target is provided, which is linked to a cell-penetrating moiety and a protecting group. In some embodiments, the biological payload, the cell-penetrating moiety, and the protecting group are a protein conjugate. In some embodiments, the biological payload, the cell-penetrating moiety, and the protecting group are included in a composition.

[0101] In another aspect, a protein conjugate is provided, comprising: a protein carrier covalently attached to a cell-penetrating moiety; a biological payload that interacts with an intracellular target; and a linker between the protein carrier and the biological payload, wherein the biological payload lacks a disulfide bond that, upon cleavage, reduces interaction with the intracellular target; the linker comprises a biocleavable bond; the cell-penetrating moiety comprises a plurality of amine groups, at least a portion of which are bound to protecting groups; and the protecting groups are stable at pH values ​​greater than 7 and can be dissociated from a portion of the amine groups at pH values ​​less than 7. In some embodiments, at least a portion of the amine groups are coupled to protecting groups to yield protected amines, and the molar ratio of protected to unprotected amines is such that the protein conjugates of the invention are characterized by a negative zeta potential of at least -0.1 mV, at least -0.5 mV, at least -1 mV, at least -2 mV, at least -3 mV, at least -5 mV, -0.1 to -50 mV, -0.5 to -50 mV, or -0.5 to -30 mV (including any range therebetween). In some embodiments, the molar ratio of protected to unprotected amines in the protein conjugates of the invention is at least about 7:10, at least about 8:10, at least about 9:10, at least about 1:1, or about 1:1 to 100:1 (including any range therebetween).

[0102] In another aspect, a protein conjugate is provided that includes a protein carrier covalently attached to a cell-penetrating moiety; a biological payload that interacts with an intracellular target; and a linker between the protein carrier and the biological payload; wherein the cell-penetrating moiety comprises a plurality of amine groups; and (i) a linker that is at least about 30%, at least about 40%, at least about 50%, about 40 to about 95%, about 40 to about 100%, about 40 to about 70%, about 40 to about 80%, about 40 to about 90%, about 40 to about 95%, or about 40 to about 99% (wherein (ii) the biological payload is attached to one or more protecting groups, or both (i) and (ii); the protecting groups are stable at pH values ​​above 7 (e.g., 7.0 to 10, or 7.2 to 10) and can be cleaved at pH values ​​below 7 (e.g., about 5 to about 6.8, about 3 to about 6.8, about 5 to about 7.0, about 3 to about 7.0 (including any range therebetween)), and the protein conjugate is characterized by a negative zeta potential.

[0103] In another aspect, a protein conjugate is provided, comprising: a protein carrier covalently attached to a cell-penetrating moiety; a biological payload that interacts with an intracellular target; and a linker between the protein carrier and the biological payload; the cell-penetrating moiety comprises a plurality of amine groups; (i) at least about 40%, or at least about 50% of the amine groups are bonded to protecting groups, (ii) the biological payload is bonded to one or more protecting groups, or both (i) and (ii); each protecting group is independently represented by Formula 2; and the protein conjugate is characterized by a negative zeta potential of at least −0.1 mV, at least −0.5 mV, at least −1 mV, at least −2 mV, at least −3 mV, at least −5 mV, −0.1 to −50 mV, −0.5 to −50 mV, or −0.5 to −30 mV (including any range therebetween).

[0104] In another aspect, a protein conjugate is provided, comprising: a protein carrier covalently attached to a cell-penetrating moiety; a biological payload that interacts with an intracellular target; and a linker between the protein carrier and the biological payload; the cell-penetrating moiety is or comprises one or more PEI molecules (e.g., 3-10 PEI molecules per single protein carrier); (i) the one or more PEI molecules are bound to one or more protecting groups, whereby the protein conjugate is characterized by a negative zeta potential, (ii) the biological payload is bound to one or more protecting groups, or both (i) and (ii); and the protecting groups are derived from citraconic anhydride.

[0105] In another aspect, a protein conjugate is provided, comprising: a protein carrier covalently attached to a cell-penetrating moiety; a biological payload that interacts with an intracellular target; and a linker between the protein carrier and the biological payload; the cell-penetrating moiety is or comprises one or more PEI molecules (e.g., 3-10, or 4-10 PEI molecules per single protein carrier); (i) at least about 40%, at least about 50%, about 40 to about 95%, about 40 to about 100%, about 40 to about 70%, about 40 to about 80%, about 40 to about 90%, about 40 to about 95%, or about 40 to about 99% of the amine groups of the one or more PEI molecules are covalently attached to protecting groups, whereby the protein conjugate is characterized by a negative zeta potential; (ii) the biological payload is attached to one or more protecting groups, or both (i) and (ii); and the protecting groups are derived from citraconic anhydride.

[0106] Another embodiment provides a protein conjugate comprising: a protein carrier covalently attached to a cell-penetrating moiety; a biological payload that interacts with an intracellular target; and a linker between the protein carrier and the biological payload; the protecting group undergoes cleavage at a pH below 7; and the protein conjugate is characterized by a negative zeta potential.

[0107] In some embodiments, a linker is attached to the protein carrier and the biological payload. In some embodiments, the linker is covalently attached to the carrier. In some embodiments, the linker is covalently attached to the carrier. In some embodiments, the linker is covalently attached to the payload. In some embodiments, the linker is covalently attached to the payload. In some embodiments, the linker comprises a bond. In some embodiments, the linker is a bond. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is long enough so as not to cause steric hindrance between the payload and the carrier. In some embodiments, the linker is long enough to allow access to the biocleavable bond. In some embodiments, the access is by an agent that cleaves the biocleavable bond. In some embodiments, the agent is an enzyme. In some embodiments, the agent is a reactive species. In some embodiments, the agent is a reducing agent.

[0108] In some embodiments, the linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 atom bond lengths. Each possibility represents a separate embodiment of the invention. As used herein, the term "atom bond" refers to the length of a carbon-carbon (C-C) bond, e.g., the length of a single C-C bond. In some embodiments, the linker comprises a length of at least 2, 4, 5, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (Å). Each possibility represents a separate embodiment of the present invention.

[0109] In some embodiments, the linker comprises a biocleavable bond. In some embodiments, the covalent bond is a biocleavable bond. In some embodiments, the linker is a biocleavable bond. In some embodiments, the linker is attached to the payload by a biocleavable bond. In some embodiments, the linker is attached to the payload by a biocleavable bond. In some embodiments, the protein conjugate is characterized by a negative zeta potential. In some embodiments, the protein conjugate is configured to release the biological payload intracellularly. In some embodiments, the protein conjugate is configured to release the biological payload in the cytosol.

[0110] In some embodiments, the linker is long enough so that the carrier does not interfere with the function of the payload. In some embodiments, the linker is long enough so that the carrier does not interfere with payload binding. In some embodiments, the binding is to an intracellular target. In some embodiments, the linker is long enough so that it does not interfere with the cell-penetrating moiety. In some embodiments, the linker is long enough so that the carrier does not form a steric hindrance to payload binding.

[0111] In some embodiments, the protein carrier is a protein with a long serum half-life. In some embodiments, the protein carrier is a protein found in blood. In some embodiments, the carrier protein comprises a molecular weight of at least 60 kDa. In some embodiments, the carrier protein comprises a molecular weight of at least 65 kDa. In some embodiments, the carrier protein comprises a molecular weight of at least 70 kDa. In some embodiments, the carrier protein comprises a molecular weight of less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, or 70 kDa. Each possibility represents a separate embodiment of the invention. In some embodiments, the carrier protein comprises an isoelectric point of at most 7. In some embodiments, the protein carrier is a human protein. In some embodiments, the protein carrier is albumin. In some embodiments, the albumin is serum albumin. In some embodiments, the serum albumin is human serum albumin (HSA). In some embodiments, the protein carrier is human serum albumin (HSA).

[0112] In some embodiments, the protein carrier is covalently bound to one or more cell-penetrating moieties. In some embodiments, the protein carrier is covalently bound to a single cell-penetrating moiety. In some embodiments, the protein carrier is covalently bound to multiple cell-penetrating moieties. In some embodiments, the biological payload is covalently bound to one or more cell-penetrating moieties. In some embodiments, the biological payload is covalently bound to a single cell-penetrating moiety. In some embodiments, the biological payload is covalently bound to multiple cell-penetrating moieties.

[0113] In some embodiments, the cell-penetrating moiety comprises a cell-internalizing molecule. In some embodiments, the cell-penetrating moiety is configured to internalize the protein conjugate of the present invention into a cell. In some embodiments, the cell-penetrating moiety is configured to induce or enhance cellular internalization of the protein conjugate of the present invention. In some embodiments, the cell-penetrating moiety is configured to enhance cell penetration or cellular internalization of the protein conjugate of the present invention compared to a control (e.g., a protein conjugate without the cell-penetrating moiety). In some embodiments, cellular internalization comprises crossing a cell membrane. In some embodiments, cellular internalization comprises delivery to the cytosol. In some embodiments, cellular internalization comprises delivery to the cytoplasm. In some embodiments, cellular internalization comprises endosomal escape.

[0114] In some embodiments, enhance is an enhance of at least 20%, at least 50%, at least 100%, at least 1000%, at least 10,000%, at least 100,000% (including any range therebetween) relative to a control, with each possibility representing a separate embodiment of the present invention.

[0115] In some embodiments, the dissociation is debinding. In some embodiments, the dissociation is cleavage of PG. In some embodiments, the protecting group is capable of undergoing cleavage at a pH value less than 7. In some embodiments, the protecting group is cleaved at a pH value less than about 7 (e.g., 0 to about 7, 3 to about 7, about 5 to about 7, about 5 to about 6.8, about 3 to about 7, including any range therebetween). In some embodiments, at least 50%, at least 70%, at least 80%, at least 90%, or at least 95% of the protecting groups undergo cleavage at a pH value less than about 7 (e.g., 0 to about 7, about 3 to about 7, about 5 to about 7, about 5 to about 7, including any range therebetween) within a maximum of 0.1 hours, a maximum of 0.5 hours, or a maximum of 1 hour (including any range therebetween). One of skill in the art will appreciate that some cleavage of the protecting group may also occur at higher pH values ​​(lower reaction rates and, therefore, longer times required to achieve efficient cleavage). Furthermore, it should be apparent that lowering the pH (below 7) will accelerate the cleavage of the protecting group.

[0116] In some embodiments, cleavage is accelerated at pH values ​​below 7. In some embodiments, the protecting group is not sufficiently cleaved at pH values ​​above 7. In some embodiments, dissociation deprotects the amine groups. Those skilled in the art will appreciate that the protecting group protects the amine groups at neutral and basic pH, but becomes deprotected at a pH below 7. The deprotection results from dissociation of the PG from the amine. In some embodiments, dissociation is induced by cleavage of the PG. In some embodiments, dissociation produces an unmasked conjugate. In some embodiments, the unmasked conjugate is characterized by a positive zeta potential. In some embodiments, the unmasked conjugate comprises a positive zeta potential.

[0117] In some embodiments, the cell-penetrating moiety of the present invention is a charge-masked moiety. In some embodiments, the protecting group is a masking. In some embodiments, the cell-penetrating moiety of the present invention is a charge-masked moiety comprising an alkylamine, a cationic polymer, and a derivative or any combination thereof, wherein the derivative comprises an alkylamine and / or a cationic polymer bound to an amine protecting group. In some embodiments, the charge-masked moiety comprises a cationic polymer bound to a protecting group (PG) of the present invention, wherein PG is an amine protecting group that can undergo cleavage at a pH value below 7. In some embodiments, the charge-masked moiety comprises an alkylamine protected by PG, wherein the protected amine can undergo deprotection at a pH value below 7.0, below 6.9, below 6.8, below 6.7, below 6.5, below 6.3, below 6.0, below 5.5, below 5, or below 3 (including any range therebetween). In some embodiments, the protected amine comprises an amine salt (e.g., a deprotonated amine) covalently bound to PG.

[0118] In some embodiments, the cationic polymer (e.g., an unprotected cationic polymer) comprises multiple amine groups. In some embodiments, the cationic polymer comprises primary amine groups, secondary amine groups, tertiary amine groups, or any combination thereof. In some embodiments, the cationic polymer can undergo ionization (positive ionization) in a solution having a pH value below the pKa value of the amine groups of the cationic polymer. In some embodiments, the cationic polymer can undergo protonation in a solution having a pH value below the pKa value of the amine groups of the cationic polymer (e.g., at a pH below 9 or below 8).

[0119] In some embodiments, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% by weight of the cationic polymer (e.g., unprotected cationic polymer) is positively charged (or protonated) in a solution having a pH value less than the pKa value of the amine groups of the cationic polymer, e.g., a pH less than 9, less than 8, less than 7.5, less than 7, less than 6, or less than 5 (including any range therebetween). In some embodiments, the cationic polymer (e.g., unprotected cationic polymer) undergoes multiple protonations in solution, resulting in multiple positive surface charges, and the solution is as described herein.

[0120] In some embodiments, the cationic polymer comprises a polyamine. In some embodiments, the cationic polymer comprises polyethyleneimine (PEI). In some embodiments, the polyamine comprises a primary amine. In some embodiments, the polyamine comprises a secondary amine. In some embodiments, the polyamine comprises a tertiary amine. In some embodiments, the polyamine comprises primary, secondary, and tertiary amines. Polyamines are well known in the art and include, for example, polyethyleneimine and polypropyleneimine, to name a few.

[0121] In some embodiments, the cationic polymer (eg, an unprotected cationic polymer) is or comprises polyethyleneimine (PEI).

[0122] In some embodiments, the PEI comprises linear PEI. In some embodiments, the PEI comprises branched PEI. In some embodiments, the PEI (e.g., branched or linear PEI) is characterized by a number average molar mass (Mn) of less than 5000 Da, less than 4000 Da, less than 3000 Da, less than 2000 Da, less than 1500 Da, less than 1000 Da, or less than 800 Da (including any range therebetween).

[0123] In some embodiments, the cell-penetrating moieties of the present invention are characterized by a MW (e.g., average molecular weight) of 100-2000 Da, 200-5000 Da, 200-3000 Da, 500-5000 Da, 500-2000 Da, 500-3000 Da, 100-300 Da, 300-400 Da, 400-500 Da, 500-600 Da, 600-700 Da, 700-1000 Da (including any range therebetween).

[0124] In some embodiments, the PEI is characterized by a Mn of 100-2000 Da, 200-5000 Da, 200-3000 Da, 500-5000 Da, 500-2000 Da, 500-3000 Da, 100-300 Da, 300-400 Da, 400-500 Da, 500-600 Da, 600-700 Da, or 700-1000 Da (including any range therebetween). In some embodiments, the cell-penetrating moiety of the invention comprises a branched PEI characterized by a Mn of 500-700 Da, or 500-2000 Da.

[0125] In some embodiments, the cell-penetrating moiety of the invention comprises a plurality of PEIs, wherein the plurality of PEIs comprises 3-10, 4-10, 2-10, 4-20, 4-50, 4-100, 2-100, 2-50, 2-20, 4-8, 6-10, 6-9, 6-8 (including any range therebetween) PEI molecules covalently attached to a single protein carrier of the invention (e.g., HSA).

[0126] In some embodiments, the cell-penetrating moieties of the present invention are charge-masked moieties comprising at least one protected amine. In some embodiments, the amine groups (e.g., primary and / or secondary amines) of the cell-penetrating moiety (e.g., PEI) are substantially protected (e.g., at least about 40%, at least about 50%, about 40 to about 95%, about 40 to about 100%, about 40 to about 70%, about 40 to about 80%, about 40 to about 90%, about 40 to about 95%, or about 40 to about 99% of the amine groups are covalently attached to a protecting group), the protecting group being represented by Formula 2 (optionally, the protecting group is derived from citraconic anhydride). In some embodiments, the cell-penetrating moieties of the present invention are covalently attached to multiple PGs, each of which has the same chemical structure. In some embodiments, the cell-penetrating moieties of the present invention are covalently attached to multiple PGs, each of which is or comprises chemically distinct PG species.

[0127] In some embodiments, the charge-masked moiety comprising the payload of the present invention is conjugated to a PG. In some embodiments, the payload is conjugated to one or more PGs. In some embodiments, the payload is conjugated to multiple PGs, where the PGs are chemically identical or chemically distinct species. In some embodiments, each payload molecule in a conjugate of the present invention is covalently conjugated to one or more PGs, such as 1, 2, 3, 4, 5, 6, 7, 8, 2-10, 2-5, 1-10, 1-5, 1-8, or 2-8 PGs (including any range therebetween).

[0128] In some embodiments, the charge-masked moiety is substantially devoid of protonation and / or positive charge (e.g., in aqueous solution) in the pH range of 7-10. In some embodiments, the charge-masked moiety and / or the protected amine groups are substantially uncharged or negatively charged (e.g., in aqueous solution) in the pH range of 7-10, and positively charged in the pH range of less than 7, less than 6.8, less than 6.5, less than 6 (including any range therebetween). In some embodiments, the amine groups of the cell-penetrating moiety are substantially uncharged or negatively charged (e.g., in aqueous solution) in the pH range of 7-10, and positively charged in the pH range of less than 7, less than 6.8, less than 6.5, less than 6 (including any range therebetween). In some embodiments, the charge-masked moiety is substantially devoid of protonatable amines (e.g., primary and / or secondary amines) in the pH range of 7-10. In some embodiments, the charge-masked moiety has a substantially reduced ability to undergo protonation in the pH range of 7-10 compared to the unmasked cell-penetrating moiety (lacking PG).

[0129] In some embodiments, the charge-masked moieties of the present invention comprise one or more protected PEIs, in which at least a portion of the amines of the PEI are conjugated to PG. In some embodiments, the protected PEI comprises one or more amines (e.g., deprotonated amines) of the PEI conjugated to a protecting group of the present invention. In some embodiments, the cell-penetrating moieties of the present invention comprise at least 1%, at least 5%, linear or branched PEI, and at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or 50-99%, 50-97%, 50-95%, 50-90%, 50-85%, 50-80% (including any range therebetween) of the amine groups (e.g., primary amines and / or secondary amines) are covalently conjugated to a protecting group of the present invention.

[0130] In some embodiments, any of the charge-masked moieties, protein conjugates of the present invention, and protein carriers of the present invention are negatively charged at a pH of 7 to 10, 7.2 to 10, or 7.5 to 10 (including any range therebetween), or at a pH above 7. In some embodiments, any of the charge-masked moieties, protein conjugates of the present invention, and protein carriers of the present invention are negatively charged at a pH of about 7.4.

[0131] In some embodiments, the cell-penetrating moiety, the protein conjugate of the invention, the payload, and any of the protein carriers of the invention are characterized by a zeta potential of less than -0.1 mV, less than -0.5 mV, less than -1 mV, less than -5 mV, less than -10 mV, less than -12, less than -14 mV, or less than -20 mV (including any range therebetween). In some embodiments, the negative zeta potential is a zeta potential of less than 0. In some embodiments, the negative zeta potential is a zeta potential of -1 mV or less. In some embodiments, the negative zeta potential is a zeta potential of -2 mV or less. In some embodiments, the negative zeta potential is a zeta potential of -3 mV or less. In some embodiments, the negative zeta potential is a zeta potential of -4 mV or less. In some embodiments, the negative zeta potential is a zeta potential of -5 mV or less.

[0132] In some embodiments, the molar ratio between PG and cationic polymer (e.g., PEI) in the charge-masked portion is between 100,000:1 and 0.8:1, 50,000:1 and 1:1, 25,000:1 and 1:1, 10,000:1 and 1:1, 8,000:1 and 1:1, 5,000:1 and 1:1, 3,000:1 and 1:1, 2,000:1 and 1:1, 1,000:1 and 1:1, 900:1 and 1:1, 800:1 and 1:1, 700:1 and 1:1, or 1, 600:1 to 1:1, 500:1 to 1:1, 400:1 to 1:1, 300:1 to 0.8:1, 250:1 to 1:1, 200:1 to 0.8:1, 150:1 to 1:1, 125:1 to 1:1, 100:1 to 0.8:1, 100:1 to 80:1, 80:1 to 50:1, 50:1 to 30:1, 30:1 to 10:1, 10:1 to 5:1, 10:1 to 0.8:1, 5:1 to 1:1, 1:1 to 0.8:1 (including any ranges between these).

[0133] In some embodiments, the molar ratio between PG and carrier (e.g., HSA) is 100,000:1, up to 1:1, 50,000:1 to 1:1, 25,000:1 to 1:1, 10,000:1 to 1:1, 8,000:1 to 1:1, 5,000:1 to 1:1, 3,000:1 to 1:1, 2,000:1 to 1:1, 1,000:1 to 1:1, 900:1 to 1:1, 800:1, up to 1:1, 700: 1 to 1:1, 600:1 to 1:1, 500:1 to 1:1, 400:1 to 1:1, 300:1 to 1:1, 250:1 to 1:1, 200:1 to 1:1, 150:1 to 1:1, 125:1 to 1:1, 100:1 to 1:1, 100:1 to 80:1, 80:1 to 50:1, 50:1 to 30:1, 30:1 to 10:1, 10:1 to 5:1, 5:1 to 1:1 (including any ranges between these).

[0134] In some embodiments, the protected amines of the charge-masked moieties are substantially stable (absent deprotection) at neutral and / or basic pH, ie, at least 50 mol%, at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol%, at least 95 mol% (including any range therebetween) of the protected amines of the charge-masked moieties remain stable at neutral and / or basic pH for the times described below.

[0135] In some embodiments, the protected amine is substantially stable at a pH in the range of 7.0 to 14, 7.0 to 7.2, 7.2 to 7.5, 7.5 to 8, 8 to 9, 9 to 12, or 12 to 14 (including any range therebetween). In some embodiments, the protected amine is substantially stable at a pH of about 7.4. In some embodiments, the protected amine is substantially stable at a pH in the range of 7.0 to 14 (including any range therebetween) for at least 1 hour, at least 2 hours, at least 10 hours, at least 24 hours, at least 48 hours, or at least 72 hours (including any range therebetween).

[0136] In some embodiments, the protected amine undergoes deprotection (or decomposition via cleavage of PG therefrom) to yield a deprotected amine (e.g., an uncharged or positively charged protonated amine). In some embodiments, the protected amine is substantially deprotected at a pH in the range of 0 to 6.9, 0 to 6.8, 6 to 6.8, 5 to 6, 0 to 3, 3 to 5, or 5 to 6.8 (including any range therebetween). In some embodiments, the protected amine is substantially deprotected at a pH of about 6.8. In some embodiments, the protected amine is substantially deprotected in a cancer microenvironment. In some embodiments, the cancer microenvironment is the tumor microenvironment (TME). In some embodiments, at least 10 mol%, at least 20 mol%, at least 30 mol%, at least 40 mol%, at least 50 mol%, at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol%, at least 95 mol% (including any range therebetween) of the protected amines are deprotected at a pH less than 7 (e.g., 0 to 6.9). In some embodiments, the protected amine is substantially deprotected at a pH of less than 7 (e.g., 0-6.9, 0-6.8, 6-6.8, 5-6, 0-3, 3-5, 5-6.8, including any range therebetween) for a time ranging from 1 second to 1 hour, 1-30 seconds, 30-60 seconds, 60 seconds to 2 minutes, 2-10 minutes, 1 minute to 1 hour, 1 minute to 24 hours, 1 minute to 12 hours, 1 minute to 8 hours, 1 minute to 6 hours, 1 minute to 4 hours, 1 minute to 3 hours, 1 minute to 2 hours, 1 second to 24 hours, 1 second to 12 hours, 1 second to 8 hours, 1 second to 6 hours, 1 second to 4 hours, 1 second to 3 hours, 1 second to 2 hours, including any range therebetween). In some embodiments, the pH less than 7 is about 6.8.

[0137] protecting group In some embodiments, the protected amine comprises a PG of the present invention covalently bonded to an amine. In some embodiments, the protected amine is obtained by reacting a PG precursor with an amine. In some embodiments, the PG precursor is reactive with amines (e.g., primary amines, secondary amines, or both). In some embodiments, the PG precursor is or includes an optionally substituted (e.g., R and R1, as described below) cyclic anhydride (e.g., a 5- to 6-membered cyclic anhydride, which may be unsaturated). In some embodiments, the PG precursor can react with an amine to form a stable protected amine. In some embodiments, the protected amine (e.g., in a charge-masked moiety) is stable under neutral and / or basic pH conditions. In some embodiments, the PG precursor can react with an amine group (primary and / or secondary amine), thereby converting the amine group to a protected amine (e.g., an amide). In some embodiments, the PG precursor has the formula: [ka] wherein n, R, R1 and R2 are as defined herein above.

[0138] In some embodiments, a PG of the present invention is covalently attached to an amine, wherein the amine is selected from (i) an amine group of the payload, (ii) an amine group of the cell-penetrating moiety, or both (i) or (ii).

[0139] In some embodiments, the PG (protecting group attached to the amine or deprotonated amine) comprises one or more moieties (e.g., 1, 2, 3, or 4 moieties) that have a negative charge at a pH above 5. In some embodiments, the PG has a negative charge at a pH of 4-8, 4-5, 5-6, 6-7, 7-8 or higher (including any range therebetween). In some embodiments, the PG is substantially negatively charged in the tissue or biological fluid of a subject, and the tissue and / or biological fluid is characterized by a pH of 4-8, or 5-9 (including any range therebetween).

[0140] In some embodiments, at least 50 mol%, at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol%, or at least 95 mol% of the PG is negatively charged at a pH above the pKa value of the moiety. In some embodiments, the moiety is or comprises carboxy or a salt thereof. In some embodiments, the PG comprises carboxy and / or a salt thereof, and the PG is negatively charged at a pH of 4-8, or 5-8, or above 8.

[0141] In some embodiments, PG has Formula 1: [ka] wherein n is an integer ranging from 0 to 5; [ka] represents the point of attachment to the amine group (e.g., the nitrogen atom of a protected amine), and [ka] represents a single or double bond; R and R are each independently H, optionally substituted alkyl (e.g., C-C 10 alkyl, or C1-C5 alkyl), halo, optionally substituted cycloalkyl, optionally substituted aryl or heteroaryl, and carboxyalkyl (e.g., C1-C 10 or R and R are joined together to form a ring.

[0142] In some embodiments, R and R are each independently H, C-C 10 Alkyl, C1-C 10Alkenyl, -NO2, -CN, -OH, -NH2, carbonyl, -CONH2, -CONR'2, -CNNR'2, -CSNR'2, -CONH-OH, -CONH-NH2, -NHCOR', -NHCSR', -NHCNR', -NC(=O)OR', -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-S2R', -SOR', -SR', -S2OR', -SON(R')2, -NHNR'2, -NNR', -NH(C1-C6 alkyl), -N(C1-C 10 Alkyl)2, C1-C 10 Alkoxy, C1-C 10 Haloalkoxy, hydroxy(C1-C 10 alkyl), hydroxy (C1-C 10 alkoxy), alkoxy(C1-C 10 alkyl), alkoxy (C1-C 10 Alkoxy), amino (C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CON(C1-C 10 represents one or more substituents selected from: -C1-C6 alkyl, ...

[0143] In some embodiments, n is 0-5, 0-1, 1-5, 1-3, 3-5 (including any range therebetween). In some embodiments, each R and / or R1 represents one or more substituents. In some embodiments, R and R1 are the same or different substituents. In some embodiments, carboxyalkyl includes -alkyl-COOH. In some embodiments, carboxyalkyl includes -(C1-C 10) alkyl-COOH, or —(C1-C5) alkyl-COOH (including any range therebetween), wherein the alkyl is optionally substituted.

[0144] In some embodiments, PG has Formula 2: [ka] wherein n, R, and R1 are as described herein, and each R2 is independently selected from H, C1-C 10 Alkyl, C1-C 10 Alkenyl, -NO2, -CN, -OH, -NH2, carbonyl, -CONH2, -CONR'2, -CNNR'2, -CSNR'2, -CONH-OH, -CONH-NH2, -NHCOR', -NHCSR', -NHCNR', -NC(=O)OR', -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-S2R', -SOR', -SR', -S2OR', -SON(R')2, -NHNR'2, -NNR', -NH(C1-C6 alkyl), -N(C1-C 10 Alkyl)2, C1-C 10 Alkoxy, C1-C 10 Haloalkoxy, hydroxy(C1-C 10 alkyl), hydroxy (C1-C 10 alkoxy), alkoxy(C1-C 10 alkyl), alkoxy (C1-C 10 Alkoxy), amino (C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CON(C1-C 10and each R' independently comprises one or more substituents (e.g., one or two substituents) selected from optionally substituted C-C alkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted phenyl, optionally substituted benzyl, or any combination thereof.

[0145] In some embodiments, one of R and R is H and the other of R and R comprises alkyl or carboxyalkyl. In some embodiments, each R and R independently comprises C-C 10 Includes alkyl (branched or straight chain), or carboxyalkyl.

[0146] In some embodiments, PG is represented by formula 2, wherein n is 0; R is selected from CH2COOH, methyl, and ethyl; and R1 is selected from H and CH2CH2COOH.

[0147] In some embodiments, PG has formula 2A: [ka] , wherein R and R1 are selected from H and methyl, and R or R1 is methyl.

[0148] In some embodiments, PG is represented by any of the formulas disclosed herein, including any salt (e.g., carboxylate), derivative, tautomer, isotope, or structural isomer thereof. In some embodiments, PG is a protecting group disclosed hereinafter. In some embodiments, PG is a maleic anhydride derivative. In some embodiments, PG is derived from citraconic anhydride. In some embodiments, PG is maleic anhydride. In some embodiments, PG is derived from aconitic anhydride. In some embodiments, PG is derived from dimethylmaleic anhydride.

[0149] As used herein, the term "derived from" encompasses molecules obtained via nucleophilic substitution of a PG precursor (e.g., a cyclic anhydride) with an amine group. In some embodiments, PG is derived from citraconic anhydride, the protecting group is represented by Formula 2A, and R or R is methyl. In some embodiments, PG is or comprises citraconic anhydride. In some embodiments, PG is derived from citraconic anhydride and has the shape of Formula 2A.

[0150] In some embodiments, the protein carrier comprises a plurality of PEI molecules covalently attached thereto, ie, 3-10, or 4-10, 2-100, 3-100, 3-90, 4-100, 4-90, 4-10, 4-40, 4-20, 20-100, 20-40, 40-60, 60-100, 6-100, 6-20, 6-40, 6-50, 4-15, 3-15, 3-10, 3-8, 4-8, 6-10 (including any range therebetween) PEI molecules covalently attached thereto.

[0151] In some embodiments, the protein carrier comprises 3-10 PEI molecules, 3-5 PEI molecules, 5-8 PEI molecules, 8-10 PEI molecules, 10-15 PEI molecules, 15-20 PEI molecules (including any range therebetween) covalently attached thereto.

[0152] In some embodiments, the protein carrier comprises a size of at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 KDa. Each possibility represents a separate embodiment of the present invention. In some embodiments, the protein carrier comprises a size of 50 KDa. In some embodiments, the protein carrier comprises a size of 60 KDa. In some embodiments, the protein carrier comprises a size of 65 KDa.

[0153] In some embodiments, the protein carrier is an endogenous blood protein. In some embodiments, the protein is naturally found in blood. In some embodiments, the blood is plasma. In some embodiments, the blood is mammalian blood. In some embodiments, the mammal is human. In some embodiments, the endogenous blood protein is albumin. In some embodiments, the endogenous blood protein is a globulin. In some embodiments, the endogenous blood protein is fibrinogen. In some embodiments, the globulin is an immunoglobulin (Ig). In some embodiments, the Ig is IgG. In some embodiments, the Ig is IgA. In some embodiments, the Ig is IgM. In some embodiments, the endogenous blood protein is selected from HSA, fibrinogen, and IgG. In some embodiments, the endogenous blood protein is not a clotting protein. Endogenous blood proteins are well known in the art and include, for example, prealbumin (transthyretin), alpha 1 antitrypsin, alpha 1-glycoprotein, alpha 1-fetoprotein, alpha 2-macroglobulin, gamma globulin, beta 2 microglobulin, haptoglobin, ceruloplasmin, complement component 3, complement component 4, C-reactive protein (CRP), lipoproteins (chylomicrons, VLDL, LDL, HDL), transferrin, prothrombin, and maltose-binding protein (MBP), to name a few. In some embodiments, the protein carrier is selected from human serum albumin (HSA), fibrinogen, IgG, fluorescent proteins (GFPs), and designed ankyrin repeat proteins (DARPins). In some embodiments, the fluorescent protein is selected from green (GFP), red (RFP), blue (BFP), and yellow (YFP). In some embodiments, the fluorescent protein is GFP. In some embodiments, the protein carrier is HSA. In some embodiments, the protein carrier is fibrinogen. In some embodiments, the protein carrier is IgG.

[0154] In some embodiments, the protein carrier is or comprises HSA. In some embodiments, the protein carrier comprises PEI-modified HSA. In some embodiments, the protein conjugate comprises a payload attached to the protein carrier via a linker, wherein the linker is as described herein, and the protein carrier is or comprises HSA covalently bound to (or modified with) 3 to 20, 3.5 to 20, 3 to 10, 3.5 to 10, 3 to 8, 3.5 to 8, 3 to 5, 5 to 8, 8 to 10, 10 to 15, 3 to 10, 3 to 15, 4 to 20, 4 to 10, 3.5 to 6, 3.5 to 15, 3.5 to 8, 3.5 to 12, 3 to 12, 3 to 17, 3.5 to 17, 3.5 to 15, 4 to 8, or 6 to 10 PEI molecules. In some embodiments, the numbers of PEI molecules described herein are average values. In some embodiments, the PEI molecules are characterized by an average MW of 100-2000 Da, 200-5000 Da, 200-3000 Da, 500-5000 Da, 500-2000 Da, 500-3000 Da, 100-300 Da, 300-400 Da, 400-500 Da, 500-600 Da, 600-700 Da, or 700-1000 Da (including any range therebetween). In some embodiments, the protein conjugate comprises PEI and is masked with 15-50, 15-45, 15-35, 15-30, 15-25, 20-50, 20-45, 20-40, 20-35, 20-25, 25-50, 25-45, 25-40, 25-35, 25-30, 30-50, 30-45, 30-40, 30-35, 35-50, 35-45, 35-40, 40-50, 40-45, or 45-50 molecules of PG (including any range therebetween).

[0155] In some embodiments, the protein carrier is covalently attached to at least two molecules of PEI. In some embodiments, the protein carrier is covalently attached to at least eight molecules of PEI. In some embodiments, the biological payload is covalently attached to one molecule of PEI. In some embodiments, the protein conjugate is covalently attached to at least three molecules of PEI. In some embodiments, the protein conjugate is covalently attached to at least eight molecules of PEI. In some embodiments, the protein conjugate is covalently attached to at least 98 molecules of PEI.

[0156] In some embodiments, the conjugate comprises a payload. As used herein, the term "payload" refers to any molecule that is delivered into the cytoplasm of a target cell. In some embodiments, the payload binds an intracellular target. In some embodiments, the payload interacts with an intracellular target. In some embodiments, the payload modulates an intracellular target. In some embodiments, the intracellular space is the cytoplasm. In some embodiments, the payload lacks a disulfide bond that, upon cleavage, reduces interaction with the intracellular target. In some embodiments, the payload lacks a disulfide bond. In some embodiments, the payload is a molecule. In some embodiments, the payload is a biological payload. In some embodiments, the payload is a biomolecule. In some embodiments, the payload is an organic compound. In some embodiments, the payload is a therapeutic molecule. In some embodiments, the payload is a detectable molecule. In some embodiments, the payload is a molecule that can bind to a target. In some embodiments, the payload is a biologic. In some embodiments, the payload is a drug. In some embodiments, the payload is a protein or peptide. In some embodiments, the peptide or protein is an isolated protein or peptide. In some embodiments, the peptide or protein is a peptide or protein moiety. The protein need not be a complete protein, but may be a portion or fragment of a protein. In some embodiments, the payload comprises or consists of amino acids. In some embodiments, the payload is a single amino acid chain. In some embodiments, the payload is a multiple amino acid chain. In some embodiments, the payload is a bioactive molecule. In some embodiments, the bioactive is a bioactive agent.

[0157] In some embodiments, the payload is a nucleic acid molecule. In some embodiments, the payload is DNA. In some embodiments, the payload is RNA. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the payload is an aptamer. In some embodiments, the payload is a primer. In some embodiments, the payload is an antisense oligonucleotide. In some embodiments, the payload is a regulatory RNA. In some embodiments, the payload is a plasmid. In some embodiments, the payload is an expression vector. In some embodiments, the vector is configured for expression in a target cell. In some embodiments, the payload is a gene therapy agent. In some embodiments, the nucleic acid molecule comprises an open reading frame. In some embodiments, the open reading frame encodes a therapeutic protein. Methods for conjugating nucleic acid molecules to chemical and amino acid linkers are well known in the art, and any such method may be employed. In some embodiments, the nucleic acid molecule comprises a nuclear localization signal (NLS). In some embodiments, the payload is selected from a protein and a nucleic acid molecule.

[0158] The term "nucleic acid" is well known in the art. As used herein, "nucleic acid" generally refers to a molecule of DNA, RNA, or a derivative or analog thereof, which comprises a nucleic acid base. Nucleic acid bases include, for example, the naturally occurring purine or pyrimidine bases found in DNA (e.g., adenine "A", guanine "G", thymine "T") or the naturally occurring purine or pyrimidine bases found in RNA (e.g., A, G, uracil "U" or C).

[0159] The term "nucleic acid molecule" includes, but is not limited to, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNAs such as miRNA, siRNA and other short interfering nucleic acids, snoRNA, snRNA, tRNA, piRNA, tnRNA, small rRNA, hnRNA, lncRNA, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, ribozymes, viral RNA or DNA, infectious nucleic acids, amplification products, modified nucleic acids, plasmids or organelle nucleic acids, and artificial nucleic acids such as oligonucleotides.

[0160] As used herein, the term "oligonucleotide" refers to a short (e.g., 100 bases or less) chemically synthesized single-stranded DNA or RNA molecule. In some embodiments, the oligonucleotide is attached to the 5' or 3' end of a nucleic acid molecule, such as by a ligation reaction.

[0161] The term "expression" refers to the biosynthesis of a gene product and includes transcription and / or translation of said gene product. Thus, expression of a nucleic acid molecule can refer to the transcription of a nucleic acid fragment (e.g., transcription produces mRNA or other functional RNA) and / or the translation of RNA into a precursor or mature protein (polypeptide).

[0162] Expression of genes in cells is well known to those skilled in the art, and delivery thereof can be carried out by the methods or compositions of the present invention, as described herein. In some embodiments, the gene is in an expression vector, such as a plasmid or a viral vector. The vector can be a viral vector. The viral vector can be a retroviral vector, a herpes virus vector, an adenoviral vector, an adeno-associated virus vector, or a poxvirus vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter.

[0163] In some embodiments, the gene or open reading frame is operably linked to a promoter or other regulatory element. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in a host cell when the vector is introduced into the host cell by the method of the present invention). In some embodiments, the regulatory element or promoter is active in the target cell.

[0164] As used herein, the term "promoter" refers to a group of transcriptional control modules clustered around an initiation site for RNA polymerase, i.e., RNA polymerase II. A promoter is composed of separate functional modules, each consisting of approximately 7-20 bp of DNA and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0165] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0166] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (which are available from Invitrogen), pCI (which is available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (which are available from Strategene), pTRES (which is available from Clontech), and derivatives thereof.

[0167] In some embodiments, expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are used in the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, bovine papillomavirus-derived vectors include pBV-1MTHA, and Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows protein expression under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters effective for expression in eukaryotic cells.

[0168] In some embodiments, recombinant viral vectors that offer advantages such as horizontal infection and target specificity are useful for in vivo expression. In one embodiment, horizontal infection, for example, is inherent in the retroviral life cycle, whereby a single infected cell produces many progeny virions that detach from the mother and infect neighboring cells. In one embodiment, the result is that a wide area becomes rapidly infected, most of which was not initially infected by the original viral particle. In one embodiment, a viral vector that cannot be horizontally transmitted is generated. In one embodiment, this feature can be useful when the desired goal is to introduce a specific gene into only a limited number of target cells.

[0169] The term "bioactive" refers to a molecule or agent that exerts an effect on a cell or tissue. Representative examples of types of bioactive agents include, but are not limited to, therapeutic drugs, vitamins, electrolytes, amino acids, peptides, polypeptides, proteins, enzymes, carbohydrates, lipids, polysaccharides, nucleic acids, nucleotides, polynucleotides, glycoproteins, lipoproteins, glycolipids, glycosaminoglycans, proteoglycans, growth factors, differentiation factors, hormones, neurotransmitters, prostaglandins, immunoglobulins, cytokines, and antigens. Various combinations of these molecules can be used. Examples of cytokines include macrophage-derived chemokines, macrophage inflammatory proteins, interleukins, and tumor necrosis factors. Examples of proteins include fibrous proteins (e.g., collagen, elastin) and adhesion proteins (e.g., actin, fibrin, fibrinogen, fibronectin, vitronectin, laminin, cadherins, selectins, intracellular adhesion molecules, and integrins). In various instances, the bioactive agent may be selected from fibronectin, laminin, thrombospondin, tenascin C, leptin, leukemia inhibitory factor, RGD peptide, anti-TNF, endostatin, angiostatin, thrombospondin, bone morphogenetic protein-1, bone morphogenetic protein, osteonectin, somatomedin-like peptide, osteocalcin, interferon, and interleukin. In some embodiments, the bioactive agent comprises a growth factor, a differentiation factor, or a combination thereof.

[0170] As used herein, the term "isolated peptide" refers to a peptide that is essentially free from contaminating cellular components, such as carbohydrates, lipids, or other proteinaceous impurities associated with the naturally occurring peptide. Typically, a preparation of isolated peptide contains the peptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.

[0171] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to a polymer of amino acid residues. In another embodiment, as used herein, the terms "peptide," "polypeptide," and "protein" encompass natural peptides, peptidomimetics (typically containing non-peptide bonds or other synthetic modifications), and peptide analogs peptoids and semipeptoids, or any combination thereof. In another embodiment, the described peptides, polypeptides, and proteins have modifications that make them more stable but more permeable to cells in the body. In one embodiment, the terms "peptide," "polypeptide," and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms "peptide," "polypeptide," and "protein" apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0172] In some embodiments, the payload binds a cytoplasmic target. In some embodiments, the payload is specific for the cytoplasmic target. In some embodiments, specific includes lesser binding to any other target. In some embodiments, the payload is a binding molecule. In some embodiments, the payload hybridizes to its target. In some embodiments, the payload is complementary to its target. In some embodiments, the payload comprises a complementarity determining region (CDR) that binds the target. In some embodiments, the payload is an antibody or antigen-binding fragment thereof. The structure of antibodies is well known, and one of skill in the art may not know which target an antibody will bind to based on its CDR sequences alone, but the general structure of an antibody and its antigen-binding region will be recognized by one of skill in the art.

[0173] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides containing at least one binding domain formed by folding of a polypeptide chain, which has an internal surface shape and a three-dimensional binding space with a charge distribution complementary to the characteristics of an antigenic determinant of an antigen. Antibodies typically have a tetrameric form, containing two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, or anti-idiotypic, and may be soluble or conjugated, labeled, or fragments containing epitope-binding fragments, variants, or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies may be derived from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2, single-chain antibodies (svFC), dimeric variable regions (diabodies), and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or fragment thereof. Those skilled in the art will further appreciate that other fusion products may be generated, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0174] The immunoglobulin molecule may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass.

[0175] In some embodiments, the antibody is a single-chain antibody (scFv). In some embodiments, the antibody is a single-domain antibody. In some embodiments, the antibody is a camelid antibody. In some embodiments, the antibody is a shark antibody. In some embodiments, the antibody is a VHH. In some embodiments, the antibody comprises a heavy chain and a light chain. In some embodiments, the antibody is a heavy-chain-only antibody. In some embodiments, the antibody is an antibody mimetic. In some embodiments, the binding molecule or antibody mimetic is a DARPin. Regardless of CDRs, the present antibodies, antibody fragments, ScFvs, nanobodies, VHHs, single-domain antibodies, DARPins, etc. can be structurally recognized by their non-variable regions. Therefore, without being limited to a particular target, it can be understood that the compositions of the present invention include these molecules as payloads.

[0176] In some embodiments, the payload comprises a C-terminal cysteine ​​amino acid. In some embodiments, the payload comprises a cysteine ​​amino acid that is proximal to the C-terminus of the payload. In some embodiments, proximal is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of the terminus. Each possibility represents a separate embodiment of the present invention.

[0177] In some embodiments, the payload comprises two or more molecules. In some embodiments, the payload comprises two or more bioactive molecules. In some embodiments, the payload is bispecific. As used herein, the term "bispecific" refers to having functionality for two different targets. In some embodiments, the payload comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 molecules. Each possibility represents a separate embodiment of the invention. In some embodiments, the payload comprises at least two molecules for two intracellular targets. In some embodiments, at least two is two. In some embodiments, the two targets are the same target. In some embodiments, the two targets are different targets. In some embodiments, the payload comprises at least two VHHs. In some embodiments, the at least two VHHs are specific for different intracellular proteins.

[0178] In some embodiments, at least two molecules are separated by a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is cleavable in the cytoplasm. In some embodiments, the linker is flexible. In some embodiments, cleavage of the linker allows each molecule to reach its target. In some embodiments, the linker is not cleavable. In some embodiments, the two molecules carry two target intracellular proteins together.

[0179] In some embodiments, the protein conjugate comprises a payload bound to a protein carrier via a linker. In some embodiments, the protein conjugate is not a fusion protein. Those skilled in the art will understand that the payload and the carrier are conjugated to each other with a separate linker. The linker is not part of the carrier or the payload, but rather is attached (conjugated) to each other, thereby connecting them.

[0180] In some embodiments, the linkers of the present invention are substantially stable in biological fluids (e.g., human blood, plasma, or serum) for at least 2, at least 10, at least 24, or at least 48 hours (including any range therebetween). Each possibility represents a separate embodiment of the present invention. In some embodiments, the linkers of the present invention are substantially stable in blood. In some embodiments, the blood is human blood. In some embodiments, the blood is mouse blood. In some embodiments, the blood is rodent blood. In some embodiments, the rodent is a rat. In some embodiments, the rodent is a mouse. In some embodiments, the linkers are substantially stable in human blood for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48, or 72 hours. Each possibility represents a separate embodiment of the present invention.

[0181] In some embodiments, the linkers and / or charge-masked moieties of the invention are unstable upon exposure to cytoplasmic conditions, hi some embodiments, the linkers of the invention are cleavable upon exposure to cytoplasmic conditions to release the biological payload into the cytosol.

[0182] In some embodiments, the charge-masked moiety undergoes cleavage or deprotection under exposure to conditions comprising a pH of 6-7 to yield a protein carrier (e.g., HSA) comprising a deprotected cell-penetrating moiety (e.g., a deprotected PEI molecule). In some embodiments, a pH of 6-7 is about 6.8.

[0183] In some embodiments, a protein carrier comprising a deprotected cell-penetrating moiety (e.g., a deprotected PEI molecule) exhibits a cleavage potential of at least 5 mV, at least 6 mV, at least 7 mV, at least 8 mV, at least 8.5 mV, at least 9 mV, at least 9.5 mV, at least 10 mV, at least 12 mV, or between 8 and 40 mV, between 8.5 and 40 mV, between 8 and 20 mV. , 8.5-20 mV, 10-40 mV, 10-20 mV, 10-30 mV, 5-50 mV, 6-50 mV, 7-50 mV, 8-50 mV, 8-40 mV, 8-30 mV, 8-20 mV, 10-50 mV, 10-40 mV, 10-30 mV, 10-20 mV, 20-50 mV, 30-50 mV (including any range therebetween). One of skill in the art will appreciate that the exact zeta potential value of a conjugate may vary depending on the MW and / or size of the protein carrier, payload, or both.

[0184] In some embodiments, a protein conjugate comprising a deprotected cell-penetrating moiety (e.g., a deprotected PEI molecule) is characterized by a positive zeta potential value of at least 6 mV, at least 7 mV, at least 8 mV, at least 8.5 mV, at least 9 mV, at least 9.5 mV, at least 10 mV, at least 12 mV, or 8-40 mV, 8.5-40 mV, 8-20 mV, 8.5-20 mV, 10-40 mV, 10-20 mV, or 10-30 mV (including any range therebetween). Each possibility represents a separate embodiment of the present invention.

[0185] Zeta potential can be measured by any method known in the art. The following protocol was used herein, which can be considered a standard method for determining whether a molecule contains a zeta potential in the range detailed herein. Zeta potential measurements were performed using a Zeta Sizer Ultra (Malvern Instruments). Samples were buffer exchanged into 1 mM NaCl at a protein concentration of 1 mg / mL. 20 μL from each sample was loaded into a Zeta Cell (DTS1070), and five replicates were measured for each sample. The average zeta potential in mV was obtained for each replicate. The average of the five measurements is reported along with the standard deviation. Measurements were performed under the following conditions: temperature 25°C; number of runs per replicate 10-40; equilibration time: 60 seconds, with no pause between sub-runs; 60-second pause between replicates; voltage was automatically selected, and a unimodal analysis method was used for data processing. In some embodiments, zeta potential was measured in approximately 1 mM salt. In some embodiments, the salt is NaCl. In some embodiments, the zeta potential is measured at a protein concentration of about 1 mg / mL.

[0186] In some embodiments, the protein conjugate of the present invention comprises a protein carrier covalently bonded to a payload via a linker. In some embodiments, the payload is covalently bonded to the linker. In some embodiments, the carrier is covalently bonded to the linker. In some embodiments, the covalent bond is not a peptide bond. In some embodiments, at least one bond between the linker and the payload and the bond between the linker and the carrier are not peptide bonds. In some embodiments, the carrier, linker, and payload are not contained in a single amino acid chain. In some embodiments, the protein conjugate of the present invention comprises a protein carrier covalently bonded to a payload via a linker, wherein the linker is a synthetic linker comprising at least one cleavable bond. In some embodiments, the protein conjugate of the present invention comprises a protein carrier covalently bonded to a payload via a linker, wherein the linker is a synthetic linker lacking a cleavable bond. In some embodiments, the carrier and payload are not derived from the same protein. In some embodiments, the linker and carrier are not derived from the same protein. In some embodiments, the linker is a peptide linker and comprises a sequence that is not present in the amino acid sequence of the protein on which the carrier is based. In some embodiments, the linker is a peptide linker and comprises a sequence that is not present in the amino acid sequence of the protein on which the payload is based. In some embodiments, the linker and the payload are not from the same protein. In some embodiments, the payload is not a naturally occurring molecule. In some embodiments, the payload is artificial. In some embodiments, the linker is not naturally occurring. In some embodiments, the linker is artificial. In some embodiments, the carrier is a naturally occurring protein or a fragment thereof.

[0187] In some embodiments, the linker is a protein linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an amino acid linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the rigid linker is an alpha helix peptide. In some embodiments, the linker is a flexible linker. In some embodiments, the flexible linker is a GGGGS linker. In some embodiments, the linker comprises a C-terminal cysteine ​​amino acid. In some embodiments, the linker comprises a cysteine ​​amino acid proximal to the C-terminus of the linker. In some embodiments, proximal is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid of the terminus. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises an N-terminal cysteine ​​amino acid and the payload comprises a C-terminal cysteine. It will be understood by those skilled in the art that the side chain of cysteine ​​contains a sulfur atom that can be used to generate a cleavable disulfide bond. For example, cysteine ​​can form a disulfide bond with cysteine ​​34 of HSA.

[0188] In some embodiments, the alpha helical peptide comprises or consists of AAASAEAAAKEAAAKEAAAKAAAGSG (SEQ ID NO: 6). In some embodiments, the alpha helical peptide comprises or consists of AAASAEAAAKEAAAKEAAAKAAAGSGLC (SEQ ID NO: 10). In some embodiments, the alpha helical peptide comprises or consists of AAASAEAAAKEAAAKEAAAKAAAGSGL (SEQ ID NO: 14). In some embodiments, the flexible linker is a GGGGS linker. In some embodiments, the flexible linker comprises 1 to 5 GGGGS repeats. In some embodiments, 1 to 5 is 1 to 3. In some embodiments, 1 to 5 is 1. In some embodiments, 1 to 5 is 2. In some embodiments, 1 to 5 is 3. In some embodiments, the linker comprises or consists of GGGGSGGGGSGGGGLC (SEQ ID NO: 4). In some embodiments, the linker comprises or consists of GGGGSGGGGSGGGLGC (SEQ ID NO: 5). In some embodiments, the linker comprises or consists of GGGGSGGGSGGGLG (SEQ ID NO:7). In some embodiments, the linker comprises or consists of GGGGSGGGGSGGGGSC (SEQ ID NO:8). In some embodiments, the linker comprises or consists of GGGGSGGGSGGGGGS (SEQ ID NO:12). In some embodiments, the linker comprises or consists of GGGGSC (SEQ ID NO:9). In some embodiments, the linker comprises or consists of GGGGS (SEQ ID NO:13). In some embodiments, the linker comprises or consists of GGGLGC (SEQ ID NO:11). In some embodiments, the linker comprises or consists of GGGLG (SEQ ID NO:15). In some embodiments, the linker comprises or consists of an amino acid sequence selected from SEQ ID NOs:4-15.

[0189] In some embodiments, the protein conjugates of the present invention substantially lack biocleavable bonds (e.g., bonds that are cleavable upon exposure of the protein conjugate to cytoplasmic conditions). In some embodiments, the linker is substantially free of biocleavable bonds. In some embodiments, the linker is attached to the protein carrier and / or payload via a bond that is not biocleavable (e.g., an amide bond, a click reaction product, a thioether bond, etc.).

[0190] In some embodiments, the linker of the invention comprises a biocleavable bond. In some embodiments, the biocleavable bond is substantially stable in a biological fluid (e.g., human blood, plasma, or serum) for at least 2 hours, at least 10 hours, at least 24 hours, or at least 48 hours (including any range therebetween). In some embodiments, cleavable means cleavable in the cytoplasm. In some embodiments, cleavable means not cleavable in serum or blood. In some embodiments, non-cleavable means substantially not cleavable. In some embodiments, biocleavable in the cytoplasm is cleaved significantly more in the cytoplasm than in blood.

[0191] In some embodiments, the biocleavable bond is cleavable upon exposure to cytoplasmic conditions, hi some embodiments, the biocleavable bond is reducible upon exposure to cytoplasmic conditions (e.g., intracellular compartments, particularly acidic pH conditions and / or containing reducing agents such as glutathione).

[0192] Biocleavable bonds are well known in the art and refer to bonds that are selectively cleaved (intracellular cleavage) after entering a cell. Preferred bonds for intracellular drug release are those that are cleavable in acidic conditions, such as those found in lysosomes. One example is a disulfide bond. Disulfide bonds are thought to be cleaved by glutathione upon cell entry. In some embodiments, the biocleavable bond is a bond that is cleaved intracellularly. In some embodiments, intracellular cleavage refers to cleavage inside the cell. In some embodiments, the inside of the cell is the cytoplasm of the cell. In some embodiments, the inside of the cell is in a vesicle of the cell. In some embodiments, the vesicle is an endosome. In some embodiments, the vesicle is a lysosome. In some embodiments, the vesicle is a Golgi vesicle. In some embodiments, the cleavage is selective cleavage. In some embodiments, selective refers to cleavage compared to extracellular cleavage. In some embodiments, extracellular is outside the cell. In some embodiments, the outside of the cell is in a biological fluid.

[0193] In some embodiments, the biocleavable bond is or comprises a disulfide bond. In some embodiments, the biocleavable bond comprises multiple disulfide bonds. In some embodiments, the biocleavable bond is sterically hindered. In some embodiments, the biocleavable bond is or comprises a sterically hindered disulfide bond.

[0194] In some embodiments, the biological fluid is a bodily fluid. In some embodiments, the biological fluid is selected from at least one of serum, plasma, gastric fluid, intestinal fluid, saliva, bile, tumor fluid, breast milk, urine, interstitial fluid, cerebrospinal fluid, and stool. In some embodiments, the biological fluid is blood. In some embodiments, the biological fluid is serum. In some embodiments, the biological fluid is plasma.

[0195] In some embodiments, the sterically hindered disulfide bond comprises a side chain or a bulky moiety adjacent thereto. In some embodiments, the side chain or bulky moiety is located adjacent to at least one sulfur atom of the disulfide bond. In some embodiments, adjacent or adjacent includes a distance ranging from 0 to 10, 0 to 2, 2 to 5, or 5 to 10 atom bonds, including any range therebetween. In some embodiments, the term "atom bond" as used herein refers to the length of a carbon-carbon (C-C) bond, e.g., the length of a single C-C bond.

[0196] In some embodiments, the sterically hindered disulfide bond comprises a side chain or a bulky moiety adjacent thereto (e.g., located at a distance ranging from 1-15 Å, 1-3 Å, 3-5 Å, 5-10 Å, or 10-15 Å from the sulfur atom of the disulfide bond).

[0197] In some embodiments, the side chain or bulky moiety is alkyl (e.g., primary, secondary, or tertiary C-C 10 In some embodiments, the side chain or bulky moiety is covalently bonded to a methylene group adjacent to the disulfide bond.

[0198] In some embodiments, the disulfide bond is located adjacent to the biological payload and / or protein carrier of the present invention, and adjacent is as described herein. In some embodiments, the biological payload and / or protein carrier of the present invention is linked to the linker via a disulfide bond. In some embodiments, the disulfide bond is proximal or adjacent to the protein carrier.

[0199] In some embodiments, the protein carrier of the present invention (e.g., HSA) is attached to the linker via a disulfide bond. In some embodiments, the HSA comprises the amino acid sequence of (SEQ ID NO: 1), or a fragment or homolog thereof. SEQ ID NO: 1 provides the sequence of HSA without the signal peptide. In some embodiments, the HSA comprises a signal peptide. In some embodiments, the HSA is a fragment of HSA. In some embodiments, the fragment comprises at least 50, 60, 70, 80, 90, 95, 99, or 100% of HSA. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fragment comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 amino acids from HSA. Each possibility represents a separate embodiment of the present invention.In some embodiments, the amino acids are consecutive amino acids. In some embodiments, HSA is a homolog of HSA. In some embodiments, the homolog of HSA comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention. In some embodiments, HSA comprises an amino acid sequence at least 70% identical to SEQ ID NO: 1. In some embodiments, HSA consists of SEQ ID NO: 1 or a fragment or homolog thereof. In some embodiments, HSA consists of an amino acid sequence at least 70% identical to SEQ ID NO: 1. In some embodiments, HSA consists of SEQ ID NO: 1. In some embodiments, HSA comprises a free cysteine. In some embodiments, the free cysteine ​​is cysteine ​​C34. In certain embodiments, the free cysteine ​​is only a single free cysteine. In some embodiments, a linker of the present invention is attached to C34 of HSA via a disulfide bond.

[0200] In some embodiments, the fibrinogen is fibrinogen alpha chain (FGA). In some embodiments, the FGA comprises the amino acid sequence of (SEQ ID NO: 2), or a fragment or homolog thereof. SEQ ID NO: 2 provides the sequence of fibrinogen without the signal peptide.In some embodiments, fibrinogen comprises a signal peptide. In some embodiments, the signal peptide comprises or consists of MFSMRIVCLVLSVVGTAWT (SEQ ID NO: 3). In some embodiments, FGA is a fragment of FGA. In some embodiments, the fragment comprises at least 50, 60, 70, 80, 90, 95, 99, or 100% of FGA. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fragment comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 850 amino acids from FGA. Each possibility represents a separate embodiment of the present invention. In some embodiments, the amino acids are consecutive amino acids. In some embodiments, FGA is a homolog of FGA. In some embodiments, homology is sequence identity. In some embodiments, a homolog of FGA comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% homology to SEQ ID NO:2. Each possibility represents a separate embodiment of the present invention. In some embodiments, FGA consists of SEQ ID NO:2 or a fragment or homolog thereof. In some embodiments, FGA consists of an amino acid sequence having at least 70% homology to SEQ ID NO:2. In some embodiments, FGA consists of SEQ ID NO:2.

[0201] In some embodiments, the fibrinogen is fibrinogen beta chain (FGB). In some embodiments, the FGB is QGVNDNEEGFFSARGHRPLDKKREEAPSLRPAPPPISGGGYRARPAKAAATQKKVERKAPDAGGCLHADPDLGVLCPTGCQLQEALLQQERPIRNSVDELNNNVEAVSQTSSSSFQYMYLLKDLWQKRQKQVKDNENVVNEYSSELEKHQLYIDETVNSNIPTNLRVLRSILENLRSKIQKLESDVSAQMEYCRTPCTVSCNIPVVSGKECEEIIRKGGETSEMYLIQPDSSVKPYRVYCD MNTENGGWTVIQNRQDGSVDFGRKWDPYKQGFGNVATNTDGKNYCGLPGEYWLGNDKISQLTRMGPTELLIEMEDWKGDKVKAHYGGFTVQNEANKYQISVNKYRGTAGNALMDGASQLMGENRTMTIHNGMFFSTYDRDNDGWLTSDPRKQCSKEDGGGWWYNRCHAANPNGRYYWGGQYTWDMAKHGTDDGVVWMNWKGSWYSMRKMSMKIRPFFPQQ (SEQ ID NO: 19), or a fragment or homolog thereof. SEQ ID NO: 19 provides the sequence of fibrinogen without the signal peptide. In some embodiments, fibrinogen includes a signal peptide. In some embodiments, the signal peptide comprises or consists of MKRMVSWSFHKLKTMKHLLLLLLCVFLVKS (SEQ ID NO: 18). In some embodiments, FGB is a fragment of FGB. In some embodiments, the fragment comprises at least 50, 60, 70, 80, 90, 95, 99, or 100% of FGB. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fragment comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 480 amino acids from FGB. Each possibility represents a separate embodiment of the present invention. In some embodiments, the amino acids are consecutive amino acids. In some embodiments, the FGB is a homolog of FGB.In some embodiments, homology is sequence identity. In some embodiments, a homolog of FGB comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% homology to SEQ ID NO: 19. Each possibility represents a separate embodiment of the present invention. In some embodiments, FGB consists of SEQ ID NO: 19 or a fragment or homolog thereof. In some embodiments, FGB consists of an amino acid sequence having at least 70% homology to SEQ ID NO: 19. In some embodiments, FGB consists of SEQ ID NO: 19.

[0202] In some embodiments, the fibrinogen is fibrinogen gamma chain (FGG). In some embodiments, FGG is YVATRDNCCILDERFGSYCPTTCGIADFLSTYQTKVDKDLQSLEDILHQVENKTSEVKQLIKAIQLTYNPDESSKPNMIDAATLKSRKMLEEIMKYEASILTHDSSIRYLQEIYNSNNQKIVNLKEKVAQLEAQCQEPCKDTVQIHDITGKDCQDIANKGAKQSGLYFIKPLKANQQFLVYCEIDGSGNGWTVFQKRLDGSVDFKKNWIQYKEGFGHLSPTGTT The fibrinogen fragment comprises the amino acid sequence of EFWLGNEKIHLISTQSAIPYALRVELEDWNGRTSTADYAMFKVGPEADKYRLTYAYFAGGDAGDAFDGFDFGDDPSDKFFTSHNGMQFSTWDNDNDKFEGNCAEQDGSGWWMNKCHAGHLNGVYYQGGTYSKASTPNGYDNGIIWATWKTRWYSMKKTTMKIIPFNRLTIGEGQQHHLGGAKQVRPEHPAETEYDSLYPEDDL (SEQ ID NO: 21), or a fragment or homolog thereof. SEQ ID NO: 21 provides the sequence of fibrinogen without the signal peptide. In some embodiments, the fibrinogen comprises a signal peptide. In some embodiments, the signal peptide comprises or consists of MSWSLHPRNLILYFYALLFLSSTCVA (SEQ ID NO: 20). In some embodiments, FGG is a fragment of FGG. In some embodiments, the fragment comprises at least 50, 60, 70, 80, 90, 95, 99, or 100% of FGG. Each possibility represents a separate embodiment of the present invention. In some embodiments, the fragment comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, or 480 amino acids from FGG. Each possibility represents a separate embodiment of the present invention. In some embodiments, the amino acids are consecutive amino acids. In some embodiments, FGG is a homolog of FGG. In some embodiments, the homology is sequence identity.In some embodiments, a homolog of FGG comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% homology to SEQ ID NO:21. Each possibility represents a separate embodiment of the present invention. In some embodiments, FGG consists of SEQ ID NO:21 or a fragment or homolog thereof. In some embodiments, FGG consists of an amino acid sequence having at least 70% homology to SEQ ID NO:21. In some embodiments, FGG consists of SEQ ID NO:21.

[0203] In some embodiments, the fibrinogen is a mixture of fibrinogen. In some embodiments, the mixture is a mixture of at least two of FGA, FGB, and FGG. In some embodiments, the mixture is a mixture of all three of FGA, FGB, and FGG. In some embodiments, the FGA, FGB, and FGG are in the ratio found in human blood. In some embodiments, the blood is plasma. Fibrinogen derived from human plasma is commercially available, such as from Sigma-Aldrich catalog number 341578. In some embodiments, the fibrinogen contains a free cysteine. In some embodiments, the fibrinogen contains a free lysine. Conjugation to fibrinogen can be performed as described herein or by any means known in the art. Conjugation can be random, as described herein, or site-specific.

[0204] In some embodiments, the linkers of the invention comprise linear or branched chains, hi some embodiments, the linkers of the invention are or comprise a backbone that optionally comprises one or more of the above chains.

[0205] In some embodiments, the linkers of the present invention are spacers (e.g., comprising natural and / or unnatural amino acids, alkyls, amide bonds, ester bonds, thioester bonds, urea bonds, and any derivatives or combinations thereof). In some embodiments, the linkers of the present invention comprise a biocompatible polymer or biocompatible moiety. In some embodiments, the biocompatible polymer is at least partially biodegradable. In some embodiments, the biocompatible polymer is or comprises a polyglycol ether, a polyester, a polyamide, a polyamino acid, a peptide, and / or a derivative thereof, or any combination thereof. In some embodiments, the polyglycol ether is or comprises polyethylene glycol (PEG). In some embodiments, the linkers of the present invention comprise PEG. In some embodiments, the linkers of the present invention comprise PEG characterized by an Mn of 100 to 5000 Da (including any range therebetween).

[0206] In some embodiments, the biocompatible moiety is or includes an amide, an ester, a glycol, an amino acid, or any combination thereof.

[0207] In some embodiments, the polyamino acid or derivative thereof comprises 2 to 50, 4 to 50, 5 to 50, 5 to 50, 4 to 20, 4 to 30, 4 to 40, 5 to 20, 5 to 30, 5 to 40, 6 to 50, 6 to 30, 6 to 40, 6 to 20, 8 to 50, 8 to 30, 8 to 20, 8 to 40 amino acids (including any range therebetween).

[0208] As used herein, the terms "peptide," "polypeptide," and "protein" encompass natural peptides, peptide derivatives such as beta-peptides, peptidomimetics (typically containing non-peptide bonds or other synthetic modifications), and peptide analogs peptoids and semipeptoids, or any combination thereof. In another embodiment, the terms "peptide," "polypeptide," and "protein" apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0209] The term "derivative" or "chemical derivative" includes any chemical derivative of a polypeptide having one or more chemically derivatized residues on side chains or any functional group within the peptide. Such derivatized molecules include, for example, peptides with one or more protecting groups (e.g., side chain protecting groups and / or N-terminal protecting groups) and / or peptides in which free amino groups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butoxycarbonyl groups, acetyl groups, or formyl groups. Free carboxyl groups can be derivatized to form amides, salts, methyl and ethyl esters, or other types of esters or hydrazides thereof. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-isobenzylhistidine. Also included as chemical derivatives are peptides containing derivatives of one or more naturally occurring amino acids of the 20 standard amino acid residues. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and Dab, Daa, and / or ornithine (O) may be substituted for lysine.

[0210] In addition, peptide derivatives may differ from the native sequences of the peptides of the invention by chemical modifications including, but not limited to, terminal -NH2 acylation, acetylation, or thioglycolic acid amidation, and by amidation of terminal and / or side chain carboxy groups with, for example, ammonia, methylamine, etc. Peptides may be either linear, cyclic, branched, etc., with any conformation that can be achieved using methods known in the art.

[0211] In some embodiments, the linkers of the invention further comprise a spacer (e.g., including natural and / or unnatural amino acids, alkyls, amide bonds, ester bonds, disulfide bonds, thioester bonds, urea bonds, and any derivatives or combinations thereof). In some embodiments, the linkers of the invention further comprise a disulfide bond. In some embodiments, the linkers of the invention comprise a Click reaction product (e.g., a cyclization reaction product formed via a Click reaction and / or a succinimide-thioether moiety).

[0212] Click reactions are well known in the art and include, among others, the Michael addition of maleimides and thiols (resulting in the formation of succinimide-thioethers); azide-alkyne cycloaddition; Diels-Alder reactions (e.g., direct and / or inverse electron demand Diels-Alder); dibenzylcyclooctyne 1,3-nitrone (or azide) cycloaddition; alkene-tetrazole photoclick reactions, and the like.

[0213] In some embodiments, the protein conjugate of the invention has Formula 1: [ka] wherein PC represents a protein carrier of the present invention (i.e., a masked protein carrier); BP represents a biological payload of the present invention; each r and m independently represents an integer in the range of 0 to 10 (including any range therebetween); l represents an integer in the range of 1 to 10 (including any range therebetween); and p represents an integer in the range of 2 to 100 (including any range therebetween); each R independently represents a bulky moiety or H; each A independently represents one or more linkers, each linker independently being selected from the group consisting of a heteroatom (e.g., O, N, NH, NR3, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NR3)NR3—, —C(NR3)O—, —C(NR3)S—), a C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10 a click reaction product comprising either a mercaptoalkyl, or any combination thereof, or A is absent.

[0214] In some embodiments, the protein conjugate of the invention has Formula 1: [ka] wherein PC represents a protein carrier (or charge-masked moiety) of the present invention; BP represents a biological payload of the present invention; each j, k, r, o, n, and m independently represent an integer in the range of 0 to 10 (including any range therebetween); l represents an integer in the range of 1 to 10 (including any range therebetween); and p represents an integer in the range of 2 to 100 (including any range therebetween); each R independently represents a bulky moiety or H; each X independently represents a heteroatom (e.g., O, N, NH, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NH)NH—, —C(NH)O—, —C(NH)S—), a spacer (e.g., C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10Alkoxy, C1-C 10 In some embodiments, at least one R is methyl.

[0215] In some embodiments, the click reaction product comprises a moiety formed via a click reaction, wherein the click reaction is as described herein above. In some embodiments, the click reaction product comprises a product formed by any of the following: Michael addition of maleimide and thiol (resulting in the formation of a succinimide-thioether); azide-alkyne cycloaddition; Diels-Alder reaction (e.g., direct and / or inverse electron demand Diels-Alder reaction); dibenzylcyclooctyne 1,3-nitrone (or azide) cycloaddition; alkene-tetrazole photoclick reaction; or any combination thereof.

[0216] In some embodiments, the protein conjugate of the invention has Formula 1A: [ka] wherein R, n, k, l, p, m, and r are as described herein, and each X independently represents a heteroatom (e.g., O, N, NH, or S), a spacer (e.g., C-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10 mercaptoalkyl, or click reaction product), or a combination thereof, or X is absent.

[0217] In some embodiments, the protein conjugate of the present invention has Formula A: [ka] wherein PC represents a protein carrier (or charge-masked moiety) of the present invention; BP represents a biological payload (i.e., payload) of the present invention; each r and m independently represent an integer ranging from 0 to 10 (including any range therebetween); each R3 independently represents a substituent or H, Het represents a heteroatom and is each independently selected from O, N, NH, and S; each A independently represents (i) a biocompatible moiety or a biocompatible polymer; and / or (ii) one or more linkers, each linker independently being selected from a heteroatom (e.g., O, N, NH, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NH)NH—, —C(NH)O—, —C(NH)S—), C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10 mercaptoalkyl, or a click reaction product comprising any combination thereof, or A is absent. In some embodiments, at least one of r or m is 1 to 10, 1 to 3, 1 to 5, or 1, 2, 3, 4, 5, 6, or 10 (including any ranges therebetween).

[0218] In some embodiments, the protein conjugate of the invention has Formula B: [ka] , or expression C: [ka] wherein PC represents a protein carrier of the present invention; BP represents a biological payload of the present invention; each r and m independently represents an integer ranging from 0 to 10 (including any range therebetween); p represents an integer ranging from 0 to 100 (including any range therebetween); Pol represents a biocompatible moiety or a biocompatible polymer; each R3 independently represents a substituent or H, Het represents a heteroatom and is each independently selected from O, N, NH, and S; each A independently represents one or more linkers, each linker independently being selected from a heteroatom (e.g., O, N, NH, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NH)NH—, —C(NH)O—, —C(NH)S—), C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10 or a click reaction product comprising any combination thereof, or A is absent. In some embodiments, the protein conjugate of the invention is represented by any one of Formulas A-C, wherein the linker has a bond length of at least 5, at least 10, at least 15, at least 20, at least 30, at least 5, at least 100, at least 300, at least 500, 5-500, 5-100, 10-100, 5-50, 50-100, or 100-500 atoms (including any range therebetween). In some embodiments, at least one p is 1-100, 1-20, 10-100, 2-20, 3-20, 3-15, 10-20, 20-50, 50-100, or 1, 2, 3, 4, 5, 6, 10, 11, 12, 15, or 20 (including any range therebetween).

[0219] In some embodiments, the protein conjugate of the invention has Formula 1: [ka] wherein PC, BP, and Pol are as described herein; each r and m independently represent an integer in the range of 0 to 10 (including any range therebetween); l represents an integer in the range of 1 to 10 (including any range therebetween); and p represents an integer in the range of 2 to 100 (including any range therebetween); each R independently represents a bulky moiety or H; each A independently represents one or more linkers, each linker independently being selected from the group consisting of a heteroatom (e.g., O, N, NR3, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NR3)NR3—, —C(NR3)O—, —C(NR3)S—), a C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10 a click reaction product comprising either a mercaptoalkyl, or any combination thereof, or A is absent.

[0220] In some embodiments, the protein conjugate of the invention has the formula: [ka] wherein PC, BP, Het, A, Pol, and p are as described herein; each r, r', m, and m' independently represent an integer ranging from 0 to 10 (including any range therebetween); each R3 and R3' independently represent one or more bulky moieties, one or more substituents, or H; and each X1 represents a click reaction product containing a heteroatom (e.g., O, N, NR3, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NR3)NR3—, —C(NR3)O—, —C(NR3)S—), or any combination thereof, and when Het is S, at least one X1 is S. In some embodiments, when Het and X1 are both S, at least one R3 is one or more bulky moieties; and at least one m' and r' is not 0.

[0221] In some embodiments, the protein conjugate of the invention has the formula: [ka] wherein PC, BP, Het, A, Pol, R, r, m, and p are as described herein, as permitted by valency. In some embodiments, when Het and X are both S, at least one R is one or more bulky moieties; and at least one m' and r' is not 0.

[0222] In some embodiments, the protein conjugate of the invention has the formula: [ka] wherein Het comprises S or NH, X represents a carbonyl derivative, a click reaction product, or a bond, and Pep represents a peptide. In some embodiments, the peptide is attached to the C-terminus of the BP. In some embodiments, Het is S and the peptide is attached to the PC via a cysteine ​​(e.g., a C-terminal cysteine).

[0223] In some embodiments, the protein conjugate of the invention has the formula: [ka] , [ka] wherein PC, BP, Pol are as described herein; each j, k, r, o, n, and m independently represent an integer in the range of 0 to 10 (including any range therebetween); l represents an integer in the range of 1 to 10 (including any range therebetween); and p represents an integer in the range of 2 to 100 (including any range therebetween); each R independently represents a bulky moiety or H; each X independently represents a heteroatom (e.g., O, N, NH, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NH)NH—, —C(NH)O—, —C(NH)S—), a spacer (e.g., C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10 In some embodiments, Pol represents a peptide, an amino acid or a dehydrated derivative thereof, PEG, or -CH2-CH2-O-. In some embodiments, a dehydrated derivative of an amino acid is: [ka] where the wavy bond represents the point of attachment to the linker or subsequent monomer, and R represents an amino acid side chain (optionally R and NH are interconnected to form a ring that results in deprotonation of the NH as in proline).

[0224] In some embodiments, the click reaction product comprises a moiety formed via a click reaction, wherein the click reaction is as described herein above. In some embodiments, the click reaction product comprises a product formed by any of the following: Michael addition of maleimide and thiol (resulting in the formation of a succinimide-thioether); azide-alkyne cycloaddition; Diels-Alder reaction (e.g., direct and / or inverse electron demand Diels-Alder reaction); dibenzylcyclooctyne 1,3-nitrone (or azide) cycloaddition; alkene-tetrazole photoclick reaction; or any combination thereof. In some embodiments, the click reaction product is a succinimide-thioether.

[0225] In some embodiments, the protein conjugate of the invention is represented by any of the formulas above, wherein X or X1 is a click reaction product, and optionally the click reaction product is a succinimide-thioether.

[0226] In some embodiments, the conjugate of the invention has the following formula 2: [ka] where Pol represents a biocompatible moiety or a biocompatible polymer (e.g., a peptide containing a C-terminal cysteine); A represents a spacer or a heteroatom (e.g., O, N, NH, or S), a carbonyl derivative (e.g., —C(O)NH—, —C(O)O—, —C(O)—, —C(O)S—, —C(NH)NH—, —C(NH)O—, —C(NH)S—), C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10or A is absent; alkyl is a C1-C aryl group optionally containing one or more of: (i) one or more heteroatoms; (ii) one or more carbonyl derivatives; (iii) one or more disulfide bonds; (iv) one or more click reaction products. 10 It is alkyl.

[0227] In some embodiments, the protein conjugate of the invention has Formula 1A: [ka] wherein R, n, k, l, p, m, Pol, and r are as described herein, and each X independently represents a heteroatom (e.g., O, N, NH, or S), a spacer (e.g., C1-C 10 Alkyl, C1-C 10 Aminoalkyl, C1-C 10 Alkoxy, C1-C 10 mercaptoalkyl, or click reaction product), or a combination thereof, or X is absent. In some embodiments, Pol represents an amino acid or a dehydrated derivative thereof, or -CH2-CH2-O-.

[0228] In some embodiments, the linker of the present invention is bound to HSA via a disulfide bond.In some embodiments, the linker of the present invention is covalently bound to the amino group or the thiol group of the biological payload of the present invention.In some embodiments, each HSA is bound to a single biological payload.In some embodiments, each HSA is bound to multiple biological payloads.

[0229] In some embodiments, the linker of the present invention is bound to HSA via a disulfide bond. In some embodiments, the linker of the present invention is covalently bound to an amino group or a thiol group of the biological payload of the present invention. In some embodiments, each HSA is bound to a single biological payload. In some embodiments, each HSA is bound to multiple biological payloads. Exemplary protein conjugates of the present invention are illustrated by Figure 9 and in the Examples section.

[0230] In some embodiments, the protein conjugates of the invention are substantially stable in biological fluids for at least 2 hours, at least 10 hours, at least 24 hours, at least 48 hours, at least 72 hours (including any range therebetween).

[0231] In some embodiments, at least 25%, at least 50%, at least 75%, at least 90% (including any range therebetween) of the protein conjugates of the invention are substantially stable.

[0232] As used herein, the term "stable" refers to the ability of a protein conjugate or linker of the invention to maintain (i) its chemical integrity (e.g., substantially lacking cleavage and / or deprotection), and (ii) its initial concentration and / or biological activity in the tissues and / or biological fluids of a subject.

[0233] In some embodiments, the protein conjugates of the invention and / or protein carriers of the invention are characterized by increased stability compared to a control (e.g., a similar protein conjugate or protein carrier lacking a protected amine). In some embodiments, the protein conjugates of the invention and / or protein carriers of the invention are characterized by increased stability in biological fluids and / or tissues (e.g., healthy tissues having a pH greater than 7) compared to a control, where increased is increased by at least 10%, at least 50%, at least 100%, at least 500%, at least 1000%, at least 10,000%, or more (including any range therebetween) compared to a control.

[0234] In some embodiments, the protein conjugates and / or protein carriers of the present invention are characterized by increased accumulation in target tissues having pH values ​​below 7, below 6.8, or below 6.5; increased being increased by at least 10%, at least 50%, at least 100%, at least 500%, at least 1,000%, at least 10,000%, or more (including any range therebetween) compared to a control (e.g., a similar protein conjugate or protein carrier lacking a protecting group).

[0235] In some embodiments, the target tissue comprises cancerous tissue, inflamed tissue, or both. In some embodiments, the target tissue comprises cancer. In some embodiments, the target tissue comprises inflammation. In some embodiments, the target cell is cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the target tissue is inflamed tissue.

[0236] Targeting section In some embodiments, the protein conjugate further comprises a targeting moiety. As used herein, the term "targeting moiety" refers to any molecule capable of specifically binding to a target antigen. In some embodiments, the targeting moiety binds to a protein expressed on the surface of a target cell. In some embodiments, the protein is a surface protein. In some embodiments, the protein is a receptor. In some embodiments, the protein is a cancer-specific antigen. In some embodiments, the protein is a surface marker for the target cell. In some embodiments, the target cell is a target cell type. In some embodiments, the cell type is a diseased cell type. In some embodiments, the binding is specific binding. In some embodiments, specific binding to a target is substantially free of binding to another target. In some embodiments, substantially means significantly. In some embodiments, substantially no binding is at most 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 7, or 10% binding to another target protein. Each possibility represents a separate embodiment of the present invention.

[0237] As used herein, the term "moiety" refers to a portion of a molecule that may include all or part of a functional group as a substructure. The term "moiety" also refers to a portion of a molecule that exhibits a particular set of chemical and / or pharmacological properties similar to the corresponding molecule. In this case, the property is binding to a target protein.

[0238] In some embodiments, the targeting moiety is an antigen-binding molecule. In some embodiments, the antigen-binding molecule is an antigen-binding molecule that binds a surface target. In some embodiments, the target is a protein. In some embodiments, the targeting moiety is selected from a single-chain antibody, a single-domain antibody, a variable heavy chain homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a designed ankyrin repeat protein (DARPin), and an antibody mimetic protein. In some embodiments, the targeting moiety is a VHH.

[0239] In some embodiments, the targeting moiety modulates the target protein. In some embodiments, modulating comprises activating the target protein. In some embodiments, modulating comprises inhibiting the target protein. In some embodiments, the targeting moiety is an agonist of the target protein. In some embodiments, the targeting moiety is an antagonist of the target protein.

[0240] In some embodiments, the targeting moiety is conjugated to a protein carrier. In some embodiments, the conjugated is conjugated by a linker. In some embodiments, the targeting moiety is conjugated to a biological payload. In some embodiments, the linker is a branched linker that conjugates the targeting moiety, the biological payload, and the protein carrier. In some embodiments, the targeting moiety and the biological payload are comprised in a single polypeptide. In some embodiments, the single polypeptide is a single chain. In some embodiments, the targeting moiety and the biological payload are separated by a linker. In some embodiments, the targeting moiety is N-terminal to the biological payload. In some embodiments, the targeting moiety is C-terminal to the biological payload. In some embodiments, the targeting moiety is at the N-terminus of the polypeptide. In some embodiments, the targeting moiety is at the C-terminus of the polypeptide. In some embodiments, the targeting moiety is separated from the C-terminus by a C-terminal cysteine ​​residue. In some embodiments, the targeting moiety is separated from the C-terminus by a C-terminal linker.

[0241] kit In another aspect, a kit is provided that includes a protein carrier covalently bound to a first moiety and a biological payload, wherein the first moiety is characterized by reactivity with the biological payload, and the protein carrier includes a charge-masked moiety of the present invention. In some embodiments, the biological payload is covalently bound to a second moiety, and the first moiety and the second moiety are reactive with each other (e.g., via a click reaction). In some embodiments, the kit further includes PG.

[0242] In some embodiments, the protein carrier bound to the first moiety has Formula 2: [ka] , or Formula 2A: [ka] wherein R, n, j, l, p, m, and r are as described herein; A is or includes a heteroatom selected from O, NR, and S; and R represents a first moiety.

[0243] In some embodiments, the biological payload covalently attached to the second moiety has Formula 3: [ka] , or Equation 4: [ka] wherein R, X, j, l, n, m, and p are as described herein; A is or includes a heteroatom selected from O, NR, and S; and R represents a second moiety.

[0244] In some embodiments, the first moiety or the second moiety is or includes a 1,3-nitrone, an azide, a diene, a tetrazine, an active ester (e.g., an ester, a pentafluorophenyl ester, an N-hydroxysuccinimide ester), an acyl halide, a chloroformate, an anhydride, an aldehyde, an epoxide, an isocyanate, an isothiocyanate, a maleimide, a carbonate, a sulfonyl chloride, an iodoacetamide, an acyl azide, an imido ester, a vinyl sulfone, an orthopyridyl disulfide, or any combination thereof.

[0245] In some embodiments, the first moiety or the second moiety is or includes a nucleophilic group (e.g., an amine, a thiol, a phosphine, a hydroxyl), a dienophile, an alkene, and an alkyne (e.g., an acetylene, a dibenzylcyclooctyne, etc.), or any combination thereof.

[0246] In some embodiments, the kits of the present invention comprise a biological payload covalently bound to a linker comprising a functional group reactive to HSA (e.g., to its cysteine ​​or lysine); and HSA. In some embodiments, the kits of the present invention comprise HSA covalently bound to a linker comprising a functional group reactive to the biological payload (e.g., to its cysteine ​​or lysine); and a biological payload. In some embodiments, the functional group is or comprises iodoacetamide, active ester, orthopyridyl disulfide, maleimide, or a combination thereof.

[0247] In some embodiments, the conjugate is a blood-stable conjugate. In some embodiments, the conjugate is a cell-penetrating conjugate. In some embodiments, the conjugate is a masked conjugate. In some embodiments, the conjugate is a maskable conjugate. In some embodiments, the conjugate is a cell membrane-crossing conjugate. In some embodiments, the conjugate is capable of entering cells. In some embodiments, the conjugate is capable of endosomal escape. In some embodiments, the conjugate is capable of intracellular delivery of a payload. In some embodiments, the intracellular delivery is cytoplasmic delivery. In some embodiments, the intracellular delivery comprises dissociation of the carrier from the payload. In some embodiments, the conjugate is configured to dissociate in the cytoplasm. In some embodiments, the dissociation is dissociation of the carrier from the payload. In some embodiments, the conjugate is for use in modulating an intracellular target. In some embodiments, the conjugate is for use in effecting a change in an intracellular target. In some embodiments, the conjugate is for use in interacting with an intracellular target.

[0248] method In another aspect, a method for producing a charge-masked protein conjugate is provided, the method comprising: providing a biological agent that binds an intracellular target to obtain a charge-masked protein conjugate comprising protected amine groups; providing a protein carrier covalently attached to a cell-penetrating moiety, the cell-penetrating moiety comprising a plurality of amine groups; providing a biological payload and the protein carrier under conditions sufficient to covalently attach the biological payload to the protein carrier via a linker to produce the protein conjugate; and providing the protein carrier under conditions sufficient to protect at least a portion of the amine groups with protecting groups that are susceptible to cleavage (deprotection) at a pH value below 7.

[0249] In some embodiments, the method further includes determining the stability of the linker in biological fluids and under cytoplasmic conditions; and selecting a charge-masked protein conjugate comprising a linker that is stable in biological fluids and unstable under cytoplasmic conditions, thereby producing a charge-masked protein conjugate capable of binding to an intracellular target. In some embodiments, the step of protecting at least a portion of the amine groups is performed (i) before performing the step of producing the protein conjugate; or (ii) after the step of producing the protein conjugate. In some embodiments, providing the protein carrier under conditions sufficient for protecting occurs before coupling a biological payload to the protein carrier. In some embodiments, providing the protein carrier under conditions sufficient for protecting occurs after coupling a biological payload to the protein carrier. In some embodiments, providing is providing an unlinked protein carrier. In some embodiments, providing is providing a protein conjugate. Those skilled in the art will understand that when a protein conjugate is protected, basic residues on the payload and linker are also protected, and therefore the entire conjugate is protected. In some embodiments, the method is for producing a charge-masked protein conjugate of the present invention. In some embodiments, the terms "charge-masked protein conjugate" and "protein conjugate" are used interchangeably herein. In some embodiments, selecting is selecting a charge-masked protein conjugate that is more stable in biological fluids than in cytoplasmic conditions. In some embodiments, more stable is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 250, 300, 350, 400, 450, or 500% greater stability. Each possibility represents a separate embodiment of the present invention.

[0250] In another aspect, a method for producing a charge-masked protein conjugate is provided, the method comprising: providing a biological agent that binds an intracellular target; providing a protein carrier covalently attached to a cell-penetrating moiety, where the cell-penetrating moiety comprises a plurality of amine groups, and then providing the protein conjugate under conditions sufficient to protect at least a portion of the amine groups with protecting groups that are susceptible to cleavage (deprotection) at a pH value less than 7, to obtain a masked protein carrier comprising the protected amine groups; providing the biological payload and the masked protein carrier under conditions sufficient to covalently attach the biological payload to the protein carrier via a linker.

[0251] In another aspect, a method of producing a charge-masked protein conjugate is provided, the method comprising: providing a biological agent that binds an intracellular target; providing a masked protein carrier covalently attached to a cell-penetrating moiety, the cell-penetrating moiety comprising a plurality of protected amine groups; and providing the biological payload and the protein carrier under conditions sufficient to covalently attach the biological payload to the protein carrier via a linker comprising at least one biocleavable bond to produce the protein conjugate.

[0252] In another aspect, a method for producing a charge-masked protein is provided, the method comprising: providing a biological agent that binds an intracellular target; attaching the biological to a cell-penetrating moiety; and providing the biological attached to the cell-penetrating moiety under conditions sufficient to protect at least a portion of the cell-penetrating moiety. In some embodiments, the cell-penetrating moiety comprises a plurality of amine groups. In some embodiments, at least a portion of the amine groups are protected. In some embodiments, the protecting group is capable of cleavage (deprotection) at a pH value of less than 7.

[0253] In another aspect, a method of producing a charge-masked protein is provided, the method comprising: providing a biological agent that binds an intracellular target; and conjugating the biological agent to a cell-penetrating moiety, wherein the cell-penetrating moiety comprises a plurality of protected amine groups.

[0254] In some embodiments, the charge-masked protein conjugate is capable of binding to an intracellular target. In some embodiments, the charge-masked protein conjugate is capable of entering the cytoplasm of a cell. In some embodiments, the charge-masked protein conjugate is capable of intracellular delivery of a biologic. In some embodiments, the charge-masked protein conjugate enables intracellular delivery of a biologic. In some embodiments, the charge-masked protein conjugate is capable of modulating an intracellular target. In some embodiments, the charge-masked protein conjugate is configured to modulate an intracellular target. In some embodiments, the biologic lacks a disulfide bond. In some embodiments, the biologic lacks a disulfide bond required for the structure of the biologic. In some embodiments, the biologic lacks a disulfide bond required for the function of the biologic. In some embodiments, the biologic lacks a disulfide bond required for binding of the biologic. In some embodiments, the biologic lacks a disulfide bond that, when cleaved, reduces the binding. In some embodiments, the binding is to an intracellular target.

[0255] In some embodiments, the charge-masked protein conjugate is a therapeutic agent. In some embodiments, the therapeutic agent is a biological therapeutic agent. In some embodiments, the therapeutic agent is a biologic. In some embodiments, the therapeutic agent is an agent against an intracellular target. In some embodiments, the therapeutic agent targets an intracellular target. In some embodiments, the therapeutic agent modulates an intracellular target.

[0256] In some embodiments, there are methods for synthesizing the charge-masked protein conjugates of the invention. In some embodiments, the methods include providing a kit of the invention and reacting a first moiety with a second moiety (e.g., under suitable conditions, optionally including a metal-based catalyst and / or UV irradiation, thermal irradiation).

[0257] In some embodiments, a method for synthesizing a charge-masked protein conjugate of the present invention includes: (i) providing a protein carrier covalently bound to a first moiety and a biological payload covalently bound to a second moiety, wherein the first moiety and the second moiety are reactive with each other (e.g., via a click reaction, via thiol-maleimide bond formation, via amine to active ester coupling, via S—S bond formation, such as the reaction of SPDP or nitro-SPDP with a thiol); (ii) providing the protein carrier and the biological payload under conditions suitable for reaction between the first moiety and the second moiety, thereby synthesizing a protein conjugate; and (iii) reacting the protein conjugate with a PG precursor under conditions suitable for subsequent protection of at least some of the amines with PG, thereby obtaining a charge-masked protein conjugate of the present invention.

[0258] In another aspect, a method for producing a charge-masked protein conjugate is provided, the method comprising: (i) providing a protein carrier covalently attached to a first moiety and a biological payload covalently attached to a second moiety; and (ii) providing the masked protein carrier and the biological payload under conditions suitable for reaction, thereby synthesizing a protein conjugate, wherein the masked protein carrier is synthesized by reacting the protein conjugate with a PG precursor under conditions suitable for protecting at least some of the amines with PG.

[0259] In some embodiments, suitable conditions for protecting include reaction conditions suitable for reacting the amine with a PG precursor, thereby yielding a protected amine. In some embodiments, suitable conditions for protecting include a neutral or basic pH; a temperature of at least -10°C, at least 0°C, at least 10°C, at least 20°C, or at least 50°C (including any range therebetween); and 90 and at least 10, at least 20, at least 50, at least 100, at least 300, or at least 500 molar equivalents of PG precursor relative to the protein conjugate (including any range therebetween).

[0260] In some embodiments, steps (i)-(iii) of the method are carried out in a solution (eg, an organic solvent, an aqueous solvent, or a combination thereof). In some embodiments, the first and second moieties are as described herein. In some embodiments, the reaction comprises a Click reaction.

[0261] In some embodiments, the method includes: (i) providing a protein carrier and a biological payload covalently attached to a linker comprising a functional group reactive to the biological payload (e.g., to its cysteines or lysines); (ii) reacting the functional group with the biological payload, thereby synthesizing a protein conjugate; and (iii) reacting the protein conjugate with a PG precursor to obtain a charge-masked protein conjugate.

[0262] In some embodiments, the method includes: (i) providing a biological payload and a protein carrier covalently attached to a linker comprising a functional group reactive to the protein carrier (e.g., to a cysteine ​​or lysine thereof); (ii) reacting the functional group with the protein carrier, thereby synthesizing a protein conjugate; and (iii) protecting the amine of the cell-penetrating moiety with PG under conditions described herein to obtain a charge-masked protein conjugate of the invention.

[0263] In some embodiments, the method includes testing the cell penetration of the masked conjugate. In some embodiments, the method includes testing the function of the biological payload upon delivery to the target cell. In some embodiments, the method includes testing the biodistribution of the masked conjugate. In some embodiments, the method includes testing the in vivo function of the biological payload in the target cell. In some embodiments, the method includes determining the stability of the linker in biological fluids and cytoplasmic conditions. In some embodiments, the method includes selecting a charge-masked protein conjugate including a linker that is stable in biological fluids and unstable in cytoplasmic conditions. In some embodiments, the method includes measuring the stability of the protected amine group at biological pH and acidic pH. In some embodiments, the biological pH is neutral pH. In some embodiments, the biological pH is neutral or basic pH. In some embodiments, the biological pH is a pH of about 7.4. In some embodiments, the acidic pH is a pH of about 6.8. In some embodiments, the acidic pH is a pH less than 7. In some embodiments, the acidic pH is a pH of 6.8 or less. In some embodiments, the method comprises selecting a charge-masked protein conjugate comprising a protected amine group that is stable at biological pH and unstable at acidic pH. In some embodiments, the method comprises selecting a protecting group that is cleaved (deprotected) at a pH below 7. In some embodiments, the method comprises selecting a biologic that binds to an intracellular target. In some embodiments, the selecting comprises determining or measuring the biologic that binds to the intracellular target. Methods for performing such tests are provided hereinafter, and any such test can be performed.

[0264] In some embodiments, the method further comprises selecting a targeting moiety. In some embodiments, the method further comprises selecting a moiety that binds to a protein of interest on the surface of the target cell. In some embodiments, the target cell is a cell of interest. In some embodiments, the target cell is a diseased cell. In some embodiments, the method comprises conjugating the selected targeting moiety to a biological payload. In some embodiments, the method comprises conjugating the selected targeting moiety to a protein carrier. In some embodiments, the method comprises conjugating the selected targeting moiety to a masked protein carrier. In some embodiments, the method comprises conjugating the selected targeting moiety to a protein conjugate. In some embodiments, the method comprises conjugating the selected targeting moiety to a masked protein conjugate. In some embodiments, the conjugating is via a linker. In some embodiments, the conjugating is constructing a single polypeptide comprising the targeting moiety and the biological payload. In some embodiments, the constructing includes inserting the targeting moiety into the biological payload.

[0265] In some embodiments, the method comprises testing binding of the targeting moiety to a target protein. In some embodiments, the method comprises testing binding of a masked protein conjugate to a target protein. In some embodiments, the target protein is to a cell that expresses the target protein on its surface. In some embodiments, testing comprises testing for specific binding. In some embodiments, testing comprises testing for lack of binding to a cell that does not contain the target protein on its surface. Methods for performing such tests are provided hereinafter, and any such test can be performed.

[0266] In another aspect, there is provided a protein conjugate produced by the method of the invention. In another aspect, there is provided a protein produced by the method of the invention.

[0267] composition In another aspect, a pharmaceutical composition comprising a protein conjugate of the invention is provided. In another aspect, a pharmaceutical composition comprising a protein of the invention is provided.

[0268] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or adjuvant. As used herein, the terms "carrier," "excipient," or "adjuvant" refer to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium such as physiological saline. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose, glycols such as propylene glycol, polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol, phosphate buffer, and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can function as carriers herein include sugar, stearic acid, magnesium stearate, calcium sulfate, polyol, pyrogen-free water, isotonic saline, phosphate buffer, and other non-toxic, pharmaceutically acceptable substances used in other pharmaceutical preparations.Wetting agents and lubricants such as sodium lauryl sulfate, as well as excipients, stabilizers, antioxidants, and preservatives may also be present.Any non-toxic, inert, and effective carrier can be used to formulate the compositions contemplated herein. Carriers in total may comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0269] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a protein conjugate of the present invention. The term "therapeutically effective amount" refers to an amount of drug effective to treat a disease or disorder in a mammal. The term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The exact dosage form and administration regimen will be determined by a physician depending on the patient's condition.

[0270] In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for administration to a subject.

[0271] In some embodiments, the pharmaceutical composition is a sustained release composition. In some embodiments, the linker lacks a biocleavable bond, and the composition is a sustained release composition. In some embodiments, the sustained release comprises payload delivery for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration. Each possibility represents a separate embodiment of the present invention. In some embodiments, the sustained release comprises payload delivery for at least 1 day after administration. In some embodiments, the sustained release comprises payload delivery for at least 3 days after administration. In some embodiments, the sustained release comprises payload delivery for at least 5 days after administration.

[0272] In some embodiments, certain compounds of the present invention may exist in free form for therapy or as a pharmaceutically acceptable salt.

[0273] As used herein, the term "pharmaceutically acceptable salt" refers to any non-toxic salt of a compound of the present invention, which, upon administration to a subject, e.g., a human, is capable of providing, directly or indirectly, a compound of the present invention or an inhibitory active metabolite or residue thereof. For example, the term "pharmaceutically acceptable" can mean approved by a federal or state regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0274] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds of the present invention. Acid addition salts can be prepared by 1) reacting the purified compound in free base form with a suitable organic or inorganic acid, and 2) isolating the salt thus formed.

[0275] Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, acetate, aspartate, benzenesulfonate, benzoate, bicarbonate, carbonate, halide (bromide, chloride, iodide, fluoride, etc.), bitartrate, citrate, salicylate, stearate, succinate, sulfate, tartrate, decanoate, edetate, fumarate, gluconate, and lactate salts, or any combination thereof.

[0276] Additional examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange.

[0277] Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxybenzoate, and 2-hydroxybenzoate. These salts include ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.

[0278] Base addition salts can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base, and 2) isolating the salt thus formed. Salts derived from appropriate bases include alkali metals (e.g., sodium, lithium, and potassium), alkaline earth metals (e.g., magnesium and calcium), ammonium, and N + (C 1-4 This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0279] Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed through the use of counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates. Other acids and bases, while not themselves pharmaceutically acceptable, can be employed in the preparation of salts which are useful as intermediates to obtain the compounds of the present invention and their pharmaceutically acceptable acid or base addition salts.

[0280] In some embodiments, the term "one or more" refers to any number selected from 1, 2, 3, 4, 5, or 6. In some embodiments, heteroatoms include any of N, O, NH, or S.

[0281] In some embodiments, the compounds described herein are chiral compounds (i.e., have asymmetric carbon atoms). In some embodiments, diastereomers, geometric isomers, and individual isomers are encompassed within the scope of the present invention. In some embodiments, the chiral compounds described herein are in the form of a racemic mixture. In some embodiments, the chiral compounds are in the form of a single enantiomer containing an asymmetric carbon atom having the R configuration. In some embodiments, the chiral compounds are in the form of a single enantiomer containing an asymmetric carbon atom having the S configuration described herein above.

[0282] In some embodiments, the chiral compound is in the form of a single enantiomer having an enantiomeric purity of greater than 70%. In some embodiments, the chiral compound is in the form of a single enantiomer having an enantiomeric purity of greater than 80%. In some embodiments, the chiral compound is in the form of a single enantiomer having an enantiomeric purity of greater than 90%. In some embodiments, the chiral compound is in the form of a single enantiomer having an enantiomeric purity of greater than 95%.

[0283] In some embodiments, compounds of the invention that contain unsaturated bonds are in the form of trans or cis isomers, hi some embodiments, compositions of the invention comprise a mixture of cis and trans isomers as described hereinabove.

[0284] In some embodiments, the compounds described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are included within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be included within the scope of the present invention.

[0285] The term "solvate" refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, etc.) that forms with a solute (a conjugate as described herein) and a solvent whereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid, and the like. The term "hydrate" refers to a solvate, as defined herein above, wherein the solvent is water.

[0286] Unless otherwise specified, structures depicted herein are intended to include all isomeric (e.g., enantiomeric, diastereomeric, geometric, and rotamer) forms of the structure. For example, R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in the present invention. As will be understood by one of ordinary skill in the art, substituents can freely rotate about any rotatable bond. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric, conformational, and rotamer mixtures of the present compounds are within the scope of the invention.

[0287] Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0288] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, the replacement of hydrogen by deuterium or tritium, or 18 Substitution of hydrogen by F, or 13 C or 14 Compounds having the present structures except for the replacement of a carbon by a C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as imaging probes.

[0289] How to use In another aspect, a method of binding an intracellular target is provided, the method comprising contacting a cell expressing the intracellular target with a protein conjugate of the invention, a protein of the invention, or a pharmaceutical composition of the invention, thereby binding the intracellular target.

[0290] In some embodiments, the method is a method of modulating an intracellular target. In some embodiments, the biological payload binds an intracellular target. In some embodiments, the biological payload modulates an intracellular target. In some embodiments, modifying is agonizing. In some embodiments, the biological payload is an agonist of the intracellular target. In some embodiments, modifying is antagonizing. In some embodiments, the biological payload is an antagonist. Modulating (i.e., antagonizing or agonizing) molecules are well known in the art, and any such molecule may be employed. In some embodiments, the biological target is specific for an intracellular target.

[0291] In some embodiments, the method is a method for detecting an intracellular target. In some embodiments, the protein conjugate comprises a detectable tag. In some embodiments, the tag is a detectable moiety. In some embodiments, the method further comprises detecting the protein conjugate. In some embodiments, the method further comprises detecting the detectable tag. In some embodiments, the detectable tag is a fluorescent tag. Detectable tags and moieties are well known in the art, and non-limiting examples include fluorophores (e.g., GFP, RFP, YFP, luciferase, etc.), radioactive tags, and colored tags. Any such known tag may be employed.

[0292] In some embodiments, the cell is in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is human. In some embodiments, the subject is suffering from a disease or condition. In some embodiments, the disease or condition is treatable by contacting an intracellular target. In some embodiments, the disease or condition is treatable by modulating an intracellular target. In some embodiments, the disease or condition is treatable by agonizing an intracellular target. In some embodiments, the disease or condition is treatable by antagonizing an intracellular target. In some embodiments, the subject is in need of modulating an intracellular target. In some embodiments, the subject is in need of treatment. In some embodiments, the subject is a subject in need thereof.

[0293] In some embodiments, the methods comprise administering to a subject a protein conjugate of the invention. In some embodiments, the methods comprise administering to a subject a pharmaceutical composition of the invention.

[0294] As used herein, the terms "administering," "administration," and the like refer to any method, in sound medical practice, of delivering a composition comprising an active agent to a subject to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, intratumoral, or intraperitoneal.

[0295] The dosage administered will depend upon the age, health, and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0296] In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is inflammation. In some embodiments, the disease or condition is ischemia. In some embodiments, the intracellular target is an oncogene and the biological payload is an antagonist. In some embodiments, the intracellular target is a tumor suppressor and the biological payload is an agonist.

[0297] In some embodiments, the contacting is not in the presence of an agent designed to induce penetration of the protein conjugate into cells. In some embodiments, the agent designed to induce penetration is an agent other than a carrier protein. In some embodiments, no other method of inducing cell penetration other than the methods of the present invention is employed. In some embodiments, the contacting continues for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration. Each possibility represents a separate embodiment of the present invention. In some embodiments, the contacting continues for at least 1 day after administration. In some embodiments, the contacting continues for at least 3 days after administration. In some embodiments, the contacting continues for at least 5 days after administration. In some embodiments, the sustained contact after administration is a sustained release of the payload / therapeutic agent.

[0298] In some embodiments, cells of a disease or condition express a target protein. In some embodiments, a protein conjugate comprising a targeting moiety is used to treat a disease or condition characterized by the expression of a target protein on cells of the disease or condition. In some embodiments, the target protein is a marker for the disease or condition. In some embodiments, the disease is cancer, and the target protein is a cancer-specific antigen. Cancer-specific antigens and antigen-binding molecules that bind them are well known in the art, and any such molecule can be used in the methods of the present invention. In some embodiments, the cancer-specific antigen is prostate-specific membrane antigen (PSMA).

[0299] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon, including straight-chain and branched-chain groups, typically containing 1 to 30, or 1 to 10, carbon atoms. As used herein, the term "alkyl" also encompasses saturated or unsaturated hydrocarbons, and thus the term further encompasses alkenyl and alkynyl.

[0300] The term "alkenyl" refers to an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be unsubstituted or substituted with one or more substituents, as described herein above.

[0301] The term "alkynyl," as defined herein, refers to an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be unsubstituted or substituted with one or more substituents, as described herein above.

[0302] The term "cycloalkyl" refers to an all-carbon monocyclic or fused ring (i.e., rings that share adjacent pairs of carbon atoms) group in which one or more rings do not have a completely conjugated pi-electron system. Cycloalkyl groups can be substituted or unsubstituted as indicated herein.

[0303] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a completely conjugated π-electron system. Aryl groups can be substituted or unsubstituted as indicated herein.

[0304] The term "alkoxy" refers to both an -O-alkyl and an -O-cycloalkyl group, as defined herein. The term "aryloxy" refers to an -O-aryl group, as defined herein.

[0305] As used herein, the alkyl, cycloalkyl, and aryl groups in the general formulas may be substituted with one or more substituents, whereby each substituent may independently be, for example, halide, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxyl, thiol, thioalkoxy, carboxy, amido, aryl, and aryloxy, depending on the substituent and its position in the molecule. Additional substituents are also contemplated.

[0306] The terms "halide," "halogen," or "halo" refer to fluorine, chlorine, bromine, and iodine. The term "haloalkyl," as used herein, refers to an alkyl group, as defined herein, further substituted with one or more halides. The term "haloalkoxy," as used herein, refers to an alkoxy group, as defined herein, further substituted with one or more halides. The term "hydroxyl" or "hydroxy," as used herein, refers to an -OH group. The term "mercapto" or "thiol" refers to an -SH group. The term "thioalkoxy" refers to both an -S-alkyl group and an -S-cycloalkyl group, as defined herein. The term "thioaryloxy" refers to both an -S-aryl and an -S-heteroaryl group, as defined herein. The term "amino" refers to an -NR'R" group or a salt thereof, where R' and R" are as defined herein.

[0307] The term "heterocyclyl" refers to a monocyclic or fused ring group containing one or more atoms such as nitrogen, oxygen, and sulfur in the ring. The ring may also have one or more double bonds. However, the ring does not have a completely conjugated pi-electron system. Representative examples include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, and the like.

[0308] The term "carboxy" refers to the group -C(O)OR', or a carboxylate thereof, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon), or heterocyclyl (bonded through a ring carbon), or "carboxylate," as defined herein.

[0309] The term "carbonyl" refers to the group -C(O)R', where R' is as defined hereinabove. The above terms also include their thio derivatives (thiocarboxy and thiocarbonyl).

[0310] The term "thiocarbonyl" refers to a -C(S)R' group, where R' is as defined herein above. The term "thiocarboxy" refers to a -C(S)OR' group, where R' is as defined herein. The term "sulfinyl" refers to a -S(S)R' group, where R' is as defined herein. The term "sulfonyl" or "sulfonic acid" refers to a -S(O)R' group, where R' is as defined herein.

[0311] The term "carbamyl" or "carbamic acid" group refers to an -O-C(O)NR'R" group, where R' is as defined herein and R" is as defined for R'. A "nitro" group refers to an -NO2 group. As used herein, the term "amido" includes C-amido and N-amido. The term "C-amido" refers to a -C(O)NR'R" terminal group or a -C(O)NR' linking group, where these terms are as defined herein above and R' and R" are as defined herein. The term "N-amido" refers to a -NR"C(O)R' terminal group or a -NR'C(O)- linking group, where these terms are as defined herein above and R' and R" are as defined herein.

[0312] The term "cyano" or "nitrile" refers to a -CN group. The terms "azo" or "diazo" refer to an -N=NR' terminal group or an -N=N- linking group, as these terms are defined herein above, and R' is as defined herein above. The term "guanidine" refers to an -R'NC(N)NR"R"' terminal group or an -R'NC(N)NR"- linking group, as these terms are defined herein above, and R', R" and R"' are as defined herein. As used herein, the term "azido" refers to an -N3 group. The term "sulfonamide" refers to a -S(O)2NR'R" group, as R' and R" are as defined herein.

[0313] The term "phosphonyl" or "phosphonic acid" group refers to the group -OP(O)-(OR')2, where R' is as defined herein above. The term "phosphinyl" refers to the group -PR'R" where R' and R" are as defined herein above. The term "alkylaryl" refers to an alkyl, as defined herein, substituted with an aryl, as described herein. An exemplary alkylaryl is benzyl.

[0314] The term "heteroaryl" refers to a monocyclic or fused ring (i.e., rings sharing adjacent pairs of atoms) group having one or more atoms in the ring, such as, for example, nitrogen, oxygen, and sulfur, and, in addition, a fully conjugated π-electron system. As used herein, the term "heteroaryl" refers to an aromatic ring in which at least one atom forming the aromatic ring is a heteroatom. Heteroaryl rings can be formed from 3, 4, 5, 6, 7, 8, 9, and more than 9 atoms. Heteroaryl groups may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C groups containing one oxygen or sulfur atom, or two oxygen atoms, or two sulfur atoms, or up to four nitrogen atoms, or combinations of one oxygen or sulfur atom and up to two nitrogen atoms, and substitutions thereof, as well as benzo- and pyrido-fused derivatives, for example, linked through one ring-forming carbon atom. 3-8 In certain embodiments, heteroaryl is selected from oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrimidinal, pyrazinyl, indolyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl.

[0315] In some embodiments, the heteroaryl group is selected from the group consisting of pyrrolyl, furanyl (furyl), thiophenyl (thienyl), imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3-oxazolyl (oxazolyl), 1,2-oxazolyl (isoxazolyl), oxadiazolyl, 1,3-thiazolyl (thiazolyl), 1,2-thiazolyl (isothiazolyl), tetrazolyl, pyridinyl (pyridyl), pyridazolyl. and n is 0, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, indazolyl, indolyl, benzothiophenyl, benzofuranyl, benzothiazolyl, benzimidazolyl, benzodioxolyl, acridinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, thienothiophenyl, 1,8-naphthyridinyl, tetrahydronaphthyridinyl, pteridinyl, or phenothiazinyl. When the heteroaryl group contains more than one ring, each additional ring is saturated (perhydro), partially unsaturated (e.g., dihydro or tetrahydro), or maximally unsaturated (non-aromatic). The term heteroaryl thus includes bicyclic radicals in which two rings are aromatic, and bicyclic radicals in which only one ring is aromatic. Examples of such heteroaryls are 3H-indolinyl, 2(1H)quinolinonyl, 4-oxo-1,4-dihydroquinolinyl, 2H-1-oxoisoquinolyl, 1,2-dihydroquinolinyl, (2H)quinolinyl. N-oxide, 3,4-dihydroquinolinyl, 1,2-dihydroisoquinolinyl, 3,4-dihydroisoquinolinyl, chromonyl, 3,4-dihydroiso-quinoxalinyl, 4-(3H)quinazolinonyl, 4H-chromenyl, 4-chromanonyl, oxindolyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydro-quinolinyl, 1H-2,3-dihydroisoindolyl, 2,3-dihydrobenzo[f]isoindolyl, 1,2,3,4-tetrahydrobenzo[g]isoquinolinyl, 1,2,3,4-tetrahydro-benzo[g]isoquinolinyl, chromanyl, isochromanonyl, 2,3-dihydrochromonyl, 1,4-benzodioxanyl, 1,2,3,4-Tetrahydroquinoxalinyl, 5,6-dihydroquinolyl, 5,6-dihydroisoquinolyl, 5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-1H-benzimidazolyl, 4,5-dihydrobenzoxazolyl, 1,4-naphthoquinolyl, 5,6,7,8-tetrahydroquinolinyl, 5,6,7,8-tetrahydroisoquinolyl, 5,6,7,8-tetrahydroquinoxalinyl, 5,6,7,8-tetrahydroquinazolyl, 4,5,6,7-tetrahydro-1H-benzimidazolyl, 4,5, 6,7-Tetrahydrobenzoxazolyl, 1H-4-oxa-1,5-diazanaphthalen-2-onyl, 1,3-dihydroimidizolo[4,5]pyridin-2-onyl, 2,3-dihydro-1,4-dinaphthoquinonyl, 2,3-dihydro-1H-pyrrole[3,4-b]quinolinyl, 1,2,3,4-tetrahydrobenzo[b][1,7]naphthyridinyl, 1,2,3,4-tetrahydrobenzo[b][1,6]naphthyridinyl, 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indolyl, 1,2,3,4-tetrahydrobenzo[b][1,7]naphthyridinyl 9H-pyrido[4,3-b]indolyl, 2,3-dihydro-1H-pyrrolo[3,4-b]indolyl, 1H-2,3,4,5-tetrahydroazepino[3,4-b]indolyl, 1H-2,3,4,5-tetrahydroazepino[4,3-b]indolyl, 1H-2,3,4,5-tetrahydroazepino[4,5-b]indolyl, 5,6,7,8-tetrahydro[1,7]napthyridinyl, 1,2,3,4-tetrahydro[2,7]naphthyridyl, 2,3-dihydro[1,4]dioxino[2,3-b]pyridyl, 2,3-dihydro b[1,4]-dioxino[2,3-b]pyridyl, 3,4-dihydro-2H-1-oxa[4,6]diazanaphthalenyl, 4,5,6,7-tetrahydro-3H-imidazo-[4,5-c]pyridyl, 6,7-dihydro[5,8]diazanaphthalenyl, 1,2,3,4-tetrahydro[1,5]napthyridinyl, 1,2,3,4-tetrahydro[1,6]napthyridinyl, 1,2,3,4-tetrahydro[1,7]napthyridinyl, 1,2,3,4-tetrahydro[1,8]napthyridinyl or 1,2,3,4-tetrahydro[2,6] napthyridinyl. In some embodiments, the heteroaryl group is optionally substituted. In one embodiment, one or more substituents are halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, C, 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, C 1-6 are each independently selected from alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl.

[0316] Examples of heteroaryl groups include, but are not limited to, unsubstituted and mono- or di-substituted derivatives of furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline and quinoxaline. In some embodiments, the substituents are halo, hydroxy, cyano, OC 1-6 -Alkyl, C 1-6 -Alkyl, Hydroxy-C 1-6 -Alkyl and Amino-C 1-6 - alkyl.

[0317] As used herein, the terms "halo" and "halide" are used interchangeably herein and refer to an atom of the halogen, which is fluorine, chlorine, bromine, or iodine, also referred to herein as fluoride, chloride, bromide, or iodide.

[0318] In some embodiments, the term "substituted" encompasses one or more substituents covalently attached to a functional group and / or molecule. In some embodiments, a substituent is C1-C6 alkyl, halo, -NO2, CN, -OH, -NH2, carbonyl, -CONH2, -CONR'2, -CNNR2, -CSNR2, -CONH-OH, -CONH-NH2, -NHCOR', -NHCSR', -NHCNR', -NC(=O)OR', -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-S02R', -SOR', -SR', -S02OR', -SON(R')2, -NHNR'2, -NNR', -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy). , alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), amino(C1-C6 alkyl), -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl), -CO2H, -CO2R', -OCOR', -OCOR', -OC(=O)OR', -OC(=O)NR', ​​-OC(=S)OR', -OC(=S)NR', ​​where each R' is independently selected from hydrogen, alkyl, alkenyl, aryl, heteroaryl, heteroatom, optionally substituted cycloalkyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or any combination thereof.

[0319] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0320] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a polynucleotide" includes a plurality of such polynucleotides, a reference to "the polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a prerequisite for using exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of "negative" limitations.

[0321] In instances where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but be limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will further be understood by those of ordinary skill in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B."

[0322] It is understood that certain features of the invention, which are for clarity described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are for brevity described in a single embodiment, may be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if all combinations were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if all such subcombinations were individually and explicitly disclosed herein.

[0323] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as described above and as claimed in the claims section below finds experimental support in the following examples.

[0324] Various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0325] Example Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature. See, for example, "Molecular Cloning: A Laboratory Manual" by Sambrook et al. (1989); "Current Protocols in Molecular Biology" Volumes I-III, Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA," Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series," Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methodologies described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057; "Cell Biology: A Laboratory Handbook," Volumes I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells—A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition; "Current Protocols in Immunology," Volumes I-III, Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated herein by reference. Other general references are provided throughout this specification.

[0326] Example 1: Direct Chemical Modification Because positive charges promote intracellular delivery via the endocytic pathway, several small polyamine derivatives were tested. GFP / IgG was directly conjugated with either tetraethylenepentamine (TEPA), triethylenetetramine (TETA), or polyethyleneimine (PEI), and protein uptake into cells was monitored. PEI proved to be far superior (data not shown), so all further experiments were performed with PEI.

[0327] PEIs, either linear or branched, typically have molecular weights exceeding 10 kDa and are characterized as having the highest cationic charge density of any known polymer. This has led to their use as gene transfer agents, where their high positive charge is used for complexation with negatively charged DNA or RNA and for the internalization of these nucleic acids into cells. While these high-molecular-weight gene transfer agents are efficient in vitro, they are less useful in vivo. Therefore, we employed very low-molecular-weight PEI moieties, i.e., branched PEI molecules with molecular weights ranging from 600 to 1800 Da (as determined by mass spectrometry). These very low-molecular-weight PEIs can be chemically and covalently conjugated to protein payloads to be internalized.

[0328] Very low molecular weight PEI, primarily 600 Da, was conjugated to IgG and GFP. Because PEI contains many primary amines, conjugation of glutamic acid and aspartic acid to the protein's carboxylic acid residues, as well as to the C-terminal carboxyl group, was carried out using carbodiimide conjugation chemistry. The reaction was performed with an excess of PEI, i.e., a 3500 molar excess, and control of the level of modification was achieved by adjusting the level of the carbodiimide reagent (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)) in the reaction system. The level of modification was measured by MALDI-ToF mass spectrometry (Figure 1). For IgG-based proteins, the average level of modification ranged from 1 to approximately 10 molecules of PEI (600 Da) per IgG molecule, which was achieved with excess levels of EDC ranging from 25 to 400 molar equivalents, respectively. Because PEI modification is not regioselective, it results in a broad distribution of MALDI-ToF molecular weight moieties, as observed in MALDI-ToF spectra (Figure 1). The average modification level was calculated based on the measured molecular weight value at the peak apex minus the measured molecular weight value at the peak apex of the unmodified protein, which gives the weight of PEI added. Dividing this value by the molecular weight of a single PEI molecule used for modification gives the average number of molecules added.

[0329] Next, PEI-modified and unmodified mouse IgG were incubated with A375 cells for 2 hours. No additional inducers for gene transfer / internalization were added. As seen in (Error! Reference source not found), unmodified mouse IgG did not enter A375 cells. In contrast, even low levels of PEI modification (an average of 3 PEI molecules per IgG molecule, X3) allowed internalization, while increasing the average level of modification proportionally increased the level of IgG observed inside the cells. Similar results were observed when internalization was measured using flow cytometry (data not shown). Similar internalization efficiencies and dependence on modification level were observed for other payloads, including other IgGs (data not shown) and green fluorescent protein (GFP) (Figures 3A-3B).

[0330] Example 2: Membrane crossing efficiency Although numerous studies using cell-penetrating peptides (CPPs) have demonstrated some degree of cellular internalization, in most cases the efficiency of initial cellular uptake was quite low, because high levels of payload in the medium were required to result in only a small proportion of molecules actually internalized into cells. To assess the efficiency of the initial internalization step, we measured the level of payload, PEI-modified mouse IgG, in the medium as a function of time using a specific ELISA. As seen in Figure 4, at high levels of PEI modification (approximately 7), almost all of the modified IgG was internalized (95%), with most internalization occurring within the first 24 hours of incubation (gray bar). Even at lower levels of modification (approximately 4.5 and 3), the internalization step was highly efficient, showing an overall uptake of approximately 75% of the total payload after 5 days of incubation (Figure 4, blue and orange bars) and following kinetics similar to that of highly modified IgG. The incomplete and slower uptake is likely related to IgGs that are modified at much lower than average modification levels. PEI modification resulted in a somewhat broad distribution of modified moieties (see Figure 1), and unmodified or lowly modified (less than 3) molecules were likely still present in the medium, even after 5 days. This cell membrane crossing efficiency was observed at initial medium concentrations ranging from 0.2 to 40 µg / mL.

[0331] Example 3: Endocytic pathways utilized by PEI-modified proteins The cationized moiety is thought to be internalized using the native endocytic mechanism. Two related endocytic mechanisms are caveolin-mediated and clathrin-mediated endocytosis. The exact mechanism of internalization was elucidated by performing internalization of PEI-modified IgG in the presence of known specific endocytosis inhibitors. A375 cells were incubated overnight with PEI-modified mouse IgG (4.5 PEI) in the presence of clathrin inhibitors (amantadine or chlorpromazine) or caveolin inhibitor (genistein). Genistein, a known caveolin inhibitor, indeed inhibited internalization of PEI-modified IgG (error, reference source not found). Interestingly, the addition of chlorpromazine, a known clathrin inhibitor, resulted in increased internalization. This demonstrates that caveolin-mediated endocytosis is utilized by PEI-modified payloads for internalization, as inhibition of clathrin-mediated endocytosis caused a compensatory increase in caveolin-mediated endocytosis in cells.

[0332] Example 4: Endosomal escape As mentioned above, perhaps the most challenging hurdle in the intracellular delivery of biologics is their endosomal escape to avoid catabolism of the therapeutic. To assess whether PEI-modified proteins can escape from endosomes, their internalization was tracked using confocal microscopy and counterstaining with endosomal and lysosomal markers. PEI-modified IgG was incubated with HEK293 cells expressing fluorescent endosomal / lysosomal markers for 5 hours. Cells were analyzed by fluorescence microscopy. Endosomes and lysosomes were transfected with a green fluorescent marker (Cell light Endosome, Molecular Probes, Catalog No. C10586 or Cell light Lysosome, Molecular Probes, Catalog No. C10596), while IgG was stained with a red fluorescent anti-mouse antibody. As seen in (Error, reference source not found), no significant colocalization was observed between internalized IgG and early endosomal or lysosomal vesicles. This demonstrates that the PEI-modified payloads indeed successfully escaped endosomes. These experiments were repeated using the endosomal / lysosomal markers EEA1, transferrin, and calcein, and the same results were observed (data not shown). Note that colocalization was not observed even with early endosomes, suggesting that endosomal escape of PEI-modified proteins occurs rapidly and very early in the endosomal pathway.

[0333] Although no colocalization was observed between PEI-modified IgG and various endosomal and lysosomal markers, the staining of these IgGs appeared punctate in nature. The punctate profile may suggest the inclusion of internalized IgG in certain vesicles. To further demonstrate the endosomal escape of PEI-modified proteins, the proteins were labeled with the pH-sensitive fluorescent dye, 5(6)-carboxynaphthofluorescein. This dye fluoresces only at pH values ​​above 7. Because the pH of early endosomes is already below 7 and only decreases as endosomes mature into lysosomes, all vesicles in the endosomal pathway are acidic. PEI-modified, pH-sensitively labeled IgG was incubated with HeLa cells, and the cells were analyzed by confocal microscopy. The cells clearly displayed the unique red fluorescence of the pH-sensitive dye, indicating that the PEI-modified proteins reside in non-acidic compartments, such as the cytoplasm (Figure 7). This provides further evidence that PEI-modified proteins efficiently escape the endosomal pathway.

[0334] Finally, functional assays were used to verify that PEI-modified proteins efficiently escape endosomes after their internalization. To this end, a functional monoclonal antibody against CD247, also known as the CD3 zeta chain, was modified with PEI and internalized into CD3+ primary cells. The intracellular domain of the CD3 receptor is involved in T cell activation via its immunoreceptor tyrosine-based activation motif (ITAM). The selected monoclonal anti-CD3 zeta chain antibody (Sigma, Clone ZT-10, catalog number SAB4200446) binds one of the ITAMs in the CD3 zeta chain.

[0335] Internalization of PEI-modified anti-CD247 and, as a negative control, PEI-modified mouse IgG was performed into CD3+ cells prestimulated with anti-CD3 / CD28 antibody-coated beads. The culture medium was monitored for interferon-gamma (IFNγ) secretion as a marker for changes in the activation level of the cells. Although the intact antibody would not be expected to be stable in the cytoplasm due to the presence of structurally essential disulfide bonds, binding of this antibody should activate a signaling cascade that would result in IFNγ secretion. Thus, even a small amount of initial activation would result in a quantifiable increase in cytokine levels. Therefore, even with its predicted cytoplasmic instability, internalization of the antibody can be expected to generate an observable IFNγ response.

[0336] Internalization of the modified antibody into CD3+ cells was demonstrated by intracellular FACS analysis (data not shown). Internalization of anti-CD247 PEI-modified mAb (4.5xPEI) resulted in dose-dependent activation of CD3+ cells, as measured by increased IFNγ secretion (Figure 8). PEI-modified mouse IgG did not cause any CD3 cell activation within the antibody concentration range used in this experiment. This result further demonstrates the endosomal escape of PEI-modified proteins and their ability to exert biological and clinical effects in target cells.

[0337] Example 5: Dispersibility in the cytosol and payload carrier solutions Although endosomal escape of PEI-modified proteins clearly occurs, microscopic images of various internalized proteins show punctate profiles (Figures 2, 3, 6, and 7). While PEI modification is highly efficient at crossing the cell membrane and endosomal escape, it was hypothesized that the same modification hinders efficient dispersal of PEI-modified proteins in the cytoplasm. This dispersal problem may be the result of strong electrostatic interactions between the strongly cationized internalized proteins and various cytoplasmic proteins, primarily cytoskeletal proteins (most of which are negatively charged). Poor cytoplasmic dispersal would negatively affect the efficacy of any biologic within cells, as the drug may not be able to reach its intracellular target, whether in the cytoplasm or other subcellular compartments or organelles.

[0338] To overcome this intracellular distribution hurdle, a disulfide bond was incorporated into the linker between the therapeutic agent and a general-purpose carrier pre-modified with PEI. The bond chosen was a chemical linker incorporating a disulfide bond. This labile disulfide bond is cleaved upon endosomal escape due to the high reduction potential of the cellular environment, primarily due to the high concentration of glutathione, thus releasing the therapeutic agent from the cationized carrier and allowing free movement of the therapeutic agent within the cell cytoplasm. The use of a carrier also has the advantage of eliminating the risk that PEI may be conjugated to a functional site and / or inhibit the function of the therapeutic agent. By using selective conjugation chemistry to link the therapeutic agent to a specific site or domain, the precise location of linker conjugation can be controlled, deflecting it from the active domain of the therapeutic agent.

[0339] A schematic diagram of this carrier and payload method is shown in Figure 9. As can be seen, the carrier protein has PEI groups, which are involved in membrane crossing and endosomal escape. These groups can be conjugated to the carrier protein either randomly or site-selectively. In the case of random conjugation, the level of conjugation can be controlled by selecting specific reaction conditions, as described previously herein.

[0340] The carrier protein itself is preferably selected from a list of human endogenous proteins to avoid immunogenicity problems.Furthermore, proteins that are commonly found in blood and have a naturally long circulating half-life are preferred.Finally, a protein that can deliver its payload to the area in the body where a therapeutic agent is desired would be advantageous.Examples of such areas in the body may include tumors and their microenvironments, as well as inflammatory sites that are important for autoimmune diseases and many other pathological conditions.Human serum albumin (HSA) was therefore selected. HSA is a circulating protein with a long half-life that has been shown to traffic to tumors and sites of inflammation and even deliver payloads to these sites, albeit only to the extracellular environment (Liu et al. BMC Biotechnology 2012, 12:68; Kratz, F., Journal of Controlled Release 132 (2008) 171-183; Um et al., Bioconjugate Chem., 2019, 10.1021 / acs.bioconjchem.9b00760, Wunder A. et al., J Immunol 2003; 170:4793-4801; Yazaki PJ et al., Nuclear Medicine and Biology, 35 (2008) 151-158).

[0341] The therapeutic agent is conjugated to a single linker at a single conjugation site along the polypeptide chain of the biologic. This conjugation site is located at a position that does not interfere with the activity of the drug. The linker can be selected from a peptide linker, a chemical linker, or a combination thereof. PEG, a polymer-based linker, was selected. As discussed above, labile bonds that are sensitive to specific conditions in the cytoplasm of cells can also be incorporated.

[0342] To evaluate the efficiency of the carrier-payload method, GFP was conjugated to PEI-cationized HSA via a PEG-based linker incorporating a disulfide bond. eGFP (Biorbyt, catalog number orb84840) was modified with NHS-PEG4-SPDP. PEI-modified HSA (11xPEI) was also further reacted with NHS-PEG4-SPDP after reduction of this SPDP to free thiols using DTT. SPDP-activated eGFP was reacted with HSA-PEI-free thiols to generate a GFP-HSA conjugate with a labile disulfide bond incorporated into the linker.

[0343] In contrast to the punctate profile of internalized PEI-modified GFP (Figure 3), when GFP disulfide-linked to PEI-cationized HSA was internalized into cells, it produced a fully dispersed profile without spots (Figure 10). To verify the efficient distribution of the internalized therapeutic payload, a fully therapeutic anti-TNFα monoclonal antibody (Humira®) was linked to PEI-modified HSA via a PEG-based linker incorporating a disulfide bond. Similar to GFP, the antibody was modified with NHS-PEG4-SPDP. PEI-modified HSA (11xPEI) was also further reacted with NHS-PEG4-SPDP after reduction of SPDP to free thiols using DTT. The SPDP-activated antibody was reacted with HSA-PEI-free thiols to generate an antibody-HSA conjugate with a labile disulfide bond incorporated into the linker. As seen in Figure 11, the internalized monoclonal antibody dispersed throughout the cytoplasm, again confirming the hypothesis that direct cationization prevented free dispersion in the cytoplasm and demonstrating the effectiveness of the solution in separating the payload from its cationized carrier.

[0344] Example 6: Cytosolic stability Therapeutics must not only enter the cytoplasm of target cells but also exert their biological activity. Even molecules transported to other intracellular locations (nucleus, ER, mitochondria, etc.) must still pass through the cytoplasm. Many biological therapeutics are based on antibody scaffolds or their derivatives. In many cases, these therapeutics bind specific targets and either antagonize or agonize them. The binding activity of these agents is entirely dependent on their tertiary and quaternary structures. In antibody-based molecules, either intact IgG or their truncated derivatives (Fab, scFv, etc.), these structures are based on and stabilized by disulfide bonds, either intra- or inter-chain. However, as mentioned above, the cytoplasm, as well as other intracellular organelles and compartments, are characterized by a highly reducing environment. Glutathione, the major reducing agent, has a cytosolic concentration in the 1-11 mM range. In contrast, its plasma concentration is low, at micromolar levels. This property of the cytoplasm has hindered the use of antibody-based drugs, as well as other biologics that utilize disulfide bonds in their structure as an efficient intracellular route of therapy.

[0345] In vitro experiments showed that exposure of antibodies to cytosolic levels of glutathione (GSH) resulted in disulfide bond reduction after just a few hours, as evidenced by the appearance of multiple bands on SDS-PAGE Western blots detected with an anti-light chain antibody (lanes 5-7, Figure 12). Overnight exposure resulted in a lack of antibody detection on Western blots, likely due to loss of 3D structure or aggregation and precipitation (lanes 1-4, Figure 12). The latter is a known phenomenon for intracellularly expressed antibodies and their derivatives (Kabayama, H. et al., Nature Communications 2020, (11), 336). Any such effects on intracellular biologics would be detrimental to their ability to bind biological targets and exert therapeutic effects.

[0346] In light of these intracellular stability issues, single-domain antibodies were selected as therapeutic payloads. Single-domain antibodies are single-chain protein-based molecules capable of binding to other proteins. Their structure lacks all essential disulfide bonds, making them resistant to the reducing environment of the cytosol. Such single-domain binding proteins include truncated heavy-chain antibodies (HcAbs), camelid-based variable heavy-chain homodimers (VHHs), also known as nanobodies, or shark-based immunoglobulin novel antigen receptors (IgNARs). Other examples of such single-domain binding proteins include designed ankyrin repeat proteins (DARPins) and genetically engineered antibody mimetic proteins.

[0347] Single-domain binding proteins, and VHHs in particular, are well suited as payloads. Their lack of structurally essential disulfide bonds makes them resistant to cytosolic conditions. They are extremely small—VHHs approximately 15 kDa and DARPins 20 kDa—a fact that supports their cytosolic dispersion. Their single-domain nature ensures that they are unlikely to cause any accidental intracellular cross-linking effects. They can be easily engineered to contain two or more moieties in different configurations, allowing for more complex binding profiles. They are not considered immunogenic and have a good safety profile. A key feature is their compatibility with site-selective conjugation to carriers. For example, in both VHHs and DARPins, their C-termini are positioned away from their antigen-binding regions (CDRs), allowing the use of this site for conjugation without affecting antigen binding. Furthermore, the C-termini can be easily engineered to contain a single cysteine ​​amino acid with a free sulfhydryl group for conjugation. This group can be conjugated to a carrier equipped with a thiol-reactive group. The free sulfhydryl at the C-terminus can be conjugated to a carrier directly or via a linker.

[0348] A commercially available anti-vimentin VHH (QVQ, Q60c) containing a single cysteine ​​amino acid at its C-terminus was conjugated to a PEI-modified HSA carrier, which was further modified with a thiol-reactive group. The PEI-modified HSA was then conjugated to a 2-pyridyldithio group (NHS-PEG). n The VHHs were conjugated to an NHS-activated PEG linker with a PEG-1000-binding domain (-SPDP, n=4). SPDP readily reacts with the free thiol of the VHH to generate the VHH-HSA moiety, and the linkage between the VHH payload and the HSA carrier involves a disulfide bond. Figure 13 shows the profiles of the reaction mixture before and after purification, as well as the ability of the linkage between the carrier and the payload to be cleaved under reducing conditions. Conjugation to the VHH was similar regardless of the PEI level. Furthermore, in all reactions, the payload was efficiently cleaved after treatment with 5 mM GSH, which mimics the reducing conditions of the cytosol.

[0349] The cellular internalization efficiency of anti-vimentin-PEI-modified HSA conjugates was assessed by measuring the disappearance of conjugation from the medium upon incubation of A375 cells with the conjugates. VHHs were conjugated to HSA modified with PEI at two levels (3.5 and 8 PEI molecules per HSA on average). As previously observed with PEI-modified IgG (Figure 4), VHH-carrier conjugates were efficiently internalized by cells (Figure 14), with nearly 80% of the conjugate internalized during the first 24 hours of incubation. Conjugate levels were measured using a proprietary two-sided ELISA that measured only VHHs conjugated to HSA, utilizing an anti-VHH antibody as a capture antibody and an anti-HSA antibody for detection. Surprisingly, both PEI modification levels showed very similar internalization efficiencies and kinetics, with the 8-PEI modification having a very slight advantage. The levels of these VHH-HSA conjugates in cell-free culture media were assessed to ensure that the observed decrease in their levels was not due to degradation. Only a slight decrease was observed in the absence of cells (Figure 14), suggesting that the dramatic decrease in VHH levels in the presence of cells was indeed due to VHH internalization.

[0350] To further evaluate the efficiency of endosomal escape and the distribution of the payload VHH in the cytoplasm, anti-vimentin VHH conjugated to PEI-modified HSA (an average of 11 PEI molecules per HSA molecule) was incubated with A375 cells for 24 hours, and the cells were analyzed by confocal microscopy (Figures 15A-15C). Using an anti-VHH antibody conjugated to AlexaFluor 647, the distribution profile showed the presence of VHH inside the cells (Figure 15A). The cells were co-stained for vimentin using a standard fluorescently labeled anti-vimentin antibody (Figure 15B). Careful inspection of the two images clearly showed that the profile obtained by anti-VHH staining was virtually identical to that of vimentin staining, suggesting that the anti-vimentin VHH was successfully delivered to the cell cytoplasm, where it could be released from its carrier and find and bind its target (Figure 15C). Further evidence for this binding can be seen in Figure 16A, where a close-up of one of the cells exposed to anti-vimentin VHH conjugated to PEI-modified HSA clearly shows the vimentin cytoskeletal pattern visualized by anti-VHH antibody staining. Figure 16B shows that cells exposed to anti-vimentin VHH alone, without carrier conjugation, show no intracellular VHH staining.

[0351] The binding of the internalized anti-vimentin VHH to its vimentin target (Figures 15A and 16A) also demonstrates that the VHH agent maintained its structural stability inside the cytoplasm. As with all binding biologics, the ability to bind to a target is strongly dependent on its structure and the stability of this structure. This data therefore supports the selection of single-domain binding proteins as payload agents of the present invention.

[0352] Another important characteristic of the delivery system of the present invention is the efficiency and uniformity of internalization, in that all cells in the culture exhibit internalization. This can be seen throughout the different microscopic images, including particularly Figures 15A-15C. Many attempts at intracellular delivery of proteins known in the literature result in only a fraction of cells internalizing the protein, suggesting very low efficiency.

[0353] Example 7: In vitro functional proof of concept Further evaluation of the functionality of intracellularly delivered biologics was performed using VHHs against the E7 protein of human papillomavirus (HPV). Nearly all cervical cancers are associated with human papillomavirus (HPV) infection, with two types, HPV16 and HPV18, accounting for 70% of cases. One of the primary oncoproteins of HPV is the E7 protein. E7 induces and maintains the malignant phenotype through its interaction with the retinoblastoma protein (RB1). E7 disrupts the function of the host RB1 protein, leading to stimulation of uncontrolled cell proliferation. E7 also interferes with host histone deacetylation mediated by HDAC1 and HDAC2, resulting in transcriptional activation. Previous studies have suggested that inhibition of E7 function suppresses the proliferation of HPV-positive cervical cancer cells. Li et al. (Molecular Immunology, 2019, 109, 12-19) showed that transfection of a plasmid encoding a VHH against the E7 protein in HPV-positive cells (previously used due to the lack of an efficient intracellular delivery system for the protein itself) could interfere with E7 activity (disrupting the E7-RB1 interaction) and result in reduced proliferation of HPV-positive cells. The inventors expressed and purified the same anti-E7 VHH with an additional C-terminal cysteine ​​and conjugated it to the intracellular carrier of the present invention. HPV-positive HeLa cells were incubated with unmodified anti-E7 VHH or VHH conjugated to PEI-modified HSA with an average of 3.5 PEI molecules per HSA molecule via a cleavable linker. After incubation, cell viability was measured using a standard MTT viability assay. Unmodified VHH had no effect on proliferation, whereas cells incubated with VHH conjugated to a PEI-modified carrier showed a dose-dependent decrease in cell viability, indicating that the anti-E7 VHH was successfully internalized and was able to inhibit the effect of E7 in HeLa cells (Figure 17).

[0354] To confirm the magnitude of the intracellular effect of E7 inhibition in HPV-positive cell lines, we employed a live cell analysis system such as the IncuCyte®. Because E7 affects cell cycle regulation, we used a specific HPV-positive HeLa cell line called the Fluorescent Ubiquitination-Based Cell Cycle Indicator (FUCCI) reporter system. The FUCCI reporter system allows tracking of different cell cycle stages. Cells in G1 fluoresce red, while cells in S, G2, or M fluoresce green. Cells were synchronized with thymidine for 24 hours before the introduction of different treatments into the cell culture medium. Cells were treated with anti-E7 VHH conjugated to PEI-modified HSA via a cleavable linker, as well as the following controls: no treatment, modified HSA, modified HSA conjugated to an irrelevant VHH (anti-vimentin), unmodified anti-E7 VHH, anti-E7 VHH conjugated to unmodified HSA, and a cell cycle inhibitor (DP, a CDK4 / 6 inhibitor). As can be seen in Figure 18, all controls had no effect on the cell cycle, which remained similar to untreated cells, whereas anti-E7 VHH conjugated to PEI-modified HSA (3.5PEI) had a dramatic effect, resulting in cell cycle arrest similar to the use of direct cell cycle inhibitors. Because no commercially available E7 inhibitors exist, the CDK4 / 6 inhibitor palbociclib isethionate (PD0332991, Sigma, catalog no. PZ0199), known to cause cell cycle arrest, was used as a positive control. Anti-E7 VHH conjugated to modified HSA produced an effect similar to the inhibitor, only with different kinetics. Furthermore, as can be seen in Figure 19, this arrest led to the death of HPV-positive cells.

[0355] Both anti-vimentin and anti-E7 VHHs demonstrated their durability in the reducing environment of the cytoplasm. To further illustrate the suitability of single-domain binding proteins that do not rely on disulfide bonds to stabilize and maintain their structure, we used designed ankyrin repeat proteins (DARPins) as binders. DARPins are genetically engineered antibody-mimetic proteins that typically exhibit high-specificity and high-affinity target protein binding. They are derived from the natural ankyrin protein, one of the most common classes of binding proteins in nature, and are involved in diverse functions such as cell signaling, cell regulation, and structural integrity. DARPins consist of several repeat motifs, and their molecular weights are approximately 14–18 kDa (kilodaltons). They are also characterized by their lack of reliance on disulfide bonds for their structural integrity, similar to VHHs.

[0356] For this purpose, a DARPin against K-RAS was selected. RAS proteins play an important role in signal transduction as molecular switches. RAS is a key target in cell transformation, contributing to cell proliferation and differentiation through the RAF-MEK-ERK cascade and cell survival through activation of PI3K. Mutations in RAS proteins (K-, H-, or N-RAS) create constitutively activated GTP-bound forms that promote cell transformation in a signal-independent manner. Activating RAS gene mutations are found in as many as 30% of human cancers, most frequently in pancreatic, colon, and lung adenocarcinomas. Oncogenic RAS has been shown to be essential for early-onset tumors and required for maintaining tumor viability. The most central RAS mutations are at glycine 12, such as G12D and G12V.

[0357] Guillard et al. (Guillard, S. et al. Nat. Commun. 2017, 8, 16111) generated an antibody mimetic, DARPin K27, which inhibits nucleotide exchange of RAS. K27 selectively binds to the inactive Ras GDP form with a Kd of 4 nM, and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 by transfection of a DARPin-encoding vector significantly reduced the amount of active Ras, inhibited downstream signaling, particularly the level of phosphorylated ERK, and slowed the growth of HCT116 cells in soft agar. The group stated that "the barrier arises from the fact that Ras is intracellular." DARPin K27 does not have the intrinsic ability to enter cells, and therefore cannot access Ras when added extracellularly. Although there have been reports of DARPin delivery to the cytoplasm of cells, a significant increase in efficiency will be required to make this approach viable for therapeutic applications. Developing small-molecule inhibitors that bind at the same site as DARPin K27 can be challenging because the scaffold binds over a large surface rather than defining a pocket.

[0358] DARPin K27 was expressed based on the reported sequence and conjugated to an HSA carrier modified with an average of 3.5 PEI molecules. Internalization and KRAS binding were assessed by confocal microscopy (Figure 20). K27 DARPin was not distributed throughout the cytoplasm but rather localized to the inner side of the plasma membrane, the primary location of KRAS. Interestingly, some cells showed both a punctate profile and intracellular localization of the internalized DARPin. The punctate localization could be the result of DARPin that had not yet escaped the endosome or had not yet separated from the carrier to freely find its target.

[0359] In further studies, pancreatic ductal carcinoma cells (SU8686) were incubated with the anti-KRAS DARPin K27 conjugated to an HSA carrier. The carrier was modified with 8 or 3.5 PEI molecules. The apoptotic state of the cells was monitored using a traditional Annexin V assay and visualized using an IncuCyte® continuous cell monitoring system. Cells exposed to DARPins conjugated to PEI-modified HSA via a cleavable linker showed a dramatic increase in apoptosis, especially for DARPins conjugated to carriers with high levels of PEI modification (Figure 21). This is likely due to enhanced internalization and more rapid kinetics. In contrast, cells untreated, treated with unmodified DARPins, or treated with PEI-modified HSA (8 molecules) alone showed baseline levels of apoptosis, highlighting the intracellular effect of the DARPins.

[0360] Guillard et al. showed that KRAS DARPin K27 binds primarily to native, non-mutated, inactive KRAS and to KRAS with different mutations at the G12 position. Therefore, anti-KRAS DARPin K27 was evaluated for its intracellular effects on proliferation and apoptosis in the HeLa cell line. This cell line is characterized by constitutive expression of GFP in its nucleus, allowing for easy tracking of these cells using continuous imaging monitoring methods such as IncuCyte®. Two different preparations of anti-KRAS DARPin were conjugated to HSA with 8 PEI molecules, and their effects on HeLa-GFP cells were compared with the following control treatments: no treatment as a negative control, treatment with a pan-Ras inhibitor as a positive control, treatment with an unmodified DARPin, treatment with a DARPin conjugated to unmodified HSA, treatment with an HSA carrier modified with 8 PEI molecules, and treatment with an anti-vimentin VHH conjugated to HSA modified with 8 PEI molecules. As can be seen in Figures 22A-22B, both preparations of anti-KRAS DARPin conjugated to HSA with 8 PEI molecules dramatically affected both proliferation (Figure 22A) and apoptosis (Figure 22B) of HeLa-GFP cells. None of the control treatments had any effect on these parameters. The effect on apoptosis can also be seen in Figure 23, where the red staining of cells treated with anti-DARPin conjugated to PEI-modified HSA (apoptotic cells) and the loss of GFP associated with cell death are clearly observed.

[0361] Example 8: Pharmacokinetics and Biodistribution (PK and BD) PEI modification confers a concentrated and strong positive charge to the carrier protein. In contrast, plasma components and cell membranes are usually negatively charged. Positively charged proteins are known to be "sticky" due to electrostatic binding with these negatively charged components. This "stickiness" can lead to short half-lives and biodistribution problems. This phenomenon is known for proteins that are naturally positively charged and characterized by a slightly basic isoelectric point (pI). The pharmacokinetic and biodistribution profiles of PEI-modified proteins may be affected by their strong positive charge. Furthermore, such stickiness may also lead to "trapping" of the administered positively charged protein at the injection site. To avoid or at least minimize these effects, the influence of the level of PEI modification was examined.

[0362] Identifying and utilizing the lowest level of PEI modification that is still effective in crossing the cell membrane and enabling endosomal escape can improve the PK and biodistribution of the proteins of the present invention. To identify this modification level, HSA carriers were produced with the following average levels of PEI modification: 2.0, 3.6, 5.2, 6.2, 8.0, and 9.4. These levels were confirmed by analysis of the resulting modified HSA using MALDI-ToF mass spectrometry (Figure 24). These carriers were further conjugated to anti-vimentin VHHs, and the internalization of these conjugates was assessed using confocal microscopy (Figure 25). As can be seen from the confocal microscopy results, even VHHs conjugated to HSA with 2.0 and 3.6 PEI molecules show clear intracellular presence. Although the use of carriers modified with higher levels of PEI appears to lead to apparently higher levels of intracellular VHH, in these cases the profile of internalized VHH was more punctate and less dispersed, likely due to slower desorption of VHH from the carrier due to the higher positive charge. The stronger staining of intracellular VHH in the case of HSA modified above 3.6 PEI / HSA may be the result of stronger imaging due to the concentrated punctate profile and may not necessarily represent higher levels in absolute terms.

[0363] The confocal microscopy results were also correlated with the internalization levels of these conjugates by measuring the residual levels of conjugates in the cell culture medium using a specific ELISA (Figure 26). Unlike the results observed with IgG directly modified with PEI (Figure 4), the levels and kinetics of HSA carriers (with conjugated VHHs) were not significantly affected by the level of modification. With 3.5 and 8 molecules of PEI, internalization was efficient, and its magnitude and rate were very similar. These results suggest that HSA used as a carrier can indeed be modified with just 3.5 or even fewer PEI molecules, a fact that would reduce the stickiness of HSA carriers in the circulation.

[0364] Example 9: Charge Masking (Non-Covalent Bonding) Non-covalent charge masking can be achieved by neutralizing the positive surface charge of PEI, such as by electrostatic binding of negatively charged molecules to PEI. Specifically, the inventors hypothesized that molecules with multiple negative charges, such as phytic acid, could electrostatically interact with the multiple positive charges of PEI to establish efficient charge masking in vivo.

[0365] To evaluate the effect of charge masking on the pharmacokinetics and biodistribution of PEI-modified HSA, HSA-PEIx8 was treated with 20 mM phytic acid, dialyzed to remove excess phytic acid, and intravenously injected into mice. Untreated HSA-PEIx8 was administered as a control. HSA concentrations were measured in the plasma of Balb-c mice using an in-house ELISA, and results are presented as a percentage of the injected dose. We observed that HSA-PEIx8 rapidly cleared from the circulation, as evidenced by the fact that only 35% of the injected dose was quantifiable even 5 minutes after administration. At 30 minutes, 95% of HSA-PEIx8 was not found in the plasma of treated mice. Although masking resulted in faster clearance from the circulation, phytic acid-masked HSA-PEIx8, however, behaved quite differently from unmasked HSA-PEIx8.

[0366] To better understand these effects, near-infrared fluorescently labeled (VivoTag 750-S, PerkinElmer) versions of the test particles were prepared and used in PK studies using an IVIS imaging system (PerkinElmer). Athymic nude mice were administered two levels of HSA-PEIx8 (125 and 250 nmol / kg) and phytic acid (5 mM)-masked HSA-PEIx8 administered at 125 nmol / kg. Animals were imaged by the IVIS system at different time points up to 48 hours after administration (Figure 27A).

[0367] Although the fluorescent labeling appears to have affected the PK and biodistribution of the labeled protein (data not shown), comparison of phytate-masked HSA-PEIx8 to the unmasked protein demonstrates a dramatic effect of masking. HSA-PEIx8 rapidly concentrates in the kidneys and liver of animals, whereas phytate-masked HSA-PEIx8 avoids this outcome. These effects are also evident when imaging different organs of animals harvested 48 hours after injection (Figure 27B).

[0368] Based on these and other results, the inventors understand that while the temporary effect of noncovalent phytic acid masking in an in vitro setting was quite satisfactory (results not shown), the masking effect of phytic acid is lost very rapidly in an in vivo setting. It is hypothesized that phytic acid's inability to establish efficient charge masking in vivo is likely due to intense competition from various divalent metal ions, such as calcium and magnesium, which have a high affinity for phytic acid. Furthermore, although zeta potential measurements showed that phytic acid dramatically reduced the charge of PEI-modified HSA, it should be noted that the positive charge was essentially neutralized so that the surface charge of the masked HSA-PEI was nearly zero. Proteins with a surface charge of approximately zero are known to be unstable in solution, and furthermore, zero-charge proteins are not known to have efficient pharmacokinetic profiles. These insights and shortcomings of non-covalent masking led the inventors to search for a masking solution based on covalent masking.

[0369] Example 10: Charge Masking (via Covalent Protecting Groups) Maleic anhydride and its derivatives have been demonstrated by the inventors as amino-protecting groups for PEI (and, in some cases, for lysine residues in carriers). The following scheme shows the general reaction of a primary amine with a maleic anhydride derivative. As can be seen from the scheme, the amine group, which has a positive charge at physiological pH, becomes neutral when it is converted to an amide group. At the same time, the reaction produces a free carboxyl group, which is characterized by a negative charge at physiological pH. [ka]

[0370] Such anhydride derivatives can react with the primary amines of the PEI modifications on the HSA carrier of the present invention, as well as with the amines of its lysine side chains and the N-terminal free amine.

[0371] The stability of these protecting groups can be controlled by the substituents on the double bond, with more and larger substituents resulting in lower stability. This lower stability can be exploited in the present invention to generate the desired transient effect of charge masking. Because these potential covalent masking agents are less stable at lower pH values, this property can be used to result in higher accumulation of masked PEI-modified carriers in the tumor microenvironment (TME). This is because such TMEs are typically characterized by a lower pH than normal, healthy extracellular or plasma environments. While the latter have a pH of approximately 7.3-7.4, the TME can have a pH of approximately 6.8-7.0 and even lower. Enhanced removal of these masking agents in the TME due to the lower pH would expose the cations of PEI, leading to cell adhesion and internalization.

[0372] The inventors have investigated the various anhydride derivatives (maleic acid-R 1 =H, R 2 = H; Citraconic acid - R 1 =Me, R 2 =H and dimethylmaleic acid -R 1 =Me, R 2 = Me, cis-aconitic acid (R 1=CH2COOH, R 2 =H), 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propanoic acid (R 1 =Me, R 2 =CH2CH2COOH), and 3-ethylfuran-2,5-dione (R 1 =CH2CH3, R 2 The effect of charge masking by dimethylmaleic anhydride (=H) was evaluated using an isoelectric focusing (IEF) gel showing the pI of the protein. Native HSA is characterized by a pI of approximately 4.8, and PEI modification resulted in an extremely high pI of over 8, which could not be measured using this gel. Both maleic anhydride and citraconic anhydride dramatically reduced the pI of PEI-modified HSA, even showing a somewhat lower pI than native. Dimethylmaleic anhydride, with its known instability even at a pH of approximately 7, gave a smear, possibly indicating removal of this masking prior to and during the analysis. Due to this instability, further experiments were not performed with dimethylmaleic anhydride. The IEF results were corroborated by zeta potential measurements (Zeta Sizer Ultra (Malvern Instruments)). These analyses showed a zeta potential value of -14.4 for native HSA. PEI modification (x8) increased the zeta potential to nearly +13, whereas masking with citraconic anhydride reduced the zeta potential of PEI-modified HSA to -12 when a molar excess of 85 equivalents was used.

[0373] The masking procedure is as follows: To a solution of the protein to be masked at a concentration greater than 1 mg / mL, the anhydride is added in a molar excess ranging from 50 to 500 equivalents. The reaction is spontaneous and does not require additional catalysts or reagents. The reaction can be maintained for minutes to hours.

[0374] Various anhydride derivatives (maleic acid, citraconic acid, cis-aconitic acid (R 1 =CH2COOH, R 2 =H), 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propanoic acid (R 1 =Me, R 2=CH2CH2COOH), and 3-ethylfuran-2,5-dione (R 1 =CH2CH3, R 2 The masking of HSA-PEIx8 by citraconic anhydride (=H) was calibrated using different molar excesses of the anhydride derivatives. We then tested the masking efficiency of the above-mentioned anhydride derivatives using IEF gel electrophoresis. The resulting IEF profiles (data not shown) of HSA-PEIx8 masked with different molar excesses of citraconic anhydride, ranging from 75 to 200 equivalents, were compared with those of native, unmodified HSA. We concluded that no further change in the pI of the masked protein could be observed beyond a molar excess of 100 equivalents. Similar results were obtained with other anhydrides (data not shown).

[0375] HSA-PEIx8 masked with an 85% molar excess of citraconic anhydride exhibited an IEF profile similar to that of native HSA. This masked HSA-PEIx8 was further used to evaluate the stability of the mask in different pH environments. Citraconic anhydride-masked HSA-PEIx8 was incubated at different pHs, and samples were collected at 0, 24, and 72 hours for IEF analysis. We found that the masking at pH 4 was extremely unstable compared to HSA-PEIx8 and was practically removed within 24 hours. We also noticed that this masking agent exhibited instability at pH 6. An interesting observation was that this agent was gradually removed at pH 6.8, which is characteristic of the TME, and the level of removal was much greater than that observed at pH 7.4, where only trace removal was observed within 72 hours. This suggests that citraconic anhydride has potential as a covalent masking agent for the PEI-modified carriers of the present invention. Maleic anhydride, in contrast, exhibits high stability at all pHs except pH 4, where complete removal was observed at 96 hours, but only slight removal was observed at early exposure times to this pH.

[0376] Citraconic anhydride is an asymmetric anhydride because it has one methyl substitution. Given this asymmetry, the reaction of citraconic anhydride with amines can have two structural products, known as the kinetic product and the thermodynamic product. The former is predicted to be somewhat less stable due to the resulting steric hindrance. HSA-PEIx8 was masked with citraconic anhydride under thermodynamic conditions (20°C, 2 h) and kinetic conditions (5°C, 10 min). The product under the kinetic conditions is likely not entirely the kinetic product, but rather is enriched in the kinetic product. Both products were further maintained at 2–8°C and their stability was further evaluated by IEF at pH 7.4 and 6.8, levels relevant to in vivo settings.

[0377] The thermodynamic and kinetic products (48 h incubation at the corresponding pH) were analyzed in parallel on IEF gels at each time point (data not shown). The IEF profiles of both products appear very similar at the beginning of the experiment, but the masked preparation enriched in the kinetic product is significantly less stable at all time points.

[0378] HSA-PEIx8 was compared to citraconic anhydride-masked HSA-PEIx8 in an in vivo setting for evaluation of their pharmacokinetic profiles and biodistribution. Masked HSA-PEIx8 was produced under the kinetic conditions described above using a molar excess of 150 equivalents. MALDI-ToF MS analysis was used to quantify the number of masking agents covalently attached to HSA-PEIx8. Based on MALDI-ToF MS, we concluded that masking resulted in a significant mass shift calculated to correspond to the addition of approximately 44 moles of citraconic anhydride (see Table 2).

[0379] HSA-PEIx3.5 and HSA-PEIx8 were modified with several maleic anhydride derivatives. The level of masking was controlled by the equivalent amount of masking agent in the reaction system. The level of masking and its effect on the molecular charge were evaluated by mass spectrometry and zeta potential, respectively (see zeta potential results in Table 1 and MS results in Table 2). To investigate the potential of the masking agents to be cleaved within the tumor microenvironment (reported pH ∼6.8), the stability of the masked carriers (HSA-PEIx3.5 or HSA-PEIx8) was tested in vitro by incubation at different pHs at 37 °C. IEF gels were used to assess masking removal. Masking removal resulted in an increase in the pI of the protein.

[0380] Based on the experimental results, the masking group derived from citraconic anhydride was unexpectedly found to be advantageous due to its stability at pH 7.4 and gradual deprotection over time at pH 6.8. Other protecting groups showed poor stability at pH 7.4 (greater deprotection) or pH 6.8 (less deprotection). [Table 1] [Table 2]

[0381] As shown in Table 1, dimethylmaleic anhydride-masked HSA-PEI has a strongly positive zeta potential, nearly identical to that of unmasked HSA-PEI. The inventors hypothesize that this is due to the instability (rapid deprotection) of dimethylmaleic anhydride. A similar phenomenon was observed with aconitic anhydride-masked HSA-PEI, which showed partial deprotection, as confirmed by the zeta potential values ​​shown in Table 1. Therefore, the inventors hypothesize that citraconic anhydride, along with maleic anhydride, is characterized by sufficient chemical stability. Furthermore, as disclosed above, citraconic anhydride appears to be preferred due to its stability at neutral or higher pH (greater than about 7 or 7.4) and substantial deprotection at slightly acidic pHs of about 6.8 or less.

[0382] This deprotection, which occurs at acidic pH, was found to be essential for intracellular delivery. Anti-E7 VHH (described previously) was conjugated to both citraconic anhydride-masked and unmasked HSA-PEIx3.5. Both conjugates were evaluated for their ability to internalize into cells. The unmasked conjugate exhibited clear cellular internalization (Figures 28A-28C), whereas the masked conjugate was not internalized at all (Figures 28E-28F). This indicates that without the positive charge of the PEI modification, the conjugate completely loses its ability to attach to the cell membrane and utilize the endocytic mechanism for internalization.

[0383] To further test the necessity of unmasking for internalization, fluorescently labeled carriers (HSA) masked with citraconic anhydride (CA, a transient acid-sensitive masking agent) or methyl succinic anhydride (MSA, a stable masking agent) were used. PEIx8 Fluorescent labeling was performed using a 2.5 molar excess of Atto-542 (ATTO-TEC, catalog no. AD542) with a maleimide moiety directed towards modification on the free cysteine ​​at position 34 of HSA. PEIx8Mass spectrometry showed that only one modification was observed (data not shown). Unmasking was achieved by incubation at 37°C, pH 4 for 8 hours on all three carriers (HSA PEIx8 ;HSA PEIx8 CA;HSA PEIx8 MSA). The labeled proteins before and after unmasking were tested for their ability to internalize into B16 melanoma cells during a 24-hour incubation. Detection of internalization was performed by confocal microscopy as before. As can be seen in Figures 29A-29F, intracellular fluorescence was observed only after incubation with unmasked carriers (Figures 29A, 2D) or CA-masked carriers after unmasking (Figure 29E). Indeed, carriers masked with stable, non-transient masking failed to enter cells, even after acid treatment (Figures 29C, 29F).

[0384] Next, we investigated whether the payload-masked carrier conjugates could be internalized into cells as well as function after in vitro unmasking (8-hour incubation at pH 4 and 37°C). This in vitro treatment was used to mimic the effects of acidic conditions found in the tumor environment as well as other acidic environments that occur in various health states. To test functionality, we used the anti-BRAF VHH, 1C5 (1C5-Hel-masked HSA), conjugated via a linker to a citraconic anhydride-masked carrier with an average of 3.5 PEI modifications. PEIx3.5) was used. Different cancer cell lines were treated with this agent before and after in vitro unmasking (pH 4, 37°C, 1 or 8 hours). Its unmasked counterpart was used as a control. As seen in Figure 30A, the unmasked conjugate had a cytotoxic effect on treated MEL-526 melanoma cells, as predicted by intracellular delivery of the BRAF drug. Unmasking had no effect in this case because there was no mask to remove. The masked conjugate, however, had no effect on treated cells because the masking effectively prevented internalization (Figure 30A). In vitro acidic treatment was able to remove the masking, exposing the PEI modification and once again allowing internalization of the conjugate into the cells. This could be seen by cell death, which was very similar to that achieved with the unmasked conjugate (Figure 30A). Similar results were observed with SK-MEL-28 melanoma cells, as seen in Figure 30B. Again, anti-BRAF 1C5 irreversibly conjugated to a masked carrier (3.5PEI) had no effect on these cells, whereas the same drug after in vitro acid treatment had a dramatic cytotoxic effect on these cells (an effect similar in magnitude to that produced by the unmasked conjugate).

[0385] In vivo experiments in mice were performed to confirm the effectiveness of masking. HSA-PEIx3.5, HSA-PEIx8, and citraconic anhydride-masked HSA-PEIx8 were injected IV (58 nmol / kg) into C57 mice subcutaneously implanted with B-16 murine melanoma cancer cells. Engraftment was performed for 2 weeks before administration of the different HSA-PEI derivatives. Each derivative was injected into 15 mice, and three animals were bled at each time point. At several time points, animals were also sacrificed, and different organs were obtained for biodistribution analysis. HSA-PEI levels were measured in plasma and organ lysates using an in-house ELISA. Because citraconic anhydride masking also interferes with ELISA detection, samples from animals injected with masked HSA-PEI were treated at pH 4 for approximately 1 hour before their ELISA analysis to completely remove the masking agent.

[0386] The pharmacokinetic profiles of the resulting HSA-PEI derivatives can be seen in Figure 31A. HSA-PEIx8 is characterized by extremely rapid clearance from plasma. Any residual amounts are eliminated more slowly, resulting in an apparent half-life of approximately 15 hours. In contrast, the same HSA-PEIx8 masked with citraconic anhydride is eliminated much more slowly, resulting in a half-life of approximately 27 hours. The masking effect is even more dramatic when examining plasma exposure, calculated as the area under the curve (AUC). The plasma exposure of masked HSA-PEIx8 is 50-fold higher than the exposure level of unmasked HSA-PEIx8. HSA-PEIx3.5 exhibits a half-life of 13 hours, and its PK profile appears more favorable than HSA-PEIx8. However, its plasma exposure is still 5-fold lower than that of masked HSA-PEIx8 (Figure 31B).

[0387] Examination of the levels of different HSA-PEI derivatives in animal organs provides further insight into the low plasma exposure of PEI-modified HSA. As seen in Figure 32A, the organ exposure levels (AUC) for both HSA-PEIx3.5 and HSA-PEIx8 appear to undergo some sequestering in clearance organs, namely the liver, kidney, and spleen. Most of the injected dose of PEI-modified HSA can be found in these organs. In contrast, citraconic anhydride masking removes this sequestering, allowing the masked HSA-PEIx8 to circulate longer in the animal's bloodstream and distribute more homogeneously among different organs and tissues. This finding is highly unexpected and important. The ability to direct a composition away from unwanted organs is essential for therapeutic efficacy. It also allows for the delivery of much lower doses. Both of these aspects will reduce unwanted side effects and off-target effects. Furthermore, although the distribution of the masked agent appears rather homogeneous among organs, one tissue appears to be more exposed to the masked agent: the tumor (Figure 32A). The tumor was exposed to 2-7 times more masked HSA-PEI than other organs (Figure 32B). This is due to the sensitivity of the masking agent to the acidic environment of the tumor, which leads to its reversal, exposing the positively charged PEI modification, which in turn leads to the initiation of cell adhesion and cytosolic delivery. We have shown above that even at the neutral pH of plasma, masking undergoes slow reversal, but this effect is more rapid in the more acidic tumor environment, which results in the concentration of the carrier of the present invention in this particular environment.

[0388] To ensure that the low amount of masked HSA-PEIx8 in the kidneys was not the result of clearance via urine, animals in this in vivo study were treated in metabolic cages, and their urine was collected over the first 14 hours of the experiment. As shown in Figure 33, only extremely low amounts of the injected carrier, less than 0.1% of the initial dose, were detected in the urine of treated animals. Collectively, all of this data emphasizes that while protection of PEI is necessary to ensure sufficient serum half-life and adequate biodistribution, deprotection of PEI is necessary in target cells (e.g., tumors) to facilitate intracellular delivery.

[0389] Example 11: In vivo results of protein conjugates containing additional protein carriers To evaluate the in vivo compatibility of alternative carriers (non-HSA carriers), an IgG protein (Humira®, Abbvie) was modified with PEI (600 Da) in the presence of EDC to obtain 4PEI modifications, designated IgG-PEIx4. The modified IgG protein was further reacted with citraconic anhydride to mask the positive charges of PEI, designated masked IgG-PEIx4.

[0390] The PK, zeta potential, and mass of two IgG carriers, IgG-PEIx4 and masked IgG-PEIx4, were evaluated. As seen in Table 3, PEI modification dramatically increased the zeta potential value of IgG, while masking changed this value to a much less negative value than that of the unmodified parent IgG. [Table 3]

[0391] Balb-C mice were intravenously injected with the test sample IgG-PEIx4 or masked IgG-PEIx4 at a dose of 120 nmol / kg. At the test time points, the mice were bled, and the blood sample concentrations were assessed by sandwich ELISA (coating: goat anti-human Fab2 (Jackson, catalog no. 109-005-097); detection: donkey anti-human FC HRP (Jackson, catalog no. 709-035-098)). As seen in Figure 34 (and Table 3), the PEI-modified IgG carrier exhibited similar PK parameters (AUC and CL) to HSA-PEIx3.5, demonstrating the characteristic rapid elimination, low AUC, and high clearance. Similar to the HSA carrier, the masked IgG carrier regains its long half-life, extremely high AUC and low clearance, and again, masking with maleic anhydride derivatives, and particularly citraconic anhydride, confers a clinically favorable pharmacokinetic profile to the PEI-modified carriers of the present invention, such as IgG.

[0392] Example 12: In vivo results of PEI-modified antibodies without a protein carrier To further test the ability of transient masking to overcome the high clearance and short plasma half-life of the antibody, the single-domain antibody directly modified with PEI was further masked with citraconic anhydride. Specifically, the active anti-E7 VHH described above was produced without a carrier and irreversibly modified with PEI (1800 Da) at the C-terminus of the payload (adding a GGGGSC linker to the C-terminus), which is referred to herein as VHH αE7-PEI1800. The PEI-modified VHH was further masked with citraconic anhydride to obtain a masked PEI-modified protein, designated masked VHH αE7-PEI1800.

[0393] The zeta potentials of two compounds, VHH αE7-PEI1800 and masked VHH αE7-PEI1800, were compared with those of native, unmodified VHH. Furthermore, the PK of the PEI-modified VHH and its masked counterpart was evaluated in mice. Balb-C mice were intravenously injected with the test samples (VHH αE7-PEI1800 or masked VHH αE7-PEI1800) at a dose of 120 nmol / kg. At the test time points, the mice were bled, and the blood sample concentrations were assessed by sandwich ELISA (coating: streptavidin (Prospec, catalog no. Pro-791-b), followed by rabbit anti-VHH + biotin (A2S, catalog no. A01995-200), detection: rabbit anti-VHH cocktail-HRP (A2S, catalog no. A02016-200)).

[0394] As can be seen in Table 4 below and Figure 35, the PEI-modified VHHs had extremely high clearance from mouse plasma and extremely low AUC. Masking the positive charge, which was shown to restore the adverse PK effects of PEI modification in the carriers of the present invention, improved the PK parameters of the PEI-modified VHHs. The effect of masking on larger carriers, such as HSA and IgG, was quite significant, whereas the effect on small PEI-modified proteins was less pronounced. Although masking itself prevented the effect of the strong positive charge, i.e., sequestration in the liver, spleen, and kidney, their small size still resulted in high clearance for simple size-related reasons.

[0395] To this end, the inventors envisioned that in addition to the use of a masking agent, the protein conjugates of the present invention must include a carrier to facilitate delivery of the payload to a target site within a subject. [Table 4]

[0396] Example 13: In vivo results of anti-HPV-E7 VHH The previously described anti-HPV-E7 VHH was conjugated to HSA-PEIx3.5 via a non-cleavable linker containing a PEG11 chain and bearing two terminal maleimide groups, both of which are reactive toward thiol groups. The VHH bearing a C-terminal cysteine ​​(a GGGGSC linker at the C-terminus) was treated with a reducing agent (TCEP) to liberate the terminal cysteine, which was then reacted with a bis-Mal agent. After chromatographic purification, the VHH, now bearing terminal maleimide groups, was reacted with HSA (an average of 3.5 per HSA) previously modified with PEI and citraconic acid for masking to yield an exemplary protein conjugate of the invention (αE7-VHH-S-Mal-PEG), as shown in the schematic diagram below. 11 -Mal-S-HSAx3.5) was obtained: [ka]

[0397] The resulting masked αE7-VHH-S-Mal-PEG11-Mal-S-HSAx3.5 was used to treat athymic nude mice subcutaneously implanted with HeLa-GFP cells. The same molecule without PEI modification was used as a negative control. The conjugate of the present invention without PEI modification showed no effect on tumor growth, as expected (Figure 36A). The PEI-modified conjugate had minimal effect on tumor growth, suggesting that without masking, drug availability to tumor cells is minimal. This is likely due to the stickiness of the positively charged conjugate, as exemplified above in the poor pharmacokinetic and biodistribution profiles exemplified for the unmasked carrier (Figures 31A-31B and 32A).

[0398] Next, the masked αE7-VHH-S-Mal-PEG11-Mal-S-HSAx3.5 was injected into the tumors approximately 100 mm after tumor engraftment. 3The mice were intravenously injected with αE7-VHH-S-Mal-PEG11-Mal-S-HSAx3.5 when the average tumor volume reached 100 μg (250 nmol / Kg). Treatment included regular administration of drugs and controls. Citraconic anhydride-masked HSAx3.5 and vehicle (PBS) were administered as controls. Mice treated with masked αE7-VHH-S-Mal-PEG11-Mal-S-HSAx3.5 showed significant tumor growth inhibition compared to controls (Figure 36B, two-tailed t-test p<0.05 masked vehicle vs. masked αE7-vehicle). Furthermore, treatment was well tolerated by all animals, as evidenced by the lack of any adverse events and the animals' normal weights (data not shown).

[0399] Somewhat limited tumor growth inhibition was observed with anti-E7 VHHs with estimated K values ​​above 1 μM. D This can be attributed to the fact that the IL-16A1 fusion protein has a limited affinity for the E7 protein.

[0400] The experiment was repeated following the same protocol with the following modifications: 1) the drug dose was increased to 350 nmol / Kg, and 2) injections were performed daily for 15 consecutive days. Animals continued to be monitored for tumor volume for an additional 24 hours. As seen in Figure 36C, tumor growth inhibition similar to that of the first study was observed, with an initial tumor volume of 97 mm at the start of treatment. 3 This was observed when examining tumor volumes in treated animals that were less than 100%. As expected from the higher doses and more frequent administration, tumor growth inhibition was first observed at earlier time points, and the magnitude of the effect was also greater.

[0401] Surprisingly, the inhibitory effect persisted even after drug administration was stopped (Figure 36D). In fact, the maximum effect was maintained for an additional 5 days and was present 24 days after cessation of treatment. The anti-E7 drug used here was permanently linked to the carrier. We previously observed that the carrier formed clusters or aggregates upon escape from endosomes (Figure 28B). This may explain the persistence of the inhibitory effect, as the aggregates could act as a kind of depot, slowly releasing additional drug into the cytoplasm over time.

[0402] Tumors from treated animals in the initial study were obtained and analyzed for the presence of VHH drug and HSA carrier using immunohistochemistry. Cross sections were analyzed by H&E and for VHH or HSA carrier. PEI Tumors from animals treated with PBS (Figures 37A-37B) showed no staining for the VHH or carrier. Tumors from animals treated with the masked carrier showed clear and strong staining for the carrier but no staining for the VHH payload (Figures 37A-37B, center). Tumors from animals treated with anti-E7 VHH conjugated to a masked carrier showed clear staining for both the carrier and the VHH payload (Figures 37A-37B, right). Most interestingly, staining was more pronounced in tumor necrotic areas. Furthermore, the presence of both the carrier and intact conjugate was observed throughout the tumor and was not restricted to specific areas, such as near major blood vessels. This latter observation suggests efficient distribution of the payload-carrier conjugate throughout the tumor tissue.

[0403] To confirm that the staining observed in tumor tissues was indeed specific and not background staining of necrotic areas, isotope control staining was performed on all tissues tested and compared across all treatments. Negative staining in all samples was observed when using an isotype control (anti-rabbit IC rabbit (DA1E) mAb IgG XP isotype control #3900, normal goat IgG, Sigma NI02-100UG) (data not shown). This confirms specific necrotic area staining.

[0404] It should be emphasized that the detection of the carrier in these sections was achieved using a polyclonal antibody raised against PEI-modified HSA, which does not cross-react with unmodified HSA. Furthermore, the masking of PEI-modified HSA by citraconic anhydride actually renders the masked carrier undetectable by this polyclonal antibody. Therefore, the fact that the carrier, PEI-modified HSA, is clearly detected in these tumor sections indicates that the masking is indeed removed in the tumor microenvironment, allowing the agent of the present invention to enter cells using its exposed PEI modification.

[0405] Example 14: In vivo results of anti-BRAF VHHs The in vivo efficacy of intracellular delivery using masked drugs was also tested using a second VHH. Anti-BRAF VHH (1C5) was irreversibly conjugated to masked HSA (3.5xPEI). 1C5-Mal-PEG for mice 11 The toxicity of -Mal-masked-HSA-PEIx3.5 was evaluated and compared with the effect of the carrier alone (masked HSA-PEIx3.5). Two strains of mice (C57BL and NOD-Scid) underwent a dose-escalation routine. The test drug was intravenously infused at a drug administration volume of 200 μL per infusion. Dose escalation began with 50, 100, 250, and 350 nmol / kg every other day, followed by three 350 nmol / kg every other day IV infusions, and finally, five days of daily 350 nmol / kg IV infusions. Mice were monitored for clinical signs of morbidity or mortality, including changes in skin, fur, eyes, mucous membranes, gait, secretory excretion, weight loss, and general health. No clinical signs were observed in any of the mice tested. In addition, at the end of the dose-escalation and repeated administration, all mice were terminally anesthetized with ketamine-xylazine cocktail (IP). Animals were perfused transcardially with PBS followed by 4% PFA. Organs (liver, heart, kidney, lung, brain, spleen) were harvested and stained with hematoxylin and eosin (H&E). No pathological changes were observed between the two groups.

[0406] The payload used in this evaluation was a nonselective anti-BRAF agent, which has been shown to inhibit both wild-type and mutated BRAF of both human and mouse origin. The inventors have previously demonstrated that the payload-masked carrier conjugate of the present invention biodistributed to virtually all organs and tissues after administration. Therefore, it is somewhat surprising that such a nonselective inhibitor of a critical cellular enzyme has no apparent toxic effects on treated animals. It should be noted, however, that masking counteracts the effect of the positive charge and thus prevents intracellular delivery unless the masking agent is removed. Because masking is pH-sensitive, biodistribution results indicated that more of the conjugate reached tumor tissue, which is characterized by a lower pH environment than normal, healthy organs and tissues. It is therefore the inventors' hypothesis that although the conjugate reaches all organs and tissues, it encounters neutral or slightly higher-than-neutral pH conditions (physiological pH) there, thereby preventing the masking and internalization of the conjugate and its toxic payload. Because these animals did not have inoculated tumors, the conjugates did not encounter the acidic tumor microenvironment and did not exhibit cellular internalization. Even if some unmasking occurs to a small extent in healthy organs and tissues, the amount that enters such cells is likely to be extremely small and ineffective.

[0407] To further investigate this point, the carrier (masked HSA PEIx3.5 ) and 1C5-Mal-PEG, which suppressed tumor growth. 11 -Mal-masked HSA PEIx3.5 The ability of this compound to induce BRAF-mediated BRAF replication was evaluated in both mouse strains. C57BL mice were subcutaneously injected with B16 tumor cells (a murine melanoma) and NOD-Scid mice were subcutaneously injected with MEL-526 (human BRAF-overexpressing melanoma cells). Testing in C57BL mice was performed on mice with well-established tumors (115 mm 3 ), while the NOD-Scid mice were tested when tumors were in the early growth stage (approximately 6 mm 3These models therefore demonstrate the ability to treat as well as prevent cancer. In both cases, the agent tested, 1C5-Mal-PEG 11 -Mal-masked HSA PEIx3.5 is just a masked HSA with no payload PEIx3.5 The 1C5-Mal-PEG showed suppression of tumor growth compared to the 1C5-Mal-PEG (Figures 38A-38B). 11 -Mal-masked HSA PEIx3.5 (αBRAF-M-carrier) had a slight inhibitory effect on B16 growth (Figure 38A). Note that B16 tumors were highly aggressive and had a rapid growth rate. Therefore, this slight growth inhibition still indicates a significant functional effect of the masked conjugate on tumor growth. In the case of MEL-526 tumors inoculated into NOD-Scid mice, the masked conjugate had a more significant inhibitory effect on tumors, showing a 47% reduction in tumor volume at the end of the measurement period (Figure 38B).

[0408] This data further demonstrated selective unmasking in acidic environments, such as those found in tumors. While nonspecific anti-BRAF VHHs had no measurable effect on healthy mice, exposure to the acidic TME resulted in antitumor effects. This allows for the use of site-specific and nonselective payloads, conferring cytotoxicity or biological effects only on cells found in these low-pH environments. Furthermore, this suggests that while the conjugates of the present invention reach all organs and tissues, they do not actually penetrate into the cells of these organs and tissues. Because the payload is directed to an intracellular target, these conjugates do not exhibit activity in healthy tissues, greatly reducing the risk of off-target effects.

[0409] Example 15: PK and biodistribution of different constructs with αE7 VHH payload The pharmacokinetic profiles and biodistribution of different masked constructs were evaluated in mice inoculated with B16 tumors. All constructs carried an anti-E7 VHH (with a single repeat G4SC linker as in the MAL construct or a triple repeat G4SC linker as in the PEP construct) as the payload. The VHH was conjugated to a carrier modified with 3.5 or 8 PEI and masked with the transient masking agent citraconic anhydride (CA). The VHH was conjugated to these carriers via a reversible disulfide bond or an irreversible bond using maleimide chemistry. The anti-E7 VHH was also irreversibly conjugated to a carrier modified with 8 PEI that had been pre-masked with the irreversible masking agent methyl succinic anhydride (MSA), which is similar in structure to citraconic anhydride but lacks a double bond.

[0410] The biodistribution and pharmacokinetic profiles of the above drugs were evaluated using B16 tumor-bearing C57BL mice. 3 Once the mice reached an average volume of 1000 mg / kg, they received a single IV infusion of the various drugs at 250 nmol / kg. Plasma and organ samples were collected 5 and 30 minutes, and 2, 6, 24, 48, and 72 hours after administration. Analysis of the constructs in both plasma and organs was performed using an in-house ELISA for the VHH versus the payload alone. Figure 39 shows the pharmacokinetic profiles of the different constructs in inoculated animals. Table 5 summarizes the pharmacokinetic parameters of each construct. [Table 5]

[0411] The pharmacokinetic profiles and parameters are very similar between the different constructs. This is likely a result of the similar pI of the final constructs and their virtually identical size. Importantly, the effect of masking is virtually the same regardless of the level of PEI modification. Similarly, different linkers do not appear to have any effect. Note that the MSA-masked construct actually exhibits a somewhat longer plasma residence time and lower clearance, likely due to the fact that its masking is not removed and therefore its uptake into cells is minimal or nonexistent. It therefore persists at higher levels in plasma.

[0412] Different organs, including tumors, were harvested and VHH levels were analyzed by appropriate in-house VHH ELISAs. Figure 40 summarizes the organ exposure of the different conjugates. These biodistribution data show no dramatic differences between the conjugates. Note that the kidney exposure data for the reversible linker conjugates is an overestimate because the material contained contaminating free VHH. Although this free VHH is rapidly cleared from the circulation via the kidney, the initial measurement in this organ measures this free VHH and influences the overall AUC analysis. In both cases, it can be seen that there is an enrichment of the payload in the tumor compared to other organs. This is consistent with the enrichment observed with the carrier alone.

[0413] While pharmacokinetic and biodistribution data show no dramatic differences between different masked conjugates with different PEI levels and linker reversibility, ELISA only indicates the total amount of drug present in the tissue / tumor but cannot distinguish between external and internalized payloads. To this end, the fluorescently labeled carrier molecules described above were tested in vivo. HSA PEIx8 CA and HSA PEIx8MSA was injected into athymic nude Foxn1 mice bearing HeLa-GPF tumors. The different carriers were injected at a dose of 180 nmol / kg in 200 μL. After 6 hours, the tumors were harvested and the distribution of the different carriers was evaluated by confocal microscopy. As can be seen in Figure 41A, the fluorescently labeled CA-masked carrier (HSA) PEIx8 CA) was clearly detected inside tumor cells (tumor cells were identified by GFP fluorescence). In fact, the carrier appeared to coat the nuclei of the cells, indicating that it was dispersed throughout the cytoplasm of these cells. In contrast, fluorescently labeled MSA-masked carriers (HSA PEIx8 MSA) was detected only outside and between cells, not in their cytoplasm (Figure 41B, arrows indicate the cytoplasm around tumor cells, which is not stained red). The effectiveness of transient masking is clearly demonstrated, as the carrier cannot efficiently penetrate into tumor cells with permanent masking. Similar results were observed in B16 tumors in mice treated with CA-masked conjugates (Figure 41C), MSA-masked conjugates (Figure 41D), and unmasked conjugates (Figure 42E). Although tumor cells did not emit fluorescence in this tumor, it is clear that the conjugate significantly penetrated cells only with transient masking, but not with permanent masking. Furthermore, masking clearly concentrated the amount of conjugate that reached the tumor.

[0414] Furthermore, as can be seen in Figures 42A-42B, the MSA-masked carrier is virtually undetectable in tumor cells. Tumor cells are characterized by double staining (green and blue) of their nuclei. However, the carrier is readily detectable only in areas containing non-tumor mouse cells (characterized by single blue nuclear staining). Based on their shape and morphology, these cells are vascular endothelial cells. This suggests that due to the non-transient nature of this masking, the negatively charged masked carrier is unable to efficiently cross the endothelial barrier and distribute into tumor tissue and tumor cells. When non-tumor tissues, i.e., tissues without an acidic microenvironment (e.g., liver), were examined, the localization of the conjugate was similar between the two masking agents in that it was unable to enter cells and was primarily localized intercellularly (Figures 42C-42D). Taken together, this data clearly demonstrates that although the pharmacokinetic and biodistribution profiles of transiently masked carriers and conjugates are very similar to those of the corresponding non-transiently masked conjugates, there are dramatic differences in their distribution in tumor tissue and their ability to be internalized into tumor target cells while avoiding exposure to healthy cells. These differences cannot be confirmed using detection tools such as ELISA, which cannot distinguish between moieties that are inside or outside the cell.

[0415] Example 16: Complete masking Up until now, only masking of the carrier itself has been tested. To further enhance the selectivity of activity, masking of the entire conjugate, including the payload, was tested. Such masking may reduce or completely inhibit the activity of the payload until the masking is removed near the site of action, or just after internalization into the cell itself after further masking removal in the endosome, thus avoiding off-target effects. Masking of the entire conjugate may also have advantages related to various processes. For this purpose, 1C5-S-Mal-PEG 11 -Mal-S-HSA-PEIx3.5 and 1C5-S-Mal-PEG 11-Mal-S-HSA-PEIx8 was masked at different masking levels, controlled by the excess citraconic anhydride (CA) used in the masking step. The resulting masking levels were assessed by IEF and zeta potential measurements. Furthermore, the number of masking moieties conjugated to the drug was also analyzed and quantified by MALDI-ToF mass spectrometry. As seen in Table 6 below, supported by the corresponding IEF gels (data not shown), masking of all conjugates can also counteract PEI modification and generate negative charges, thus lowering the zeta potential of these conjugates. [Table 6]

[0416] As with the masking of the carrier alone, the masking of the complete conjugate can be removed in vitro by incubation with citrate buffer at pH 4 at 37° C. After 8 h, complete unmasking was achieved regardless of the PEI level on the carrier (IEF results not shown).

[0417] The activity of the masked payload was further tested after unmasking. To this end, a BRAF binding ELISA was performed on the anti-BRAF payload (1C5) after unmasking. In this assay, the presence of the carrier (HSA-PEI) was observed to give a high irrelevant signal, likely due to adhesion of the highly positively charged protein to the ELISA plate. Therefore, only in this case was the payload, 1C5-Hel-LC, used and masked with citraconic anhydride (masking via the positive lysine residue in the payload). The masked payload was tested for its ability to bind BRAF before and after unmasking; the binding ability of the anti-BRAF VHH was virtually lost after masking (Figure 43A). However, unmasking completely restored binding. In addition, the change in payload charge after masking and unmasking was assessed by IEF (data not shown) and zeta potential (Table 6). A clear decrease in the zeta potential of the payload after masking was observed, which is associated with the excess masking agent used. Furthermore, it can be clearly seen that the acidic conditions completely removed the masking, as the zeta potential of the payload was restored to its original value.

[0418] Similar to the carrier-masked conjugates (Figures 30A-30B), the fully masked conjugates were also shown to recover their in vitro cytotoxic activity after unmasking. As seen in Figure 43B, the 1C5 payload expressed with a rigid helical linker and further conjugated to a carrier with 3.5 PEI regained its activity after unmasking. Furthermore, fully masked 1C5-HSA-PEIx3.5 and fully masked 1C5-HSA-PEIx8 had no effect on MEL-526 cells. These conjugates regained their activity (equivalent to that of the fully unmasked 1C5 conjugate) after in vitro unmasking (Figure 43B; data for PEIx3.5 not shown).

[0419] Example 17: Partial Masking To identify the level of masking that prevents internalization, or in other words, the level of positive charge required for internalization, HSA-PEIx3.5 was labeled with an appropriate fluorescent dye (ATTO542 or ATTO647N). The labeled protein was then masked with the irreversible masking agent, methyl succinic anhydride (MSA), at different molar equivalents to obtain carriers with various levels of masking. The level of masking for each protein was assessed by IEF and zeta potential. The ability of carriers labeled with different masking levels to internalize into cells was assessed by 16-hour incubation with A375 cells, followed by detection of cells with fluorescent signal by flow cytometry. Fluorescent labeling was performed on HSA using a 2.5 molar excess of ATTO-542 (ATTO-TEC, catalog no. AD542), which has a maleimide moiety directed toward modification on the free cysteine ​​at position 34 of HSA. PEIx3.5 or a 5 molar excess of ATTO-647N (ATTO-TEC, Cat. No. AD647-41) bearing a maleimide moiety to HSA PEIx3.5 For this assay, irreversible masking rather than reversible masking (with citraconic anhydride) was used to avoid potential unmasking during the internalization assay. IEF analysis showed a strong correlation between the pI and the level of MSA reagent used in the masking reaction (data not shown). The results for ATTO542 are summarized in Table 7. [Table 7]

[0420] Zeta potential measurements were performed on ATTO647N-labeled constructs (unmasked and masked with various molar excesses of MSA) and yielded similar results, with up to a 60 molar excess of MSA exhibiting a positive zeta potential, and an 80 or more molar excess (up to 200 molar equivalents) of MSA exhibiting a negative zeta potential.

[0421] A375 cells (0.5x10 per well) 6) were seeded for 12 hours, and then 20 (or 50) ng of labeled carrier (HSA PEIx3.5 ATTO647N) was added to the wells. The samples were incubated for 16 hours. At the end of the uptake period, the upper medium was washed away, and the cells were detached from the plate and washed with chilled PBS. The amount of carrier internalized into the cells was assessed by flow cytometric detection of ATTO647N, compared with an isotope control sample. Internalization decreased with increasing masking levels (Figure 44). Furthermore, internalization levels equivalent to the at least 20% internalization achieved with unmasked HSA-PEIx3.5 were achieved when up to a 60 molar excess of MSA was used in the reaction. All these samples gave positive values ​​in zeta potential analysis, while samples with negative values ​​did not even show 10% internalization. Therefore, a zeta potential below zero is required for functional masking. Based on the fact that citraconic anhydride masking introduces a negative charge to the conjugate, it is estimated that approximately 50% or more of the amine groups (e.g., the amine groups of PEI) need to be masked. Thus, the inventors estimate that the molar ratio of protected to unprotected amines in the protein conjugates of the invention (e.g., within the cell-penetrating moiety) is at least about 7:10, at least about 1:1, or includes an excess of protected amines, such as from about 1:1 to 100:1 (including any range therebetween).

[0422] Example 18: Tumor Targeting - Anti-PSMA While masking results in the enrichment of the conjugate in acidic target regions, additional targeting may be beneficial. To this end, the inventors investigated the use of an additional targeting moiety. This could be any protein domain or antibody-like structure selected for its ability to bind extracellular markers on target cells. A second VHH was selected as the targeting moiety and expressed in tandem with the payload VHH targeting an intracellular target. Masking allows the use of a targeting moiety because it eliminates strong electrostatic binding, allowing the masked conjugate to move around plasma and organs and find its target.

[0423] Prostate-specific membrane antigen (PSMA), known as JVZ-007, was selected as the targeting moiety. PSMA is known to be presented on prostate cancer cells and has been widely used to target various imaging agents to prostate tumors. Tandem agents were generated containing JVZ-007 and anti-BRAF, 1C5, VHH. Two tandemly expressed agents were generated, one with the anti-PSMA VHH expressed at the N-terminus and one at the C-terminus, creating αPSMA(JVZ-007)-Hel-αBRAF(1C5)-Hel-L-Cys and αBRAF(1C5)-Hel-αPSMA(JVZ-007)-Hel-L-Cys, respectively. Both ends with C-terminal cysteines allow for conjugation to carriers.

[0424] The binding of the two constructs (before conjugation to carrier) to PSMA-positive cells (LNCaP clone FGC, prostate cancer, ATCC No. CRL-1740) was assessed by FACS and compared to the binding of these constructs to PSMA-negative cells (PC-3, prostate cancer, ATCC No. CRL-1435). The binding of the active VHH, 1C5, alone to these cells was also assessed.

[0425] As expected, the anti-PSMA VHH strongly bound PSMA-positive cells and showed no binding to PSMA-negative cells. Interestingly, both agents, including the anti-PSMA VHH expressed in tandem with the anti-BRAF VHH, also showed extremely strong binding to PSMA-positive cells and no binding to PSMA-negative cells (Figure 45A). Both constructs showed similar levels of binding to PSMA-positive cells. Also as expected, the anti-BRAF 1C5 VHH showed no binding to either PSMA-negative or -positive cells. These results indicate that tandem expression of a targeting moiety with another active moiety does not adversely affect the ability of the targeting moiety to recognize and bind to its target.

[0426] The described agents were further conjugated to the masked carrier of the present invention, and their binding to PSMA-positive cells was evaluated by FACS as before. The anti-PSMA containing conjugate (JVZ-1C5-HSA-PEIx3.5-CA) showed binding to PSMA-positive cells (LNCaP prostate cancer cells) but not to negative cells (MEL-526 melanoma cancer cells) (Figure 45B). This indicates that conjugation to the masked carrier does not inhibit target binding. The unconjugated payload, JVZ-1C5, bound to PSMA-positive cells equivalently to the conjugated payload, indicating that the carrier did not affect the binding of the targeting moiety. Another anti-PSMA containing conjugate (1C5-JVZ-HSA-PEIx3.5-CA) also showed binding to LNCaP cells but not to PSMA-negative cells (data not shown). None of the tested agents showed binding to PC3 prostate cancer cells, which are PSMA-negative (data not shown).

[0427] Having shown that expressing the anti-PSMA moiety in tandem with the anti-BRAF moiety does not interfere with its ability to bind PSMA, we tested the ability of the tandem constructs to still recognize and bind BRAF via the anti-BRAF moiety. This was performed by recombinant BRAF binding ELISA. As can be seen in Figure 46, although binding was slightly weakened compared to the parent anti-BRAF VHH alone, both constructs expressing the anti-BRAF and anti-PSMA moieties in tandem maintained their ability to bind BRAF.

[0428] Next, we evaluated the ability of the tandem constructs with unmasked carriers to inhibit BRAF in cells and cause cell death. The cytotoxic activity of anti-BRAF agents expressed with targeting moieties was evaluated in two different cell lines: MEL-526 (PSMA-negative) and LNCaP (PSMA-positive). In both cell lines, the tandem agents exhibited cytotoxicity, indicating that the constructs were internalized, escaped endosomes, and were able to modulate BRAF regardless of binding to surface moieties (Figure 47). This cytotoxicity was similar in magnitude to that exhibited by anti-BRAF VHHs alone conjugated to the same carriers. These results demonstrate the potential of these agents, which incorporate both targeting and active moieties to function as delivery agents.

[0429] The tandemly expressed anti-PSMA-anti-BRAF (JVZ-1C5) is further conjugated to a masked carrier and its in vivo biodistribution is evaluated. Athymic nude Foxn1nu mice are injected with PSMA-positive cancer cells (e.g., LNCaP tumor cells). The cells are injected at 10 7 The 1C5-masked HSA-PEIx3.5 or JVZ-1C5-masked HSA-PEIx3.5 is injected subcutaneously at 250 nmol / kg in 200 μL. Organs and tumors are harvested at different time points (e.g., 2, 24, and 48 hours) after injection to assess the amount of conjugate in the tumor and other organs. ELISA (e.g., anti-VHH ELISA) is used to assess the total payload of the present invention. Imaging is also performed as described above to assess distribution within the tumor and tumor cells. Total tumor weights of two sets of treated mice are also monitored. Increased delivery to tumors and into tumor cells is observed with the tandem JVZ-1C5-masked HSA-PEIx3.5 agent as the delivery moiety increases tumor targeting. Increased tumor volume also leads to a concomitant decrease in other healthy tissues. Increased tumor cell killing, as measured by a reduction in tumor burden, is also observed with tandem molecules containing targeting moieties.

[0430] While the present invention has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations are possible. Accordingly, the present invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. a. a protein carrier covalently attached to a cell-penetrating moiety comprising a plurality of amine groups, wherein the cell-penetrating moiety is low molecular weight polyethyleneimine (PEI); b. a biological payload that interacts with an intracellular target; and c. a linker between the protein carrier and the biological payload; A protein conjugate comprising: at least a portion of the amine groups are bound to protecting groups; the protecting group is capable of undergoing cleavage at a pH value of less than 7; and protein conjugates characterized by a negative zeta potential.

2. The protein conjugate described in claim 1, wherein at least 50% of the multiple amine groups are bound to the protecting group.

3. The protein conjugate of claim 1 , wherein the linker is covalently linked to the carrier, the payload, or both.

4. The protein conjugate of any one of claims 1 to 3, wherein the protecting group comprises a moiety that is negatively charged at a pH between 6 and 8.

5. The protein conjugate of claim 4 , wherein the moiety comprises a carboxy group.

6. The protecting group has the formula 1: 【Chemistry 1】 wherein n is an integer ranging from 0 to 5; 【Chemistry 2】 represents the point of attachment to the amine group, and 【Transformation 3】 represents a single or double bond; R and R １ each independently represent a substituent selected from H, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aryl or heteroaryl, and carboxyalkyl, or a combination thereof; or R and R １ are linked together to form a ring.

7. R and R １ is H and one of R and R １ The protein conjugate of claim 6 , wherein another one of the groups comprises alkyl or carboxyalkyl.

8. The protecting group is 【Chemistry 4】 and R and R １ is selected from H and methyl, and R or R １ is methyl, and optionally the protecting group is derived from citraconic anhydride.

9. The protein conjugate according to any one of claims 1 to 3, wherein the low molecular weight PEI is a PEI having a molecular weight of 100 to 1000 daltons.

10. a. the biological payload is an antigen-binding molecule that binds the intracellular target; b. the biological payload lacks a disulfide bond that, upon cleavage, reduces interaction with the intracellular target; and c. The antigen-binding molecule is selected from a single-chain antibody, a single-domain antibody, a variable heavy chain homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a designed ankyrin repeat protein (DARPin), and an antibody mimetic protein; The protein conjugate according to any one of claims 1 to 3, which is at least one of:

11. 4. The protein conjugate of claim 1, wherein the protein carrier or biological payload comprises a plurality of PEI molecules, optionally the protein carrier comprises 2 to 30 PEI molecules.

12. 4. The protein conjugate of claim 1, wherein the protein carrier is human serum albumin (HSA). Optionally, the HSA comprises 3 to 10 PEI molecules.

13. The protein conjugate of claim 1 , wherein the linker comprises a biocompatible polymer, a biodegradable polymer, or both.

14. 14. The protein conjugate of claim 13, wherein the biocompatible polymer comprises polyethylene glycol (PEG), and optionally the biodegradable polymer comprises a polyamino acid, and the linker further comprises a spacer covalently bonded to (i) the biocompatible polymer or the biodegradable polymer and (ii) the protein carrier, and wherein the covalent bond is via a click reaction product.

15. 4. The protein conjugate of claim 1, wherein the linker comprises a biocleavable bond, optionally wherein the biocleavable bond comprises a disulfide bond, and optionally wherein the biocleavable bond is sterically hindered.

16. 13. The protein conjugate of claim 12, wherein the HSA comprises the amino acid sequence of SEQ ID NO: 1, or a fragment or homolog thereof comprising cysteine ​​34 (C34).

17. 17. The protein conjugate of claim 16, wherein the linker is attached to the HSA via a disulfide bond, and optionally the linker is attached to the C34 of the HSA.

18. 18. The protein conjugate of claim 17, wherein the disulfide bond is proximal to the C34, proximal being a distance from the C34 in the range of 5 to 15 angstroms.

19. The protein conjugate of any one of claims 1 to 3, wherein the protein carrier lacks DNA or the biological payload does not bind a cell surface protein.

20. The protein conjugate according to any one of claims 1 to 3, wherein the protein conjugate is characterized by a negative zeta potential of less than -1 mV.

21. 4. The protein conjugate of claim 1 or 3, further comprising a detectable tag, optionally conjugated to the biological payload.

22. 4. The protein conjugate of claim 1, wherein the protecting group is derived from citraconic anhydride and optionally the click reaction product is a succinimide thioether.

23. The protein conjugate of any one of claims 1 to 3, further comprising a targeting moiety that binds to a protein expressed on the surface of a target cell.

24. a. the targeting moiety is selected from a single chain antibody, a single domain antibody, a variable heavy chain homodimer (VHH), a nanobody, an immunoglobulin novel antigen receptor (IgNAR), a designed ankyrin repeat protein (DARPin) and an antibody mimetic protein; b. the targeting moiety is conjugated to the protein carrier via a linker; and c. the targeting moiety and the biological payload are contained in a single polypeptide, optionally the targeting moiety and the biological payload are separated by a linker; 24. The protein conjugate of claim 23, wherein the protein conjugate is at least one of:

25. 1. A method for producing a charge-masked protein conjugate capable of binding an intracellular target, comprising: a. providing a biological payload that binds to said intracellular target; b. providing a protein carrier covalently attached to a cell-penetrating moiety comprising a plurality of amine groups, wherein the cell-penetrating moiety is low molecular weight polyethyleneimine (PEI); c. providing the biological payload and the protein carrier under conditions sufficient to covalently bond the biological payload to the protein carrier via a linker to produce a protein conjugate; and d. providing the protein carrier under conditions sufficient to protect at least a portion of the amine groups with protecting groups that are susceptible to cleavage at a pH value less than 7, to obtain the charge-masked protein conjugate comprising protected amine groups; Including, A method for producing a charge-masked protein conjugate capable of binding an intracellular target.

26. At least one of the following: a) providing the protein carrier under conditions sufficient to protect occurs before or after conjugating the biological payload to the protein carrier; b) the protein carrier comprises HSA; c) the charge-masked protein conjugate is characterized by a negative zeta potential; d) the protecting group comprises a moiety that is negatively charged at a pH between 6 and 8, optionally the moiety comprises a carboxy group; e) the protein carrier or biological payload is covalently bound to at least two molecules of PEI, optionally the protein carrier is covalently bound to at least eight molecules of PEI, or the biological payload lacks a disulfide bond that, when cleaved, reduces binding to the intracellular target; f) the linker comprises a biocompatible polymer or a biocleavable bond; g) said covalently linking is via a click reaction or comprises disulfide bond formation; and h) The low molecular weight PEI is a PEI having a molecular weight of 100 to 1000 Daltons.

26. The method of claim 25.

27. A pharmaceutical composition comprising the protein conjugate of any one of claims 1 to 3 and a pharmaceutically acceptable carrier, excipient or adjuvant.

28. 10. An in vitro method of binding an intracellular target, comprising contacting a cell expressing said intracellular target with a protein conjugate of any one of claims 1 to 3, wherein said biological payload binds said intracellular target, thereby binding said intracellular target.

29. a. A method of detecting an intracellular target, wherein the protein conjugate comprises a detectable tag, and further comprises detecting the detectable tag; b. A method of modulating the intracellular target and the biological payload is an agonist or antagonist of the intracellular target; c. the cell expresses a target surface protein and the protein conjugate comprises a targeting moiety that binds to the target surface protein; 29. The method of claim 28, wherein the at least one of

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