Intracellular delivery composition

Abstract

A protein conjugate is provided that includes a protein carrier that includes polyethyleneimine, a payload that interacts with an intracellular target, and a linker. Pharmaceutical compositions that include the protein conjugate, and 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 of U.S. Provisional Patent Application No. 63 / 177,144, filed on April 20, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of intracellular delivery of proteins.

Background Art

[0003] In particular, in the search for new and potent therapeutic targets in the field of oncology, targeting intracellular targets, especially those considered "undruggable", is very difficult. Such targets cannot be targeted by small molecule drugs. This is mainly because the required therapeutic intervention involves interfering with protein - protein interactions and small molecule drugs have not been able to exhibit efficacy and / or sufficient selectivity. Such targets and biological processes are classically and efficiently addressed by biological agents such as monoclonal antibodies, receptors, interleukins, and their derivatives and combinations, etc., but such agents cannot easily penetrate clinically relevant cellular compartments such as living cells, especially the cytoplasm, nucleus, ER, etc. This is because the outer cell membrane is impermeable to protein - based molecules.

[0004] When a protein-based molecule is internalized into living cells by a natural endocytosis-based mechanism, this protein-based molecule enters the endosomal pathway, which causes its lysosomal degradation. Thus, this pathway is, of course, only relevant for the treatment of lysosomal disorders or for antibody-drug conjugates (ADCs). In the former case, the therapeutic protein-based agent is intended to exert its activity in endosomes or lysosomes and is generally designed to withstand or is naturally suitable for the endosomal / lysosomal conditions. In the latter case, the antibody is purely used to target / deliver a small molecule drug to specific cells, and the lysosomal degradation of the antibody carrier releases the small molecule drug, usually a cytotoxin.

[0005] However, when the protein is itself the therapeutic moiety and not just a targeting moiety, the technology of the present invention must further enable so-called "endosomal escape". This important step is to escape the therapeutic biological agent from the various stages of the endosomal pathway, namely the vesicles of early or late endosomes and lysosomes, in order to avoid the catabolism of the therapeutic agent by this cellular mechanism.

[0006] Upon successful escape from the endosome, the therapeutic agent is released into the cytoplasm of the cell. However, the cytoplasm is a crowded environment and is not suitable for most of the currently used biological therapeutic agents, such as monoclonal antibodies and their derivatives. The therapeutic biological agent needs to be efficiently dispersed within the cytoplasm in order to find and engage its therapeutic target or to reach target intracellular organelles such as the nucleus, endoplasmic reticulum (ER), mitochondria, etc.

[0007] The most potent biological agents designed to block protein-protein interactions and clinically used, or that can be used as agonists, are based on antibodies, mainly monoclonal antibodies or their derivatives, such as Fab, scFv, and combinations thereof. All of these agents are structurally and functionally dependent on disulfide bonds, either intra-chain or inter-chain. These disulfide bonds are unstable in the reducing environment of the cytoplasm. This reducing environment is a result of high concentrations of reducing agents, the most prominent of which is glutathione, which causes reduction of the disulfide bridges in such therapeutic agents, causing them to lose their structure and thus their ability to engage the target. Efficient intracellular delivery technology is therefore needed to either utilize agents that are stable in the cytoplasm or provide some way to protect the agent being delivered from this reducing environment.

[0008] In addition to this, intracellular delivery technology needs to have appropriate pharmacokinetic and pharmacodynamic behavior that enables efficient delivery of the therapeutic payload to various organs and tissues throughout the body.

[0009] One of the classical and most studied intracellular delivery technologies involves the use of enhanced positive charge of the payload. The most classical approach has evolved from an understanding of the intracellular uptake mechanisms that can be used by viruses. The latter uses 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 given rise to a number of cell-penetrating peptides (CPPs) with various charges, amino acid sequences, additional modifications, and structures (linear, cyclic, etc.), which have been fused to or chemically conjugated to various payloads or used to modify various nanoparticles. CPPs show the ability to internalize into cells, but their endosomal escape efficiency remains debated and their overall efficiency seems insufficient for use in actual pharmaceutical applications.

[0010] There is a great need for a more efficient method of delivering protein-based therapeutic agents into the cytoplasm of cells that exceeds standard CPP and has improved in vivo distribution and pharmacodynamics.

SUMMARY OF THE INVENTION

[0011] The present invention provides a protein conjugate comprising a protein carrier containing polyethyleneimine, a payload that interacts with an intracellular target, and a linker that links them. Also provided are pharmaceutical compositions containing the protein conjugate, as well as methods of using and manufacturing the protein conjugate.

[0012] According to a first aspect, a. a protein carrier covalently bound to a plurality of polyethyleneimine (PEI) moieties; b. a payload that interacts with an intracellular target; c. a linker covalently bound to the protein carrier and the payload via a covalent bond; A protein conjugate is provided, wherein the payload lacks a disulfide bond that reduces interaction with the intracellular target when cleaved, and the protein conjugate is not a fusion protein.

[0013] According to another aspect, a. HSA covalently bound to a plurality of polyethyleneimine (PEI) moieties and characterized by a positive zeta potential of at least 8 mV; b. a single domain antibody; c. a linker covalently bound to HSA and the single domain antibody via a covalent bond; A protein conjugate is provided, wherein the protein conjugate is not a fusion protein.

[0014] According to another aspect, a method of producing a protein conjugate capable of binding to an intracellular target is provided, the method comprising a. providing a payload that binds to an intracellular target and reduces binding to the intracellular target when cleaved, and that has no disulfide bonds; b. providing a protein carrier that is covalently bound to a plurality of PEI moieties; c. covalently linking a selected payload to a selected protein carrier via a linker to produce a protein conjugate; d. determining the stability of the linker in human blood, plasma, or serum; e. selecting a protein conjugate that contains a linker that is stable in human blood, plasma, or serum, thereby producing a protein conjugate capable of binding to an intracellular target; comprising.

[0015] According to another aspect, there is provided a protein conjugate produced by the method of the present invention.

[0016] According to another aspect, there is provided a pharmaceutical composition comprising the protein conjugate of the present invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0017] According to another aspect, there is provided a method of binding to an intracellular target, the method comprising contacting a cell expressing the intracellular target with the protein conjugate of the present invention or the pharmaceutical composition of the present invention, wherein the biological payload binds to the intracellular target, thereby binding the intracellular target.

[0018] According to another aspect, there is provided a method of binding to an intracellular target, the method comprising: a. producing a protein conjugate capable of binding to an intracellular target by the method of the present invention; b. contacting a cell expressing the intracellular target with the produced protein conjugate; thereby binding to the intracellular target comprising.

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

[0020] According to some embodiments, the plurality of PEI moieties comprises 3 to 90 molecules.

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

[0022] According to some embodiments, the antigen-binding molecule is selected from single-chain antibodies, single-domain antibodies, variable heavy chain homodimers (VHHs), nanobodies, immunoglobulin new antigen receptors (IgNARs), designed ankyrin repeat proteins (DARPins), and antibody mimetic proteins.

[0023] According to some embodiments, the antigen-binding molecule is selected from VHHs and DARPins.

[0024] According to some embodiments, the protein carrier is an endogenous plasma protein.

[0025] According to some embodiments, the protein carrier is at least 60 KDa in size.

[0026] According to some embodiments, the protein carrier is selected from human serum albumin (HSA), fibrinogen, IgG, green fluorescent protein (GFP), and designed ankyrin repeat proteins (DARPins).

[0027] According to some embodiments, the protein carrier is HSA.

[0028] According to some embodiments, the protein carrier is HSA and the protein carrier comprises 3 to 10 PEI molecules.

[0029] According to some embodiments, the linker comprises a biocompatible polymer.

[0030] According to some embodiments, the biocompatible polymer comprises polyethylene glycol (PEG).

[0031] According to some embodiments, the linker comprises a biologically cleavable bond.

[0032] According to some embodiments, the biologically cleavable bond comprises a disulfide bond.

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

[0034] According to some embodiments, stability comprises less than 25% cleavage in blood after 24 hours.

[0035] According to some embodiments, the biologically cleavable bond is sterically hindered.

[0036] According to some embodiments, HSA comprises the amino acid sequence of SEQ ID NO: 1, or a fragment or homolog thereof comprising cysteine 34 (C34).

[0037] According to some embodiments, the linker is bound to HSA via a disulfide bond.

[0038] According to some embodiments, the linker is bound to C34 of HSA.

[0039] According to some embodiments, the disulfide bond is proximal to C34.

[0040] According to some embodiments, proximal is at a distance in the range of 5-15 angstroms from C34.

[0041] According to some embodiments, the linker is a peptide linker.

[0042] According to some embodiments, the linker is selected from SEQ ID NOs: 4-15.

[0043] According to some embodiments, the protein carrier does not contain DNA.

[0044] According to some embodiments, the biological payload does not bind to cell surface proteins.

[0045] According to some embodiments, the protein carrier covalently bound to a plurality of PEI moieties is characterized by a positive zeta potential of at least 8 mV.

[0046] According to some embodiments, HSA contains 3 to 10 PEI molecules.

[0047] According to some embodiments, the protein conjugate further comprises a detectable tag, and optionally the tag is conjugated to the biological payload.

[0048] According to some embodiments, the protein conjugate is a blood-stable conjugate.

[0049] According to some embodiments, the protein conjugate is a cell-permeable conjugate.

[0050] According to some embodiments, determining is performed before or after the formation of the protein conjugate.

[0051] According to some embodiments, the method includes confirming binding of the biological payload to an intracellular target.

[0052] According to some embodiments, the protein carrier bound to a plurality of PEI moieties is characterized by a positive zeta potential of at least 8 mV.

[0053] According to some embodiments, the protein carrier comprises HSA.

[0054] According to some embodiments, the protein carrier is covalently bound to at least three molecules of PEI.

[0055] According to some embodiments, the method further comprises contacting a selected protein conjugate with a cell and confirming that the biological payload enters the cytoplasm of the cell.

[0056] According to some embodiments, stable comprises less than 25% cleavage in the blood after 24 hours, and unstable comprises at least 50% cleavage in the cytoplasmic state after 24 hours.

[0057] According to some embodiments, the linker comprises a biocompatible polymer.

[0058] According to some embodiments, linking by covalent bond is via a click reaction.

[0059] According to some embodiments, the payload is covalently bound to a linker comprising a first reactive group, wherein the protein carrier is covalently bound to a linker comprising a second reactive group having reactivity towards the first reactive group.

[0060] According to some embodiments, linking by covalent bond is by reacting a first reactive group with a second reactive group, thereby covalently linking the payload and the protein carrier.

[0061] According to some embodiments, the linker comprises a biologically cleavable bond.

[0062] According to some embodiments, linking by covalent bond comprises formation of a disulfide bond.

[0063] According to some embodiments, (i) the 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 payload, where the attachment is optionally via a disulfide bond.

[0064] According to some embodiments, the method further comprises determining the stability of the linker in the cytoplasmic state and selecting a protein conjugate comprising a linker that is unstable in the cytoplasmic state.

[0065] According to some embodiments, the pharmaceutical composition is formulated for systemic administration.

[0066] According to some embodiments, the method is a method for detecting an intracellular target, the protein conjugate comprises a detectable tag, and the method further comprises detecting the detectable tag.

[0067] According to some embodiments, the method is a method for modulating an intracellular target, and the payload is an agonist or antagonist of the intracellular target.

[0068] According to some embodiments, the cell is in a subject, and contacting comprises administering the protein conjugate to the subject.

[0069] According to some embodiments, the method is a method for treating a condition in a subject in need thereof, where the condition is treatable by modulation of an intracellular target.

[0070] According to some embodiments, the condition is cancer, the intracellular target is oncogenic, and the biological payload is an antagonist.

[0071] According to some embodiments, contacting is not in the presence of an agent other than a carrier protein designed to induce permeation of the protein conjugate into the cell.

[0072] Further embodiments and the full scope of applicability of the present invention will become apparent from the following detailed description. However, while the detailed description and specific examples represent preferred embodiments of the present invention, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description, and these are provided by way of illustration only.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0074] In some embodiments, the present invention provides a protein conjugate comprising a protein carrier covalently attached to a cell-penetrating moiety; a payload; and a linker attached to the protein carrier and the payload via a covalent bond.

[0075] In some embodiments, the protein carrier is a protein having a long serum half-life. In some embodiments, the protein carrier is a protein found in blood. In some embodiments, the carrier protein has a molecular weight of at least 60 kDa. In some embodiments, the carrier protein has a molecular weight of at least 65 kDa. In some embodiments, the carrier protein has a molecular weight of at least 70 kDa. In some embodiments, the carrier protein has 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 has an isoelectric point of up to 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).

[0076] 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 one cell-penetrating moiety. In some embodiments, the protein carrier is covalently bound to multiple cell-penetrating moieties.

[0077] In some embodiments, the cell-penetrating moiety comprises a cell internalization molecule. In some embodiments, the cell-penetrating moiety is configured to internalize the protein conjugate of the invention into the cell. In some embodiments, the cell-penetrating moiety is configured to induce or enhance the cell internalization of the protein conjugate of the invention. In some embodiments, the cell-penetrating moiety is configured to enhance the cell penetration or internalization of the protein conjugate of the invention as compared to a control (e.g., a protein conjugate without a cell-penetrating moiety).

[0078] In some embodiments, the enhancement is at least 20%, at least 50%, at least 100%, at least 1000%, at least 10000%, at least 100000% (including any range therebetween) compared to the control. Each possibility represents a separate embodiment of the invention.

[0079] In some embodiments, the cell-penetrating moiety of the invention comprises an alkylamine, a cationic polymer, or a combination thereof. In some embodiments, the cell-penetrating moiety comprises a cationic polymer. In some embodiments, the cell-penetrating moiety comprises an alkylamine.

[0080] In some embodiments, the cationic polymer comprises a plurality of 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 be ionized (positively ionized) in a solution having a pH value lower than the pKa value of the amine groups of the cationic polymer. In some embodiments, the cationic polymer can be protonated in a solution having a pH value lower than the pKa value of the amine groups of the cationic polymer.

[0081] In some embodiments, at least 50% by weight of the cationic polymer is positively charged (or protonated) in a solution having a pH value lower than the pKa value of the amine groups of the cationic polymer. In some embodiments, the cationic polymer undergoes multiple protonations in solution, resulting in multiple positive surface charges, where the solution is as described herein.

[0082] 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.

[0083] In some embodiments, the cationic polymer of the present invention is polyethyleneimine (PEI) or comprises polyethyleneimine (PEI).

[0084] 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, less than 800 Da (including any range therebetween).

[0085] In some embodiments, the cell-penetrating moiety of the present invention is characterized by an 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).

[0086] In some embodiments, the PEI is characterized by an 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, 700 - 1000 Da (including any range therebetween).

[0087] In some embodiments, the cell-penetrating moiety of the present invention comprises branched PEI characterized by an Mn of 500 - 700 Da, or 500 - 2000 Da.

[0088] In some embodiments, the protein carrier comprises a plurality of cell-penetrating moieties covalently attached thereto. In some embodiments, the plurality of cell-penetrating moieties includes 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).

[0089] In some embodiments, the protein carrier comprises a plurality of PEI molecules covalently attached thereto. In some embodiments, the protein carrier comprises 3 - 10, 3 - 5, 5 - 8, 8 - 10, 10 - 15, 15 - 20 PEI molecules (including any range therebetween) covalently attached thereto.

[0090] In some embodiments, the protein carrier includes 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 includes a size of at least 50 KDa. In some embodiments, the protein carrier includes a size of at least 60 KDa. In some embodiments, the protein carrier includes a size of at least 65 KDa.

[0091] In some embodiments, the protein carrier is an endogenous blood protein. In some embodiments, the protein is naturally found in the blood. In some embodiments, the blood is plasma. In some embodiments, the blood is mammalian blood. In some embodiments, the mammal is a human. In some embodiments, the endogenous blood protein is albumin. In some embodiments, the endogenous blood protein is globulin. In some embodiments, the endogenous blood protein is fibrinogen. In some embodiments, the globulin is 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), alpha1-antitrypsin, alpha-1-acid glycoprotein, alpha1-fetoprotein, alpha2-macroglobulin, gamma globulin, beta2-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). In some embodiments, the protein carrier is selected from human serum albumin (HSA), fibrinogen, IgG, fluorescent protein (GFP), and designed ankyrin repeat protein (DARPin). 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.

[0092] 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 conjugated to the protein carrier via a linker, the linker being as described herein, and the protein carrier is HSA that is covalently bound to (or modified by) 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, 6 to 10 (including any range therebetween) PEI molecules, or comprises HSA. In some embodiments, the number of PEI molecules described herein represents an average value. In some embodiments, the PEI molecules are characterized by an average MW of 100 to 2000 Da, 200 to 5000 Da, 200 to 3000 Da, 500 to 5000 Da, 500 to 2000 Da, 500 to 3000 Da, 100 to 300 Da, 300 to 400 Da, 400 to 500 Da, 500 to 600 Da, 600 to 700 Da, 700 to 1000 Da (including any range therebetween).

[0093] In some embodiments, the conjugate comprises a payload. As used herein, the term "payload" refers to any molecule that is delivered to the cytoplasm of a target cell. In some embodiments, the payload binds to 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 is the cytoplasm. In some embodiments, the payload lacks a disulfide bond that, when cleaved, reduces its 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 biological molecule. In some embodiments, the payload is organic. 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 capable of binding 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 a peptide. In some embodiments, the peptide or protein is an isolated protein or peptide. In some embodiments, the peptide or protein is a portion of a peptide or protein. It will be understood that the protein need not be a complete protein and can be a part 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 multiple amino acid chains. In some embodiments, the payload is a bioactive molecule. In some embodiments, the bioactive molecule is a bioactive agent.

[0094] 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 to be expressed in a target cell. In some embodiments, the payload is gene therapy. In some embodiments, the nucleic acid molecule contains an open reading frame. In some embodiments, the open reading frame encodes a therapeutic protein. Methods of conjugating nucleic acid molecules to chemical linkers and amino acid linkers are well known in the art and any such method can be used. In some embodiments, the nucleic acid molecule contains a nuclear localization signal (NLS). In some embodiments, the payload is selected from a protein and a nucleic acid molecule.

[0095] The term "nucleic acid" is well known in the art. As used herein, "nucleic acid" generally refers to a molecule (i.e., strand) of DNA, RNA, or a derivative or analog thereof that contains nucleobases. Nucleobases include, for example, naturally occurring purine or pyrimidine bases found in DNA (e.g., adenine "A", guanine "G", thymine "T" or cytosine "C") or RNA (e.g., A, G, uracil "U" or C).

[0096] 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, other short interfering nucleic acids, snoRNAs, snRNAs, tRNA, piRNA, tnRNA, low rRNA, hnRNA, lncRNA, circulating nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, ribozymes, viral RNA or DNA, nucleic acids of infectious agents, amplification products, modified nucleic acids, plasmid nucleic acids or organellar nucleic acids, and artificial nucleic acids such as oligonucleotides.

[0097] 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, an oligonucleotide is attached to the 5' or 3' end of a nucleic acid molecule by means such as a ligation reaction.

[0098] As used herein, the term "expression" refers to the biosynthesis of a gene product and includes transcription and / or translation of the above-mentioned gene product. Thus, the expression of a nucleic acid molecule can refer to the transcription of a nucleic acid fragment (e.g., transcription resulting in mRNA or other functional RNA) and / or the translation of RNA into a precursor or mature protein (polypeptide).

[0099] The expression of genes in cells is well known to those skilled in the art, and herein its delivery can be carried out by the methods of the present invention or by the use of the compositions of the present invention. In some embodiments, the gene is in an expression vector such as a plasmid or viral vector. The vector can be a viral vector. The viral vector can be a retroviral vector, herpesvirus vector, adenovirus vector, adeno-associated virus vector or poxvirus vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter.

[0100] In some embodiments, a gene or open reading frame is operably linked to a promoter or other control element. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the control element(s) in such a way as to enable expression of that nucleotide sequence (e.g., when a vector is introduced into a host cell by the methods of the invention, in an in vitro transcription / translation system or in a host), such as in a manner that allows the nucleotide sequence to be expressed. In some embodiments, the control element or promoter is active within the target cell.

[0101] As used herein, the term "promoter" refers to a group of transcriptional control modules that assemble around the start site for RNA polymerase, i.e., RNA polymerase II. A promoter is composed of individual functional modules, each of which consists of about 7-20 bp of DNA and contains one or more recognition sites for transcriptional activator proteins or transcriptional repressor proteins.

[0102] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAPII 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 snRNA and microRNA.

[0103] 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 (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (available from Strategene), pTRES (available from Clontech), and derivatives thereof.

[0104] In some embodiments, expression vectors containing control elements derived from eukaryotic viruses such as retroviruses are used according to the present invention. Examples of SV40 vectors include pSVT7 and pMT2. In some embodiments, examples of vectors derived from bovine papillomavirus include pBV-1MTHA, and examples of vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that enables 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 any other promoter shown to be effective for expression in eukaryotic cells.

[0105] In some embodiments, recombinant viral vectors that provide advantages such as lateral infection and target specificity are used for in vivo expression. In one embodiment, lateral infection is, for example, inherent in the life cycle of a retrovirus and is the process by which one infected cell produces many progeny virions that bud and infect adjacent cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which were not initially infected with the original virus particles. In one embodiment, viral vectors that cannot spread laterally are produced. In one embodiment, this feature can be useful if the desired goal is to introduce a specific gene into only a local number of target cells.

[0106] The term "bioactive" refers to a molecule or agent that affects cells or tissues. Representative examples of types of bioactive agents include therapeutic agents, 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, cadherin, selectin, intracellular adhesion molecule, and integrin). In various cases, the bioactive agent can be selected from fibronectin, laminin, thrombospondin, tenascin C, leptin, leukemia inhibitory factor, RGD peptide, anti-TNF, endostatin, angiostatin, thrombospondin, osteogenic 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.

[0107] As used herein, the term "isolated peptide" refers to a peptide that is essentially free of contaminating cellular components such as carbohydrates, lipids, or other proteinaceous impurities that are associated with the peptide by nature. Typically, a preparation of an isolated peptide comprises the peptide in a highly purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, more than 95% pure, or more than 99% pure.

[0108] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably to refer to polymers of amino acid residues. In another embodiment, the terms "peptide", "polypeptide", and "protein" as used herein include natural peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications), as well as 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 or more permeable into cells while they are 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.

[0109] In some embodiments, the payload binds to a cytoplasmic target. In some embodiments, the payload is specific for a cytoplasmic target. In some embodiments, specific includes not significantly 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 includes a complementarity determining region (CDR) that binds to the target. In some embodiments, the payload is an antibody or an antigen-binding fragment thereof. The structure of an antibody is well known, and those skilled in the art can understand that an antibody binds to a target only by its CDR sequence, but the general structure of an antibody and its antigen-binding region can be recognized by those skilled in the art.

[0110] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides comprising at least one binding domain formed from the folding of polypeptide chains having a three-dimensional binding space with an internal surface shape and charge distribution complementary to the characteristics of the antigenic determinant of an antigen. Antibodies typically have a tetrameric form comprising two identical pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain. The mature variable regions of each light chain / heavy chain pair form the antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and soluble or labeled in bound form, and can be fragments, including epitope-binding fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies can be derived from any species. The term antibody includes, but is not limited to, binding fragments such as Fv, Fab, Fab’, F(ab’)2, single-chain antibody (scFv), dimeric variable region (diabody), and disulfide-linked variable region (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing the antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including but not limited to the Fc region or fragments thereof. Those skilled in the art will further understand that other fusion products can be generated, including but not limited to scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, and scFv-scFv-Fc fusions.

[0111] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0112] 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 camel 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 the presence of CDRs, antibodies, antibody fragments, ScFv, nanobodies, VHHs, single-domain antibodies, DARPins, etc. can be structurally recognized by their non-variable regions. Thus, although not limited to a particular target, the compositions of the present invention can be understood to include these molecules as payloads.

[0113] In some embodiments, the payload comprises a C-terminal cysteine amino acid. In some embodiments, the payload comprises a cysteine amino acid 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 from the terminus. Each possibility represents a separate embodiment of the present invention. In some embodiments, the payload comprises a tag. In some embodiments, the tag is a C-terminal tag. In some embodiments, the tag is C-terminal to the cysteine amino acid. In some embodiments, the tag is N-terminal to the cysteine amino acid. In some embodiments, the tag and the cysteine amino acid are adjacent. In some embodiments, the cysteine amino acid is within the tag. In some embodiments, the tag comprises or consists of GGGGSC (SEQ ID NO: 9). In some embodiments, the payload comprises C-terminal SEQ ID NO: 9.

[0114] In some embodiments, the protein conjugate comprises a payload conjugated to a protein carrier via a linker. In some embodiments, the protein conjugate is not a fusion protein. It will be understood by those skilled in the art that the payload and the carrier are conjugated to each other by individual linkers. The linker is not part of the carrier nor part of the payload, but is attached (conjugated) to each and thereby links them.

[0115] In some embodiments, the protein conjugate comprises a payload conjugated to a protein carrier via a linker, where the linker is as described herein, and the protein carrier (e.g., HSA) is covalently bound to a plurality of PEI molecules sufficient to obtain a protein carrier characterized by a positive zeta potential value of at least 6, at least 7, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 12 mV, or 8 - 40, 8.5 - 40, 8 - 20, 8.5 - 20, 10 - 40, 10 - 20, 10 - 30 mV (including any range therebetween). One of ordinary skill in the art will understand that the exact zeta potential value of the conjugate can vary depending on the molecular weight and / or size of the protein carrier, the payload, or both. In some embodiments, the carrier and the cell permeable moiety are characterized by a positive zeta potential value of at least 6, at least 7, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 12 mV, or 8 - 40, 8.5 - 40, 8 - 20, 8.5 - 20, 10 - 40, 10 - 20, 10 - 30 mV (including any range therebetween). Each possibility represents a separate embodiment of the invention. In some embodiments, the conjugate is characterized by a positive zeta potential value of at least 6, at least 7, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 12 mV, or 8 - 40, 8.5 - 40, 8 - 20, 8.5 - 20, 10 - 40, 10 - 20, 10 - 30 mV (including any range therebetween). Each possibility represents a separate embodiment of the invention.

[0116] The zeta potential can be measured by any method known in the art. In this specification, the following protocol is used and can be regarded as a standard for determining whether a molecule contains a zeta potential within the scope described herein. The zeta potential measurement was carried out using a Zetasizer Ultra (Malvern Instruments). The buffer of the sample was exchanged with 1 mM NaCl at a protein concentration of 1 mg / mL. 20 μL from each sample was loaded into a zeta cell (DTS1070), and 5 repetitions were measured for each sample, and the average zeta potential in mV was obtained for each repetition. The average of the 5 measurements is reported together with the standard deviation. The measurement was carried out under the following conditions: temperature 25 °C; number of runs per repetition 10 - 40; equilibration time: 60 seconds, no pause after sub-run; 60-second pause between repetitions; voltage was automatically selected, and a unimodality analysis method was used in data processing. In some embodiments, the zeta potential is measured in about 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.

[0117] In some embodiments, the protein conjugate of the present invention comprises a protein carrier covalently attached to a payload via a linker. In some embodiments, the payload is covalently attached to the linker. In some embodiments, the carrier is covalently attached to the linker. In some embodiments, the covalent bond is not a peptide bond. In some embodiments, at least one of the bond between the linker and the payload and the bond between the linker and the carrier is not a peptide bond. 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 attached 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 attached to a payload via a linker, wherein the linker is a synthetic linker not comprising at least one cleavable bond. In some embodiments, the carrier and the payload are not derived from the same protein. In some embodiments, the linker and the carrier are not derived from the same protein. In some embodiments, the linker is a peptide linker and comprises a sequence not present in the amino acid sequence of the protein serving as the basis of the carrier. In some embodiments, the linker is a peptide linker and comprises a sequence not present in the amino acid sequence of the protein serving as the basis of the payload. In some embodiments, the linker and the payload are not derived 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 does not occur naturally. In some embodiments, the linker is artificial. In some embodiments, the carrier is a naturally occurring protein or a fragment thereof.

[0118] 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 α-helix peptide. 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 payload. In some embodiments, proximal is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of the terminus. Each possibility represents a separate embodiment of the invention. In some embodiments, the linker comprises an N-terminal cysteine and the payload comprises a C-terminal cysteine. It will be understood by those skilled in the art that the side chains of cysteine contain sulfur atoms that can be used to form cleavable disulfide bonds. For example, cysteine can form a disulfide bond with cysteine 34 of HSA.

[0119] In some embodiments, the α-helix peptide comprises or consists of AAASAEAAAKEAAAKEAAAKAAAGSG (SEQ ID NO: 6). In some embodiments, the α-helix peptide comprises or consists of AAAASAEAAAKEAAAKEAAAKAAAGSGLC (SEQ ID NO: 10). In some embodiments, the α-helix 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 GGGGSGGGGSGGGLG (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 GGGGSGGGGSGGGGS (SEQ ID NO: 12). In some embodiments, the linker consists of GGGGSGGGGSC (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 to 15.

[0120] In some embodiments, the linker of the present invention 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, at least 48 hours (including any range therebetween). Each possibility represents a separate embodiment of the present invention. In some embodiments, the linker of the present invention is substantially stable in blood. In some embodiments, the blood is human blood. In some embodiments, the linker of the present invention is 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.

[0121] In some embodiments, the linker of the present invention is unstable under exposure to a cytoplasmic condition. In some embodiments, cleavable is biologically cleavable. In some embodiments, the linker of the present invention is cleavable under exposure to a cytoplasmic condition so as to release the payload into the cytosol. In some embodiments, the linker and / or protein conjugate of the present invention is substantially stable under a cytoplasmic condition, where substantially stable is as described herein.

[0122] In some embodiments, the protein conjugate of the present invention substantially lacks a biologically cleavable bond (e.g., a bond cleavable under exposure of the protein conjugate to a cytoplasmic condition). In some embodiments, the linker substantially lacks a biologically cleavable bond. In some embodiments, the linker is attached to the protein carrier and / or payload via a non-biologically cleavable bond (e.g., an amide bond, a click reaction product, a thioether bond, etc.).

[0123] In some embodiments, the protein conjugate of the present invention comprises a biologically cleavable bond. In some embodiments, the protein carrier and / or payload is linked to the linker via a biologically cleavable bond. According to some embodiments, the linker of the present invention comprises a biologically cleavable bond. In some embodiments, the biologically cleavable 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, at least 48 hours (including any range therebetween). In some embodiments, cleavable means cleavable in the cytoplasm. In some embodiments, cleavable does not mean cleavable in serum or blood. In some embodiments, not cleavable means substantially not cleavable. In some embodiments, biologically cleavable in the cytoplasm means significantly more cleavage occurs in the cytoplasm than in the blood.

[0124] In some embodiments, the biologically cleavable bond is cleavable upon exposure to a cytoplasmic state. In some embodiments, the biologically cleavable bond is reducible upon exposure to a cytoplasmic state (e.g., an intracellular compartment containing, inter alia, acidic pH conditions and / or a reducing agent such as glutathione).

[0125] Biologically cleavable bonds are well known in the art and refer to bonds that are selectively cleaved after entering the cell (intracellular cleavage). A preferred linkage for releasing a drug intracellularly is cleavable under acidic conditions such as those found in lysosomes. One example is a disulfide bond. It is hypothesized that disulfide bonds are cleaved when entering the cell by glutathione.

[0126] In some embodiments, the biologically cleavable bond is or includes a disulfide bond. In some embodiments, the biologically cleavable bond includes a plurality of disulfide bonds. In some embodiments, the biologically cleavable bond is sterically hindered. In some embodiments, the biologically cleavable bond is or includes a sterically hindered disulfide bond.

[0127] In some embodiments, the biological fluid is a body fluid. In some embodiments, the biological fluid is selected from at least one of blood, serum, plasma, gastric juice, intestinal juice, saliva, bile, tumor fluid, breast milk, urine, interstitial fluid, cerebrospinal fluid, and feces. In some embodiments, the biological fluid is blood. In some embodiments, the biological fluid is serum. In some embodiments, the biological fluid is plasma.

[0128] In some embodiments, the sterically hindered disulfide bond includes a side group or a bulky moiety adjacent thereto. In some embodiments, the side group or bulky moiety is located proximal to at least one sulfur atom of the disulfide bond. In some embodiments, adjacent or closely proximal includes a distance in the range of 0 to 10, 0 to 2, 2 to 5, 5 to 10 atomic bonds (including any range therebetween). In some embodiments, the term "atomic bond" as used herein refers to a carbon-carbon (C-C) bond length, e.g., a single C-C bond length.

[0129] In some embodiments, the sterically hindered disulfide bond includes a side group or a bulky moiety adjacent thereto (e.g., located at a distance in the range of 1 to 15 Å, 1 to 3 Å, 3 to 5 Å, 5 to 10 Å, 10 to 15 Å (including any range therebetween) from the sulfur atom of the disulfide bond).

[0130] In some embodiments, the side group or bulk moiety comprises an alkyl (e.g., primary, secondary or tertiary C1-C10 alkyl optionally containing unsaturated bonds and / or substituents), an aromatic ring, an amino acid containing a sterically hindered side chain (e.g., leucine, valine, isoleucine, phenylalanine, histidine, tyrosine, and tryptophan), a protein, or any combination thereof. In some embodiments, the side group or bulk moiety is covalently attached to a methylene group adjacent to a disulfide bond.

[0131] In some embodiments, the disulfide bond is located adjacent to the payload and / or protein carrier of the present invention, where adjacent is as described herein. In some embodiments, the payload and / or protein carrier of the present invention is attached to a linker via a disulfide bond. In some embodiments, the disulfide bond is proximal or adjacent to the protein carrier.

[0132] In some embodiments, the protein carrier of the present invention (e.g., HSA) is linked to the linker via a disulfide bond. In some embodiments, HSA comprises the amino acid sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 1) or a fragment or homolog thereof. SEQ ID NO: 1 provides the sequence of HSA without a signal peptide. In some embodiments, HSA comprises a signal peptide. In some embodiments, the signal peptide comprises or consists of MKWVTFISLLFLFSSAYSRGVFRR (SEQ ID NO: 17). In some embodiments, 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 invention. In some embodiments, the amino acids are contiguous amino acids. In some embodiments, the HSA is a homolog of HSA. In some embodiments, the homolog of HSA comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% homology to SEQ ID NO: 1. Each possibility represents a separate embodiment of the invention. In some embodiments, the HSA comprises an amino acid sequence having at least 70% homology to SEQ ID NO: 1. In some embodiments, the HSA consists of SEQ ID NO: 1, or a fragment or homolog thereof. In some embodiments, the homology is sequence identity. In some embodiments, the HSA consists of an amino acid sequence having at least 70% homology to SEQ ID NO: 1. In some embodiments, the HSA consists of SEQ ID NO: 1. In some embodiments, the HSA contains a free cysteine. In some embodiments, the free cysteine is cysteine C34. In some embodiments, the free cysteine is the only free cysteine. In some embodiments, the linker of the invention is bound to C34 of HSA via a disulfide bond.

[0133] In some embodiments, the fibrinogen is fibrinogen alpha chain (FGA). In some embodiments, FGA comprises the amino acid sequence of ADSGEGDFLAEGGGVRGPRVVERHQSACKDSDWPFCSDEDWNYKCPSGCRMKGLIDEVNQDFTNRINKLKNSLFEYQKNNKDSHSLTTNIMEILRGDFSSANNRDNTYNRVSEDLRSRIEVLKRKVIEKVQHIQLLQKNVRAQLVDMKRLEVDIDIKIRSCRGSCSRALAREVDLKDYEDQQKQLEQVIAKDLLPSRDRQHLPLIKMKPVPDLVPGNFKSQLQKVPPEWKALTDMPQMRMELERPGGNEITRGGSTSYGTGSETESPRNPSSAGSWNSGSSGPGSTGNRNPGSSGTGGTATWKPGSSGPGSTGSWNSGSSGTGSTGNQNPGSPRPGSTGTWNPGSSERGSAGHWTSESSVSGSTGQWHSESGSFRPDSPGSGNARPNNPDWGTFEEVSGNVSPGTRREYHTEKLVTSKGDKELRTGKEKVTSGSTTTTRRSCSKTVTKTVIGPDGHKEVTKEVVTSEDGSDCPEAMDLGTLSGIGTLDGFRHRHPDEAAFFDTASTGKTFPGFFSPMLGEFVSETESRGSESGIFTNTKESSSHHPGIAEFPSRGKSSSYSKQFTSSTSYNRGDSTFESKSYKMADEAGSEADHEGTHSTKRGHAKSRPVRDCDDVLQTHPSGTQSGIFNIKLPGSSKIFSVYCDQETSLGGWLLIQQRMDGSLNFNRTWQDYKRGFGSLNDEGEGEFWLGNDYLHLLTQRGSVLRVELEDWAGNEAYAEYHFRVGSEAEGYALQVSSYEGTAGDALIEGSVEEGAEYTSHNNMQFSTFDRDADQWEENCAEVYGGGWWYNNCQAANLNGIYYPGGSYDPRNNSPYEIENGVVWVSFRGADYSLRAVRMKIRPLVTQ (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 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 invention. In some embodiments, the amino acids are contiguous amino acids. In some embodiments, FGA is a homolog of FGA. In some embodiments, the homology is sequence identity. In some embodiments, the 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 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.

[0134] In some embodiments, the fibrinogen is fibrinogen beta chain (FGB). In some embodiments, FGB comprises the amino acid sequence of QGVNDNEEGFFSARGHRPLDKKREEAPSLRPAPPPISGGGYRARPAKAAATQKKVERKAPDAGGCLHADPDLGVLCPTGCQLQEALLQQERPIRNSVDELNNNVEAVSQTSSSSFQYMYLLKDLWQKRQKQVKDNENVVNEYSSELEKHQLYIDETVNSNIPTNLRVLRSILENLRSKIQKLESDVSAQMEYCRTPCTVSCNIPVVSGKECEEIIRKGGETSEMYLIQPDSSVKPYRVYCDMNTENGGWTVIQNRQDGSVDFGRKWDPYKQGFGNVATNTDGKNYCGLPGEYWLGNDKISQLTRMGPTELLIEMEDWKGDKVKAHYGGFTVQNEANKYQISVNKYRGTAGNALMDGASQLMGENRTMTIHNGMFFSTYDRDNDGWLTSDPRKQCSKEDGGGWWYNRCHAANPNGRYYWGGQYTWDMAKHGTDDGVVWMNWKGSWYSMRKMSMKIRPFFPQQ (SEQ ID NO: 19), or a fragment or homolog thereof. SEQ ID NO: 19 provides the sequence of fibrinogen without a signal peptide. In some embodiments, the fibrinogen comprises 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 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 invention. In some embodiments, the amino acids are contiguous amino acids. In some embodiments, FGB is a homolog of FGB. In some embodiments, the homology is sequence identity.In some embodiments, the 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 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.

[0135] In some embodiments, fibrinogen is fibrinogen gamma chain (FGG). In some embodiments, FGG comprises the amino acid sequence of YVATRDNCCILDERFGSYCPTTCGIADFLSTYQTKVDKDLQSLEDILHQVENKTSEVKQLIKAIQLTYNPDESSKPNMIDAATLKSRKMLEEIMKYEASILTHDSSIRYLQEIYNSNNQKIVNLKEKVAQLEAQCQEPCKDTVQIHDITGKDCQDIANKGAKQSGLYFIKPLKANQQFLVYCEIDGSGNGWTVFQKRLDGSVDFKKNWIQYKEGFGHLSPTGTTEFWLGNEKIHLISTQSAIPYALRVELEDWNGRTSTADYAMFKVGPEADKYRLTYAYFAGGDAGDAFDGFDFGDDPSDKFFTSHNGMQFSTWDNDNDKFEGNCAEQDGSGWWMNKCHAGHLNGVYYQGGTYSKASTPNGYDNGIIWATWKTRWYSMKKTTMKIIPFNRLTIGEGQQHHLGGAKQVRPEHPAETEYDSLYPEDDL (SEQ ID NO: 21), or a fragment or homolog thereof. SEQ ID NO: 21 provides the sequence of fibrinogen without a signal peptide. In some embodiments, 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 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 invention. In some embodiments, the amino acids are contiguous amino acids. In some embodiments, FGG is a homolog of FGG. In some embodiments, the homology is sequence identity.In some embodiments, the 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 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.

[0136] In some embodiments, fibrinogen is a mix of fibrinogens. In some embodiments, the mix is a mix of at least two of FGA, FGB and FGG. In some embodiments, the mix is a mix of all three of FGA, FGB and FGG. In some embodiments, FGA, FGB and FGG are in the ratios as found in human blood. In some embodiments, the blood is plasma. Fibrinogen from human plasma is available, for example, from Sigma-Aldrich catalog number 341578. In some embodiments, fibrinogen contains free cysteine. In some embodiments, fibrinogen contains free lysine. Conjugation to fibrinogen can be carried out as described herein or by any means known in the art. The conjugation may be random or site-specific as described herein.

[0137] In some embodiments, the IgG is the Fc portion of the IgG. In some embodiments, the IgG is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG is IgG1. In some embodiments, the IgG is mammalian IgG. In some embodiments, the IgG is human IgG. In some embodiments, the IgG is mouse IgG. In some embodiments, the IgG is the constant region of the heavy chain of the IgG. In some embodiments, the IgG comprises a heavy chain and a light chain. In some embodiments, the IgG comprises two heavy chains and two light chains. In some embodiments, the IgG is a protein complex. In some embodiments, the IgG is polyclonal. In some embodiments, the IgG is monoclonal. In some embodiments, the IgG is IgG raised against a non-human species. In some embodiments, the species is goat or sheep. In some embodiments, the IgG is against the heavy chain of IgG of a non-human species. Commercially available mouse IgG such as Sigma-Aldrich's anti-goat / sheep IgG antibody, mouse monoclonal (clone GT-34) can be purchased. In some embodiments, the IgG contains free cysteine. In some embodiments, the IgG contains free lysine. In some embodiments, the IgG contains a C-terminal cysteine. Conjugation to the IgG can be carried out as described herein or by any means known in the art. The conjugation may be random or site-specific as described herein.

[0138] In some embodiments, the linker of the present invention is linear or branched, or comprises a linear or branched chain. In some embodiments, the linker of the present invention is optionally a backbone comprising one or more of the above chains, or comprises this backbone.

[0139] In some embodiments, the linker of the present invention is a spacer (e.g., including natural and / or non-natural amino acids, alkyl, amide bonds, ester bonds, thioester bonds, urea bonds, any derivatives or combinations thereof). In some embodiments, the linker of the present invention includes a biocompatible polymer or a biocompatible moiety. In some embodiments, the biocompatible polymer is at least partially biodegradable. In some embodiments, the biocompatible polymer is a polyglycol ether, a polyester, a polyamide, a polyamino acid, a peptide and / or a derivative thereof, or any combination thereof, or includes them. In some embodiments, the polyglycol ether is polyethylene glycol (PEG) or includes polyethylene glycol (PEG). In some embodiments, the linker of the present invention includes PEG. In some embodiments, the linker of the present invention includes PEG characterized by an Mn of 100 to 5000 Da (including any range therebetween).

[0140] In some embodiments, the biocompatible moiety is an amide, an ester, a glycol, an amino acid, or any combination thereof, or includes them.

[0141] In some embodiments, the polyamino acid or its derivative includes 2 to 50 amino acids, 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 (including any range therebetween).

[0142] As used herein, the terms "peptide," "polypeptide," and "protein" include natural peptides, peptide derivatives such as beta-peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications), as well as 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 analog of the corresponding naturally occurring amino acid.

[0143] The term "derivative" or "chemical derivative" includes any chemical derivative of a polypeptide having one or more residues that are chemically derivatized by reaction on a side chain within the peptide or on any functional group. Such derivatized molecules include, for example, peptides carrying one or more protecting groups (e.g., side chain protecting group(s) and / or N-terminal protecting group), and / or peptides in which the free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, acetyl group, or formyl group. The free carboxyl group may be derivatized to form its amide, salt, methyl ester, and ethyl ester, or other types of esters or hydrazides. The free hydroxyl group may be derivatized to form an O-acyl or O-alkyl derivative. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are peptides containing one or more naturally occurring amino acid derivatives 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.

[0144] Furthermore, the peptide derivative may differ from the natural sequence of the peptide of the present 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. The peptide may be linear, cyclic, or branched, etc., having any three-dimensional structure, which can be achieved using methods known in the art.

[0145] In some embodiments, the linker of the present invention further comprises a spacer (including, for example, natural and / or non-natural amino acids, alkyl, amide bond, ester bond, disulfide bond, thioester bond, urea bond, any derivative or combination thereof). In some embodiments, the linker of the present invention further comprises a disulfide bond. In some embodiments, the linker of the present invention comprises a click reaction product (such as covalent linkage by a cyclization reaction product, and / or a succinimide-thioether moiety formed via a click reaction).

[0146] Click reactions are well-known in the art and include, in particular, Michael addition of maleimide and thiol (resulting in the formation of succinimide-thioether); azide-alkyne addition cyclization; Diels-Alder reaction (such as direct and / or inverse electron-demand Diels-Alder); dibenzylcyclooctyne 1,3-nitrone (or azide) addition cyclization; alkene-tetrazole photoclick reaction, etc.

[0147] In some embodiments, the protein conjugate of the present invention has the formula A:

Chemical formula

[0148] In some embodiments, the protein conjugate of the present invention has the formula B:

Chemical formula

Chemical formula

[0149] In some embodiments, the protein conjugate of the present invention has the formula 1:

Chemical formula

[0150] In some embodiments, the protein conjugate of the present invention has the formula:

Chemical formula

[0151] In some embodiments, the protein conjugate of the present invention has the formula: [Chemical formula] wherein PC, BP, Het, A, Pol, R3, r, m, and p are as described herein, as permitted by valence. In some embodiments, if both Het and X1 are S, then at least one R 3 is one or more bulky moieties; and at least one of m' and r' is not 0. In some embodiments, the protein conjugate of the present invention has the formula: [Chemical formula] wherein Het contains S or NH, X is 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 BP. In some embodiments, Het is S and the peptide is attached to PC via a cysteine (e.g., C-terminal cysteine). In some embodiments, the protein conjugate of the present invention has the formula: [Chemical formula] [Chemical formula] represented by any one of (wherein, PC, BP, and Pol are as described herein; each j, k, r, o, n, 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); p represents an integer in the range of 2 to 100 (including any range therebetween); each R independently represents a bulk 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-C10 alkyl, C1-C10 aminoalkyl, C1-C10 alkoxy, C1-C10 mercaptoalkyl, or a click reaction product) or a combination thereof, or X is absent). In some embodiments, at least one R is methyl. In some embodiments, Pol represents a peptide, an amino acid or a dehydrated derivative thereof, PEG, or -CH2-CH2-O-. In some embodiments, the dehydrated derivative of the amino acid includes: [Chemical formula] (wherein, the wavy bond represents the position of attachment to the linker or to the subsequent monomer, and R represents an amino acid side chain).

[0152] In some embodiments, the click reaction product includes a moiety formed via a click reaction, where the click reaction is as described above. In some embodiments, the click reaction product includes a product formed by any one of the following: Michael addition of maleimide and thiol (resulting in the formation of succinimide-thioether); azido-alkyne cycloaddition; Diels-Alder reaction (e.g., direct and / or inverse electron-demand Diels-Alder); dibenzylcyclooctyne 1,3-nitrone (or azide) cycloaddition; alkene-tetrazole photoclick reaction, or any combination thereof.

[0153] In some embodiments, the protein conjugate of the present invention has the formula 1A: [Chemical Formula] represented by (wherein R, n, k, l, p, m, Pol, and r are as described herein, and each X is independently a heteroatom (e.g., O, N, NH, or S), a spacer (e.g., C1-C10 alkyl, C1-C10 aminoalkyl, C1-C10 alkoxy, C1-C10 mercaptoalkyl, or a 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-.

[0154] 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 thiol group of the 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 payloads. Exemplary protein conjugates of the present invention are represented by FIGS. 4A-4B and 13A-13D, and Table 1 below.

[0155] [Table 1] TIFF0007713030000013.tif242160TIFF0007713030000014.tif146159

[0156] In some embodiments, the protein conjugate of the present invention is substantially stable in biological fluids for at least 2 hours, at least 10 hours, at least 24 hours, at least 48 hours (including any range therebetween).

[0157] In some embodiments, at least 25%, at least 50%, at least 75%, at least 90% (including any range therebetween) of the protein conjugate of the present invention is substantially stable.

[0158] As used herein, the term "stable" refers to the ability of the protein conjugate or linker of the present invention to maintain its chemical integrity (e.g., lack of cleavage).

[0159] In some embodiments, at least 25%, at least 50%, at least 75%, at least 90% of the protein conjugate of the present invention is characterized by a positive zeta potential. In some embodiments, at least 25%, at least 50%, at least 75%, at least 90% of the protein conjugate of the present invention is characterized by a negative zeta potential. In some embodiments, the protein carrier of the present invention is characterized by an average positive zeta potential of at least 1 mV, at least 5 mV, at least 10 mV, at least 15 mV, at least 20 mV, at least 30 mV (including any range therebetween). In some embodiments, the protein carrier of the present invention is characterized by an average positive zeta potential of at least 5 mV, at least 6 mV, at least 7 mV, at least 8 mV, at least 9 mV, at least 10 mV, at least 15 mV (including any range therebetween). In some embodiments, the protein conjugate of the present invention is characterized by an average positive zeta potential of 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).

[0160] In some embodiments, the substituents each independently comprise one or more substituents selected from the group consisting of: 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’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxyhaloalkoxy, 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)2, -CO2H, -CO2R’, -OCOR’, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’: wherein each R’ is independently selected from hydrogen, alkyl, alkenyl, aryl, heteroaryl, heteroatom, optionally substituted cycloalkyl, optionally substituted heterocyclyl, or any combination thereof.

[0161] Kit In another aspect, a kit is provided that includes a protein carrier covalently attached to a first moiety and a payload, the first moiety being characterized by its reactivity towards the payload. In some embodiments, the payload is covalently attached to a second moiety, where the first moiety and the second moiety are reactive with each other (e.g., via a click reaction).

[0162] In some embodiments, the protein carrier covalently attached to the first moiety has the formula 2:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0163] In some embodiments, the first moiety or the second moiety is 1,3-nitrone, azide, diene, tetrazine, active ester (e.g., thioester, pentafluorophenyl ester, N-hydroxysuccinimide ester), acyl halide, chloroformate, anhydride, aldehyde, epoxide, isocyanate, isothiocyanate, maleimide, carbonate, sulfonyl chloride, iodoacetamide, acyl azide, imido ester, vinyl sulfone, orthopyridyl disulfide, or any combination thereof or contains these.

[0164] In some embodiments, the first moiety or the second moiety is a nucleophilic group (e.g., amine, thiol, phosphine, hydroxyl), dienophile, alkene, and alkyne (e.g., acetylene, dibenzylcyclooctyne, etc.), or any combination thereof or contains these.

[0165] In some embodiments, the kit of the invention comprises a payload covalently linked to a linker comprising a functional group having reactivity with respect to HSA (e.g., with respect to its cysteine or lysine), and HSA. In some embodiments, the kit of the invention comprises HSA covalently linked to a linker comprising a functional group having reactivity with respect to the payload (e.g., with respect to its cysteine or lysine); and a payload. In some embodiments, the functional group is any one of, or comprises, iodoacetamide, an active ester, ortho - pyridyldisulfide, maleimide, or a combination thereof.

[0166] In some embodiments, the conjugate is a blood - stable conjugate. According to some embodiments, the conjugate is a cell - permeable conjugate. In some embodiments, the conjugate is a cell - membrane - crossing conjugate. In some embodiments, the conjugate can enter cells. In some embodiments, the conjugate can escape from endosomes. In some embodiments, the conjugate enables intracellular delivery of the payload. In some embodiments, the intracellular delivery is cytoplasmic delivery. In some embodiments, the intracellular delivery involves dissociation of the carrier from the payload. In some embodiments, the conjugate is configured to dissociate within the cytoplasm. In some embodiments, the dissociation is the 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 acting on an intracellular target. In some embodiments, the conjugate is for use in interacting with an intracellular target.

[0167] Method In another aspect, provided is a method for producing a protein conjugate, the method comprising providing a biological agent that binds to an intracellular target; providing a protein carrier covalently attached to a cell permeable moiety; covalently linking a selected biological agent to a selected protein carrier via a linker to produce a protein conjugate; determining the stability of the linker in human blood, plasma, or serum; selecting a protein conjugate comprising a linker that is stable in human blood, plasma, or serum; thereby producing a protein conjugate capable of binding to an intracellular target.

[0168] In some embodiments, the protein conjugate is capable of binding to an intracellular target. In some embodiments, the protein conjugate is capable of entering the cytoplasm of a cell. In some embodiments, the protein conjugate enables intracellular delivery of a biological agent. In some embodiments, the protein conjugate is capable of modulating an intracellular target. In some embodiments, the protein conjugate is configured to modulate an intracellular target. In some embodiments, the biological agent lacks disulfide bonds. In some embodiments, the biological agent lacks disulfide bonds that are required for the structure of the biological agent. In some embodiments, the biological agent lacks disulfide bonds that are required for the function of the biological agent. In some embodiments, the biological agent lacks disulfide bonds that are required for the binding of the biological agent. In some embodiments, the biological agent lacks disulfide bonds that, when cleaved, reduce binding. In some embodiments, the binding is binding to an intracellular target.

[0169] In some embodiments, the payload is known to bind to an intracellular target. In some embodiments, the payload has been shown to bind to an intracellular target. In some embodiments, the payload has been confirmed to bind to an intracellular target. In some embodiments, the method further comprises confirming that the payload binds to the intracellular target. In some embodiments, the payload is a biological agent.

[0170] In some embodiments, the method further comprises confirming delivery into the cell. In some embodiments, delivery into the cell is into the cytoplasm of the cell. In some embodiments, the method further comprises confirming the function of the biological agent inside the cell. In some embodiments, inside the cell is within the cytoplasm of the cell. In some embodiments, the function is to bind. In some embodiments, the function is to regulate.

[0171] In some embodiments, the linker comprises a cleavable bond. In some embodiments, the bond is biologically cleavable. In some embodiments, the method further comprises selecting a protein conjugate that is unstable in the cytoplasmic state. In some embodiments, the method comprises determining the stability of the linker in the cytoplasmic state. In some embodiments, the cytoplasmic state is within the cell. In some embodiments, the cytoplasmic state is in the presence of glutathione (GSH). In some embodiments, unstable means less stable than in blood, plasma, or serum.

[0172] In some embodiments, stable includes dissociation of less than 1%, 2%, 3%, 5%, 7%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. Each possibility represents a separate embodiment of the present invention. In some embodiments, stable is stable for at least 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 48 hours, 72 hours or 96 hours. Each possibility represents a separate embodiment of the present invention. In some embodiments, stable includes less than 20% dissociation after 96 hours. In some embodiments, dissociation is dissociation of the carrier from the payload (e.g., biological agent). In some embodiments, unstable is any situation that is not stable. In some embodiments, unstable includes more dissociation than in a stable state.

[0173] In some embodiments, the linker is linked away from the active site of the above-mentioned payload (e.g., biological agent). In some embodiments, away means distal. In some embodiments, away means not proximal. In some embodiments, away means at least 5, 10, 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 amino acids away. Each possibility represents a separate embodiment of the present invention. In some embodiments, away means at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 angstroms. Each possibility represents a separate embodiment of the present invention. It will be known to those skilled in the art that the C-terminus of the VHH is about 40-50 angstroms from the end of the last CDR. In some embodiments, away means at least 40 angstroms. In some embodiments, away means at least 50 angstroms. In some embodiments, the linkage is at the end of the agent. In some embodiments, the end is the N-terminus. In some embodiments, the end is the C-terminus.

[0174] In some embodiments, the 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.

[0175] In some embodiments, there is a method for synthesizing the protein conjugate of the present invention. In some embodiments, the method includes providing a kit of the present invention and reacting a first moiety and a second moiety (e.g., under suitable conditions optionally including a metal-based catalyst and / or UV, thermal irradiation).

[0176] In some embodiments, the method for synthesizing the protein conjugate of the present invention is: (i) providing a protein carrier covalently attached to a first moiety and a payload covalently attached to a second moiety, wherein the first moiety and the second moiety are reactive with respect to each other (e.g., via a click reaction, via formation of a thiol-maleimide linkage, via coupling of an amine and an active ester, via formation of an S-S bond such as by reaction of SPDP or nitro-SPDP with a thiol), and (ii) providing the protein carrier and the payload under conditions suitable for the reaction between the first moiety and the second moiety, thereby synthesizing the protein conjugate of the present invention. In some embodiments, the first moiety and the second moiety are as described herein. In some embodiments, the reaction includes a click reaction.

[0177] In some embodiments, the method includes: (i) providing a protein carrier and a payload covalently attached to a linker containing a functional group reactive with respect to the payload (e.g., with respect to its cysteine or lysine), and (ii) reacting the functional group with the payload, thereby synthesizing the protein conjugate of the present invention.

[0178] In some embodiments, the method comprises: (i) providing a payload and a protein carrier covalently attached to a linker comprising a functional group reactive with respect to the payload (e.g., with respect to its cysteine or lysine); and (ii) reacting the functional group with the protein carrier, thereby synthesizing the protein conjugate of the invention. An exemplary synthetic scheme is shown in FIG. 36.

[0179] In another aspect, there is provided a protein conjugate produced by the method of the invention.

[0180] Composition According to another aspect, there is provided a pharmaceutical composition comprising the protein conjugate of the invention.

[0181] 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 kind of formulation aid, or simply a sterile aqueous medium such as physiological saline. Some examples of materials that can act as pharmaceutically acceptable carriers are 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 and phosphate buffer, and other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances that can act as carriers herein include sugars, stearic acid, magnesium stearate, calcium sulfate, polyols, pyrogen-free water, isotonic saline, phosphate buffer, and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, and excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein.

[0182] The carrier may constitute, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical composition presented herein.

[0183] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the protein conjugate of the present invention. The term "therapeutically effective amount" refers to the amount of a drug that is effective for treating a disease or disorder in a mammal. The term "therapeutically effective amount" refers to the amount that is effective over a dosage and period necessary to achieve the desired therapeutic or prophylactic result. The exact dosage form and regimen are determined by the physician according to the condition of the patient.

[0184] 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.

[0185] In some embodiments, the compounds of the invention may exist in free form for treatment or as pharmaceutically acceptable salts.

[0186] As used herein, the term "pharmaceutically acceptable salt" refers to any non-toxic salt of a compound of the invention that can directly or indirectly provide the compound of the invention, or its inhibitory active metabolite or residue, upon administration to a subject, such as a human. For example, the term "pharmaceutically acceptable" may mean approved by a regulatory authority of the federal or state government or listed in the US Pharmacopeia or other generally recognized pharmacopeias for use in animals, more specifically in humans.

[0187] 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 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 compound. 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.

[0188] Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, acetate, aspartate, benzenesulfonate, benzoate, bicarbonate, carbonate, halide (e.g., bromide, chloride, iodide, fluoride), bitartrate, citrate, salicylate, stearate, succinate, sulfate, tartrate, decanoate, edetate, fumarate, gluconate, and lactate, or any combination thereof.

[0189] Further examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed 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 formed by other methods used in the art, such as ion exchange.

[0190] Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-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.

[0191] The basic addition salts can be prepared by: 1) reacting the purified compound in 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+(C1-4 alkyl)4 salts. The present invention also contemplates the quaternization of the basic nitrogen-containing groups of the compounds disclosed herein. Water-soluble, oil-soluble, or dispersible products can be obtained by such quaternization.

[0192] Further pharmaceutically acceptable salts are formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates, and include non-toxic ammonium, quaternary ammonium, and amine cations. Other acids and bases, although not themselves pharmaceutically acceptable, can be used in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid or basic addition salts.

[0193] In some embodiments, the term "one or more" refers to any numerical value selected from 1, 2, 3, 4, 5, or 6. In some embodiments, the heteroatom includes any of N, O, NH, or S.

[0194] In some embodiments, the compounds described herein are chiral compounds (i.e., having an asymmetric carbon atom). In some embodiments, diastereomers, geometric isomers, and individual isomers are included 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 compound is in the form of one enantiomer having an asymmetric carbon atom with the R configuration. In some embodiments, the chiral compound is in the form of one enantiomer having an asymmetric carbon atom with the S configuration as described above herein.

[0195] In some embodiments, the chiral compound is in the form of one enantiomer having an enantiomeric purity of greater than 70%. In some embodiments, the chiral compound is in the form of one enantiomer having an enantiomeric purity of greater than 80%. In some embodiments, the chiral compound is in the form of one enantiomer having an enantiomeric purity of greater than 90%. In some embodiments, the chiral compound is in the form of one enantiomer having an enantiomeric purity of greater than 95%.

[0196] In some embodiments, the compounds of the present invention containing unsaturated bonds are in the form of trans isomers or cis isomers. In some embodiments, the compositions of the present invention contain a mixture of cis isomers and trans isomers as described hereinabove.

[0197] In some embodiments, the compounds described herein can exist in unsolvated forms as well as solvated forms including hydrated forms. Generally, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention. Specific compounds of the present invention can exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the intended uses by the present invention and are intended to be within the scope of the present invention.

[0198] The term "solvate" refers to complexes of various stoichiometries (e.g., di, tri, tetra, penta, hexa, etc.) formed by a solute (the conjugate described herein) and a solvent. Thereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid, and the like.

[0199] The term "hydrate" refers to a solvate where the solvent is water, as defined above.

[0200] Unless otherwise indicated, the structures shown in this specification also mean that they include all isomeric (e.g., enantiomeric, diastereomeric, geometric, conformational, and rotational) forms of the structure. For example, the R and S configurations of each chiral center, (Z) and (E) double bond isomers, and (Z) and (E) structural isomers are included in the present invention. As will be understood by those skilled in the art, substituents can rotate freely around any rotatable bond. Accordingly, one stereochemical isomer of the compounds of the present invention, as well as enantiomeric, diastereomeric, geometric, conformational, and rotational mixtures, are within the scope of the present invention.

[0201] Unless otherwise specified, all tautomers of the compounds of the present invention are within the scope of the present invention.

[0202] Furthermore, unless otherwise specified, the structures shown in this specification also mean that they include compounds that differ only in the presence of one or more isotope-enriched atoms. For example, compounds having this structure are within the scope of the present invention, except for the substitution of hydrogen by deuterium or tritium, or the substitution of hydrogen by 18F, or the substitution of carbon by 13C- or 14C-enriched carbon. Such compounds are useful, for example, as imaging probes.

[0203] Method of Use In another aspect, a method of binding to an intracellular target is provided, the method comprising contacting a cell expressing the intracellular target with a protein conjugate of the present invention or a pharmaceutical composition of the present invention, thereby binding to the intracellular target.

[0204] In some embodiments, the method is a method of modulating an intracellular target. In some embodiments, the payload binds to an intracellular target. In some embodiments, the payload modulates an intracellular target. In some embodiments, modifying is acting. In some embodiments, the payload is an agonist of an intracellular target. In some embodiments, modifying is antagonizing. In some embodiments, the payload is an antagonist. Molecules that modulate (i.e., antagonize or stimulate) are well known in the art and any such molecule may be used. In some embodiments, the biological target is specific for an intracellular target.

[0205] In some embodiments, the method is a method of 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 coloring tags). Any such known tag may be used.

[0206] In some embodiments, the cells are in a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a 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 acting on 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.

[0207] In some embodiments, the method comprises administering to the subject a protein conjugate of the present invention. In some embodiments, the method comprises administering to the subject a pharmaceutical composition of the present invention.

[0208] As used herein, the terms "administering", "administration" and like terms refer to any method of delivering a composition comprising an active agent to a subject in a manner that provides a therapeutic effect in the context of sound medical practice. One aspect of the subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the subject matter to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, intratumoral or intraperitoneal.

[0209] The dosage will depend upon the age, health and weight of the recipient, the nature of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.

[0210] In some embodiments, the disease or condition is cancer. In some embodiments, the intracellular target is a cancer gene and the payload is an antagonist. In some embodiments, the intracellular target is a tumor suppressor and the payload is an agonist.

[0211] In some embodiments, contacting is not in the presence of an agent designed to induce permeation of the protein conjugate into the cell. In some embodiments, the agent designed to induce permeation is an agent other than a carrier protein. In some embodiments, no other method of inducing cell permeation other than the methods of the invention is used.

[0212] As used herein, the term "alkyl" includes straight and branched chain groups and represents aliphatic hydrocarbons usually containing from 1 to 30, or from 1 to 10 carbon atoms. As used herein, the term "alkyl" also encompasses saturated or unsaturated hydrocarbons and thus further includes alkenyl and alkynyl.

[0213] The term "alkenyl" represents an unsaturated alkyl as defined herein having at least two carbon atoms and at least one carbon-carbon double bond. Alkenyl may or may not be substituted with one or more substituents as described above.

[0214] The term "alkynyl" as defined herein is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. Alkynyl may or may not be substituted with one or more substituents as described above.

[0215] The term "cycloalkyl" represents a fully carbon monocyclic or fused ring (i.e., rings sharing an adjacent pair of carbon atoms) group having no fully conjugated pi electron system in one or more of the rings. Cycloalkyl groups may or may not be substituted as shown herein.

[0216] The term "aryl" represents a fully carbon monocyclic or fused ring polycyclic (i.e., rings sharing an adjacent pair of carbon atoms) group having a fully conjugated pi electron system. Aryl groups may or may not be substituted as shown herein.

[0217] The term "alkoxy" represents both O-alkyl groups and -O-cycloalkyl groups as defined herein. The term "aryloxy" represents -O-aryl as defined herein.

[0218] Each of the alkyl, cycloalkyl, and aryl groups in the general formulas herein may be substituted with one or more substituents, where each substituent may independently be, depending on the substituent and its position in the molecule, for example, a halide, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxyl, thiol, thioalkoxy, carboxy, amide, aryl, and aryloxy. Additional substituents are also contemplated.

[0219] The terms "halide", "halogen", or "halo" represent fluorine, chlorine, bromine, or iodine. The term "haloalkyl" represents an alkyl group as defined herein further substituted with one or more halides. The term "haloalkoxy" represents an alkoxy group as defined herein further substituted with one or more halides. The term "hydroxyl" or "hydroxy" represents an -OH group. The term "mercapto" or "thiol" represents an -SH group. The term "thioalkoxy" represents both -S-alkyl groups and -S-cycloalkyl groups as defined herein. The term "thioaryloxy" represents both -S-aryl groups and -S-heteroaryl groups as defined herein. The term "amino" represents an -NR’R’’ group or a salt thereof having R’ and R’’ as described herein.

[0220] The term "heterocyclyl" represents a monocyclic or fused ring group having one or more atoms, such as nitrogen, oxygen, and sulfur, in the ring(s). The ring may also have one or more double bonds. However, the ring does not have a fully conjugated pi electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, and the like.

[0221] The term "carboxy" refers to a -C(O)OR' group, or a carboxylate thereof, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (attached through a ring carbon), or heterocyclyl (attached through a ring carbon), or refers to a "carboxylate".

[0222] The term "carbonyl" refers to a -C(O)R' group, where R' is as defined above. The above terms also include their thio derivatives (thiocarboxy and thiocarbonyl).

[0223] The term "thiocarbonyl" refers to a -C(S)R' group, where R' is as defined above. The "thiocarboxy" group refers to a -C(S)OR' group, where R' is as defined herein. The "sulfinyl" group represents a -S(O)R' group, where R' is as defined herein. The "sulfonyl" or "sulfonate" group represents a -S(O)2R' group, where R' is as defined herein.

[0224] The "carbamyl" or "carbamate" group represents an -OC(O)NR'R'' group, where R' is as defined herein and R'' is as defined for R'. The "nitro" group refers to a -NO2 group. The term "amide" as used herein includes C-amides and N-amides. The term "C-amide" represents a -C(O)NR'R'' terminal group or a -C(O)NR'-linking group, these terms being defined above, and R' and R'' are as defined herein. The term "N-amide" represents a -NR''C(O)R' terminal group or a -NR'C(O)-linking group as these terms are defined above in this specification, where R' and R'' are as defined herein.

[0225] The term "cyano" or "nitrile" group refers to a -CN group. The terms "azo" or "diazo", as defined above, represent a -N=NR' end group or a -N=N- linking group, where R' is as defined above. The term "guanidine", as defined above, represents a -R'NC(N)NR''R''' end group or a -R'NC(N)NR''- linking group, where R', R'' and R''' are as defined herein. The term "azide" as used herein refers to a -N3 group. The term "sulfonamide" refers to a -S(O)2NR'R'' group, where R' and R'' are as defined herein.

[0226] The term "phosphonyl" or "phosphonate" represents an -OP(O)-(OR')2 group, where R' is as defined above. The term "phosphinyl" represents a -PR'R'' group, where R' and R'' are as defined above. The term "alkylaryl" represents an alkyl as defined herein substituted by an aryl as described herein. An exemplary alkylaryl is benzyl.

[0227] The term "heteroaryl" refers to a group of monocyclic or fused rings (i.e., rings that share a pair of adjacent atoms) that have one or more atoms, such as nitrogen, oxygen, and sulfur, in the ring(s), and in addition have a fully conjugated pi 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 of 3, 4, 5, 6, 7, 8, 9, and more than 9 atoms. Heteroaryl groups may optionally be substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C3-8 heterocyclic groups containing one oxygen or sulfur atom, or two oxygen atoms, or two sulfur atoms or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted derivatives, and benzocondensed and pyrido-condensed derivatives, for example, connected via one of the ring-forming carbon atoms. In certain embodiments, heteroaryl is selected from oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl.

[0228] In some embodiments, the heteroaryl group is selected from 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) pyridazinyl, pyrimidinyl, pyrazinyl, 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, thieno[2,3-b]thiophenyl, 1,8-naphthyridinyl, other naphthyridinyl, pteridinyl or phenothiazinyl. When the heteroaryl group contains more than one ring, each additional ring is in a saturated form (perhydro form) or a partially unsaturated form (e.g., dihydro form or tetrahydro form) or a maximally unsaturated (non-aromatic) form. Thus, the term heteroaryl includes bicyclic radicals in which two rings are aromatic and bicyclic radicals in which only one ring is aromatic. Such examples of heteroaryl include 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-dihydroisoquinoxalinyl, 4-(3H)quinazolinonyl, 4H-chromenyl, 4-chromanonyl, oxyindolyl, 1,2,3,4-tetrahydroisoquinolinyl, 1,2,3,4-tetrahydroquinolinyl, 1H-2,3-dihydroisoindolyl, 2,3-dihydrobenzo[f]isoindolyl, 1,2,3,4-tetrahydrobenzo[g]isoquinolinyl, 1,2,3,4-tetrahydrobenzo[g]isoquinolinyl, chromanyl, isochromanonyl, 2,3-dihydrochromonyl, 1,4 - benzodioxanyl, 1,2,3,4 - tetrahydro - quinoxalinyl, 5,6 - dihydro - quinolyl, 5,6 - dihydroiso - quinolyl, 5,6 - dihydroquinoxalinyl, 5,6 - dihydroquinazolinyl, 4,5 - dihydro - 1H - benzimidazolyl, 4,5 - dihydro - benzoxazolyl, 1,4 - naphthoquinolyl, 5,6,7,8 - tetrahydro - quinolinyl, 5,6,7,8 - tetrahydro - isoquinolinyl, 5,6,7,8 - tetrahydroquinoxalinyl, 5,6,7,8 - tetrahydroquinazolinyl, 4,5,6,7 - tetrahydro - 1H - benzimidazolyl, 4,5,6,7 - tetrahydro - benzoxazolyl, 1H - 4 - oxa - 1,5 - diaza - naphthalen - 2 - onyl, 1,3 - dihydroimidazolo - [4,5] - pyridin - 2 - onyl, 2,3 - dihydro - 1,4 - dinaphtho - quinonyl, 2,3 - dihydro - 1H - pyrrolo[3,4 - b]quinolinyl, 1,2,3,4 - tetrahydrobenzob - [1,7]naphthyridinyl, 1,2,3,4 - tetrahydrobenzob[1,6] - naphthyridinyl, 1,2,3,4 - tetrahydro - 9H - pyrido[3,4 - b]indolyl, 1,2,3,4 - tetrahydro - 9H - pyrido[4,3 - b]indolyl, 2,3 - dihydro - 1H - pyrrolo - [3,4 - b]indolyl, 1H - 2,3,4,5 - tetrahydro - azepino[3,4 - b]indolyl, 1H - 2,3,4,5 - tetrahydroazepino - [4,3 - b]indolyl, 1H - 2,3,4,5 - tetrahydro - azepino[4,5 - b]indolyl, 5,6,7,8 - tetrahydro[1,7]naphthyridinyl, 1,2,3,4 - tetrahydro - [2,7] - naphthyridyl, 2,3 - dihydro[1,4]dioxino[2,3 - b]pyridyl, 2,3 - dihydro[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] - naphthyridinyl, 1,2,3,4 - tetrahydro[1,6]naphthyridinyl, 1,2,3,4 - tetrahydro[1,7]naphthyridinyl, 1,2,3,4 - tetrahydro - [1,8]naphthyridinyl, or 1,2,3,Examples include 4-tetrahydro[2,6]naphthyridinyl. In some embodiments, the heteroaryl group is optionally substituted. In one embodiment, one or more substituents are each independently selected from halo, hydroxy, amino, cyano, nitro, alkylamide, acyl, C1-6-alkyl, C1-6-haloalkyl, C1-6-hydroxyalkyl, C1-6-aminoalkyl, C1-6-alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl.,

[0229] Examples of heteroaryl groups include, but are not limited to, 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, quinolidine, cinnoline, phthalazine, quinazoline and quinoxaline unsubstituted and mono- or di-substituted derivatives. In some embodiments, the substituents are halo, hydroxy, cyano, O-C1-6-alkyl, C1-6-alkyl, hydroxy-C1-6-alkyl and amino-C1-6-alkyl.

[0230] As used herein, the terms “halo” and “halide” are used interchangeably herein and refer to a halogen atom, i.e., fluorine, chlorine, bromine or iodine, and are also referred to herein as fluoride, chloride, bromide, and iodide.

[0231] As used herein, the term “about,” when combined with a value, refers to ±10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm ± 100 nm.

[0232] It should be noted that, as used herein and in 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 a "polypeptide" includes a reference to one or more polypeptides and equivalents thereof known to those of skill in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prerequisite for using exclusive language, such as "solely," "only," and the like, in connection with the description of claim elements or the use of a "negative" limitation.

[0233] Where a convention similar to "at least one of A, B, and C, etc." is applied, such a configuration 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" includes, but is not limited to, systems with 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 be further 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 consider the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0234] For the sake of clarity, it is understood that certain features of the invention described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for the sake of brevity, the various features of the invention described in the context of a single embodiment may be provided separately or in any suitable sub-combination. All combinations of embodiments of the invention are specifically embraced by the invention and are disclosed herein as if each combination were individually and explicitly disclosed. Further, all sub-combinations of the various embodiments and their elements are also specifically embraced by the invention and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

[0235] Further objects, advantages, and novel features of the invention will become apparent to those skilled in the art by considering the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the invention described above and claimed in the following claims finds experimental support in the following examples.

[0236] Each of the various embodiments and aspects of the invention described above and claimed in the following claims finds experimental support in the following examples.

[0237] Examples 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 explained in detail in the literature. For example, "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and 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" 1-4, Cold Spring Harbor Laboratory Press, New York (1998); the methods described below, 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, J.E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" Freshney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); Stites et al.(Eds.), see 「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 by reference. Other general references are provided herein.

[0238] Example 1: Direct Chemical Modification To facilitate intracellular delivery via the endocytosis 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. Since PEI was found to be far superior (data not shown), all further experiments were carried out using PEI.

[0239] PEI is a linear or branched long polymer usually having a molecular weight in the range exceeding 10 KDa and is characterized by having the highest cationic charge density among existing polymers. This has led to their use as transfection agents, and their high positive charge is used for complex formation with negatively charged DNA or RNA and further for internalizing these nucleic acids into cells. These high molecular weight transfection agents are efficient for in vitro applications, but they are not very useful for in vivo applications. Therefore, ultra-low molecular weight PEI moieties, i.e., branched PEI molecules with a molecular weight in the range of 600 - 1800 Da (measured by mass spectrometry), were used. These ultra-low molecular weight PEIs can be chemically and covalently conjugated to the internalized protein payload.

[0240] Ultra-low molecular weight PEI, mainly 600 Da, was conjugated to IgG and GFP. Since PEI contains many primary amines, conjugation to the carboxylic acid residues of glutamic acid and asparagine of the protein and to the C-terminal carboxy group was carried out using carbodiimide conjugation chemistry. The reaction was carried out with an excess of PEI, namely 3500 molar excess, and control of the modification level was achieved by adjusting the level of the carbodiimide agent (N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC)) in the reaction. The level of modification was determined by MALDI-ToF mass spectrometry (Figure 1). In the case of IgG-based proteins, the average modification level ranged from 1 to 8.5 molecules of PEI (600 Da) per IgG molecule, which was achieved with excess levels of EDC in the range of 25 to 400 molar equivalents, respectively. Since PEI modification is not site-selective, it results in a broad distribution of the molecular weight portion, as observed in the MALDI-ToF spectrum (Figure 1). The average modification level was calculated based on the value obtained by subtracting the measured peak top molecular weight value of the unmodified protein from the measured peak top molecular weight value of the added PEI that gives the weight of the added PEI. By dividing this value by the molecular weight of a single PEI molecule used for modification, the average number of added molecules was obtained.

[0241] Next, unmodified and PEI-modified mouse IgG were incubated with A375 cells for 2 hours. No additional induction for transfection / internalization was added. As can be confirmed in Figure 2, unmodified mouse IgG did not enter A375 cells. In contrast, even a low level of PEI modification (average 3 PEI molecules per IgG molecule, X3) enabled internalization, while increasing the average level of modification proportionally increased the level of IgG observed intracellularly. Similar results were also observed when internalization was measured using flow cytometry (data not shown).

[0242] Similar internalization efficiency and dependency on modification levels were also observed for other payloads, including other IgG (data not shown) and green fluorescent protein (GFP) (Figures 3A-3B).

[0243] Single-domain antibodies and other smaller payloads are ideal for intracellular activity because their size is also an advantage due to higher and easier dispersibility in the crowded cytoplasm. Small protein payloads can face functional problems when directly modified with PEI molecules. In small proteins, the likelihood that such chemical modifications interfere with the functional domains of the protein is very high. By using selective binding chemistry for linkers to therapeutic agents at specific sites or domains, the exact location of linker conjugation can be controlled and it can be diverted from the active domain of the therapeutic agent.

[0244] Even better, the use of carrier proteins for chemical modification completely eliminates this risk. Furthermore, small proteins in general and single-domain antibodies in particular are surely faced with pharmacokinetic issues and have a very short plasma half-life and high clearance, making them unsuitable for therapeutic use. Therefore, the inventors chose the delivery of protein payloads using the described carrier protein system.

[0245] Two schematic diagrams of this carrier and payload methodology are shown in Figures 4A-4B. As can be confirmed, the carrier protein bears PEI groups and is responsible for membrane passage 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 above. Figure 4A shows a configuration with a cytosolic cleavable S-S bond. It will be understood that this bond can be simply replaced with a C-C bond or an O-C bond to produce a non-cleavable linker. Another configuration of the conjugate with a non-cleavable linker is shown in Figure 4B.

[0246] The carrier protein itself is preferably selected from the list of human endogenous proteins to avoid immunogenicity issues. Further, proteins that are commonly found in the blood and have a naturally long circulation half-life are preferred. Finally, proteins that can deliver the payload to the area within the body where the therapeutic agent is desired are advantageous. Examples of such areas in the body can include tumors and their microenvironment, as well as inflammatory sites important for autoimmune diseases and many other pathological conditions. Human serum albumin (HSA) was thus selected. HSA is a circulating protein with a long half-life and has been shown to transport to tumors and inflammatory sites and deliver the payload, although only to the extracellular environment (see 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; and Yazaki P.J. et al., Nuclear Medicine and Biology, 35 (2008) 151 - 158, all of which are incorporated herein by reference in their entirety).

[0247] The therapeutic agent is conjugated to a single linker at one conjugation site along the polypeptide chain of the biological agent. This conjugation site is in a position where it does not interfere with the activity of the agent. The linker can be selected from a peptide linker, a chemical linker, or a combination. PEG, a polymer-based linker, was selected. As discussed above, labile bonds that are sensitive to specific conditions in the cytoplasm of the cell can also be incorporated.

[0248] To demonstrate the importance of the carrier, the single-domain antibody payload was conjugated directly to the PEI molecule without a carrier. To minimize interference with the CDRs of the single-domain antibody, the PEI should be attached to a specific site in the single-domain antibody, preferably as far as possible from these CDRs. The appropriate position is the C-terminus of such a payload. As described above, when using the C-terminal cysteine of VHH or DARPin for PEI modification, only 1 unit of PEI is conjugated to the payload. Therefore, to achieve a significant positive charge with only 1 PEI unit, branched PEI with a molecular weight of 1800 Da was also used in addition to the 600 Da PEI described in the present invention for carrier modification.

[0249] The conjugation of PEI was performed at a selected site at the C-terminus of the payload by either reversible or irreversible disulfide bonds. Additionally, random (not site-specific), irreversible PEI modification of the single-domain antibody was also performed. It should be noted that the inventors always obtained multiple PEI-modified payloads. Furthermore, the crude reaction mixture always contained multiple products, and they took a long time to separate. Therefore, it was hypothesized that to obtain a specific PEI modification (e.g., a conjugate having a PEI modification at a predetermined position and / or having a predetermined number of PEI moieties), it was necessary to synthesize the conjugate of the present invention by linking the PEI-modified carrier to the payload via a linker as described herein.

[0250] For this purpose, PEI (1800 or 600 Da) was modified with NHS-PEG12-SPDP or NHS-PEG8-MAL to generate thiol-reactive PEI for payload conjugation in a reversible or irreversible manner, respectively. The activated PEI was reacted with the anti-BRAF VHH, 1C5-Hel20-LC to obtain the following constructs: 1. 1C5-Hel20-LC-S-S-PEG 12 -C(O)-NH-PEI1800(S6) 2.1 C5-Hel20-LC-PEG8-C(O)-NH-PEI1800 (S7) 3.1 C5-Hel20-LC-SS-PEG 12 -C(O)-NH-PEI600 (S6A) 4.1 C5-Hel20-LC-PEG8-C(O)-NH-PEI600 (S7A)

[0251] Random modification was achieved by the reaction of 1C5-Hel20-LC with PEI1800 in the presence of EDC to obtain 1C5-Hel20-LC-rC(O)-NH-PEI1800 (S8). Various constructs of payload directly conjugated to PEI were evaluated for their ability to permeate A375 cells and induce apoptosis after BRAF inhibition. These constructs were compared with payload conjugated to the carrier of the present invention via reversible binding, named 1C5-Hel20-LC-SS-HSA-PEIx4.5 (S5C), and via irreversible binding: 1C5-Hel20-LC-HSA-PEIx4.5 (S5D).

[0252] The chemical structures of the conjugates (S5D, S6 - S7A) are shown in Table 1 above.

[0253] As can be confirmed in FIGS. 5A-5D, only the anti-BRAF VHH conjugated to the carrier by either reversible or irreversible linkage exhibited efficient BRAF inhibition resulting in a distinct apoptotic signal when treated with all agents at 1 μM (FIGS. 5A-5B) or 3 μM (FIGS. 5C-5D), while all VHHs directly modified with PEI, regardless of site-specific modification (FIGS. 5A-5B) or random modification (FIGS. 5C-5D), did not exhibit apoptosis or exhibited very limited apoptosis. The inactivity of the directly modified VHHs can be explained by a lower level of membrane penetration and a much lower efficiency of endosomal escape, as revealed by limited intracellular staining with anti-VHH in all cases lacking the carrier protein (FIGS. 5E-5L). The lower escape was due to the low level of PEI molecules, as even the use of a larger PEI modification factor (1800 Da) was insufficient to compensate for it. The zeta potential of one of these VHHs, non-reversibly modified with 1800 Da PEI at its C-terminus, was measured to be just under about +5 mV, which is considered too low for efficient internalization and endosomal escape. Furthermore, as described above, the lack of activity may also be caused by interference of the PEI conjugated closely to the active domain of the VHH or by folding of the linker on itself, hiding either the PEI, the CDR, or both.

[0254] In the parallel experiment, A375 cells were plated at 1500 cells / well in a black / clear-bottom 96-well plate. After 24 hours, anti-BRAF 1C5 VHH conjugated to HSA-PEIx3.5 (using an irreversible maleimide-based linker) diluted in PBS was added to the cells (final concentration 3.2 μM). 2A1 VHH conjugated to HSA-PEIx3.5 (anti-CD28 VHH, an irrelevant VHH here) and PEI-modified HSA, both using an irreversible maleimide-based linker, were used as negative controls (final concentration 24 μM). PF-04880594 (a selective RAF inhibitor, Sigma, catalog number PZ0294) was used as a positive control (final concentration 10 μM). Caspase reagent was added to the cells at the start of the uptake period (final dilution 1:1000). The kinetic profile of caspase 3 / 7 activation was monitored and quantified using live cell imaging with an IncuCyte® Analyzer every 2 hours for 4 days. At the end of the assay, the medium was removed and CellTiter-Glo® Luminescent reagent (Promega) was added for the evaluation of cell viability. The CellTiter-Glo® Luminescent cell viability assay is a method for measuring the number of viable cells in a culture based on the quantification of ATP present, which is an indicator of metabolically active cells.

[0255] As can be confirmed in Figure 5M, only 1C5 VHH conjugated to HSA-PEI resulted in a dramatic increase in caspase 3 / 7 activity, suggesting good and effective inhibition of BRAF in these cells, which induces apoptosis. Neither the irrelevant VHH nor any of the VHH controls caused substantial apoptosis of the cells. It should be noted that the BRAF inhibitor was a very potent cell killer. Therefore, many cells died completely before a fluorescence signal could be detected (Figures 5A, 5C, 5M). However, when the signal was normalized against the cell titer, it was clear that the inhibitor was very effective (Figures 5B, 5D).

[0256] Example 2: Membrane Permeation Efficiency Many studies using cell-penetrating peptides (CPPs) have shown some level of cellular internalization, but in most cases, the efficiency of initial uptake into cells was very low. This is because high levels of payload in the medium were required to produce only a small fraction of the molecules that were actually internalized into the cells. To evaluate the efficiency of the initial internalization step, a specific ELISA was used to measure the levels of mouse IgG modified with the payload PEI in the medium as a function of time. As can be seen in Figure 6, at high levels of PEI modification (about 7), almost all of the modified IgG was internalized (95%), and most of the internalization occurred during the first 24 hours of incubation (gray bar graph). Even at lower levels of modification (about 4.5 and 3), the internalization step was very efficient, showing an overall uptake of about 75% of the total payload after 5 days of incubation (Figure 6, darkest gray bar graph and lightest gray bar), following a reaction rate similar to that of highly modified IgG. Incomplete and slow uptake may be associated with IgG that is much less modified than the average modification level. The PEI modification results in a somewhat broad distribution of the modified moiety (see Figure 1), and unmodified or lowly modified (less than 3) molecules are likely to still be present in the medium even after 5 days. This cell membrane passage efficiency was observed at various initial medium concentrations in the range of 0.2 - 40 μg / mL of IgG.

[0257] Example 3: Endocytosis Pathways Utilized by PEI-Modified Proteins The cationized moiety is thought to be internalized using the natural endocytosis machinery. Two related endocytosis mechanisms are caveolin-mediated endocytosis and clathrin-mediated endocytosis. The exact mechanism of internalization was elucidated by performing the internalization of PEI-modified IgG in the presence of known specific endocytosis inhibitors. A375 cells were incubated overnight with PEI-modified mouse IgG (4.5PEI) in the presence of a clathrin inhibitor (amantadine or chlorpromazine) or a caveolin inhibitor (genistein). Genistein, a known caveolin inhibitor, actually inhibited the internalization of PEI-modified IgG (Figure 7). Very interestingly, the addition of chlorpromazine, a known clathrin inhibitor, resulted in an increase in internalization. This supports the use of caveolin-mediated endocytosis by the PEI-modified payload for internalization, since inhibition of clathrin-mediated endocytosis caused a compensatory increase in caveolin-mediated endocytosis within the cell.

[0258] Example 4: Endosomal Escape As described above, perhaps the most intractable difficulty in the intracellular delivery of biotherapeutics is their endosomal escape in order to avoid the degradation of the therapeutic agent. To evaluate whether proteins modified with PEI can escape from endosomes, their internalization was traced using confocal microscopy and the counterstaining of endosome and lysosome markers. IgG modified with PEI was incubated with HEK293 cells expressing a fluorescent endosome / lysosome marker for 5 hours. The cells were analyzed by confocal microscopy. Endosomes and lysosomes were transfected with a green fluorescent marker (Cell light Endosome, Molecular Probes, Cat. No. C10586 or Cell light Lysosome, Molecular Probes, Cat. No. C10596), while IgG was stained with a red fluorescent anti-mouse antibody. As can be confirmed in Figure 8, no major co-localization was observed between internalized IgG and early endosome or lysosome vesicles. This indicates that the payload actually modified with PEI successfully escaped from the endosome. These experiments were repeated using the endosome / lysosome markers EEA1, transferrin, and calcein, and the same results were observed (data not shown). It should be noted that co-localization was not observed even in early endosomes, suggesting that the endosomal escape of proteins modified with PEI occurs rapidly very early in the endosomal pathway.

[0259] Colocalization was not observed between PEI-modified IgG and various endosomal and lysosomal markers, although the staining of these IgGs appeared essentially punctate. The punctate profile may suggest the containment of internalized IgG in certain vesicles. To further support the endosomal escape of the PEI-modified protein, the protein was labeled with a pH-sensitive fluorescent dye, 5(6)-carboxynaphthofluorescein. This dye fluoresces only at values above pH 7. Since the pH of early endosomes is already less than 7 and only decreases as the endosomes mature into lysosomes, all vesicles in the endosomal pathway are acidic. PEI-modified and pH-sensitive-labeled IgG was incubated with HeLa cells, and the cells were analyzed by confocal microscopy. The cells clearly showed the unique red fluorescence of the pH-sensitive dye, indicating that the PEI-modified protein was in a non-acidic compartment such as the cytoplasm (Figure 9). This is further evidence that the PEI-modified protein efficiently escapes the endosomal pathway.

[0260] Finally, functional assays were used to verify that PEI-modified proteins efficiently escape from endosomes after their internalization. To achieve this, a 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 responsible for 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 to one of the ITAMs within the CD3 zeta chain.

[0261] The internalization of PEI-modified anti-CD247 and PEI-modified mouse IgG as a negative control was carried out in CD3+ cells pre-stimulated with beads coated with anti-CD3 / CD28 antibodies. The medium was monitored for interferon gamma (IFNγ) secretion as a marker for changes in the activation level of the cells. The complete antibody is not expected to be stable in the cytoplasm due to the presence of structurally essential disulfide bonds, but the binding of this antibody will activate the signaling cascade and ultimately lead to IFNγ secretion. Thus, even a small amount of initial activation will result in a quantifiable increase in cytokine levels. Therefore, despite its expected cytoplasmic instability, the internalization of the antibody is expected to result in an observable IFNγ response.

[0262] The internalization of the modified antibody into CD3+ cells was verified by intracellular FACS analysis (data not shown). The internalization of anti-CD247 PEI-modified mAb (4.5xPEI) resulted in dose-dependent activation of CD3 cells as measured by increased IFNγ secretion (Figure 10). PEI-modified mouse IgG did not cause any activation of CD3 cells within the range of antibody concentrations used in this experiment. This result further supports the endosomal escape of proteins modified with PEI and their ability to exert biological and clinical effects in target cells.

[0263] Example 5: Cytosolic dispersibility and payload-carrier solution The endosomal escape of PEI-modified proteins clearly occurs based on function, but the microscopic images of various internalized proteins exhibit a punctate profile (Figures 2, 3, 8, and 9). Although PEI modification is very efficient in cell membrane passage and endosomal escape, a hypothesis was put forward that the same modification hinders the efficient dispersion of PEI-modified proteins in the cytoplasm. This problem of dispersibility may be the result of strong electrostatic interactions between highly cationized internalized proteins and various cytoplasmic proteins, mostly negatively charged, mainly cytoskeletal proteins. Low cytoplasmic dispersibility can negatively affect the effectiveness of any intracellular biological agent because the biological agent may not be able to reach its intracellular target in the cytoplasm or in other intracellular compartments or organelles.

[0264] To overcome this intracellular dispersibility obstacle, disulfide bonds were incorporated into the linker between the therapeutic agent and the universal carrier pre-modified with PEI. The selected linker was a chemical linker into which disulfide bonds were incorporated. This labile disulfide bond is cleaved upon endosomal escape mainly due to the high reducing potential of the cytoplasm by high concentrations of glutathione, thus releasing the therapeutic agent from its cationized carrier and allowing free movement of the therapeutic agent in the cytoplasm of the cell. To evaluate the efficiency of the carrier payload method, GFP was conjugated to PEI-cationized HSA via a PEG-based linker incorporating disulfide bonds. eGFP (Biorbyt, catalog number orb84840) was modified with NHS-PEG4-SPDP. HSA modified with PEI (11xPEI) was also further reacted with NHS-PEG4-SPDP after reduction of this SPDP to free thiols using DTT. Activated eGFP with SPDP was reacted with the HSA-PEI free thiol to create a GFP-HSA conjugate with labile disulfide bonds incorporated into the linker.

[0265] In contrast to the punctate profile of the internalized PEI-modified GFP (Figure 3), when GFP disulfide-linked to PEI-cationized HSA was internalized into cells, it yielded a completely dispersed profile without puncta (Figure 11). To verify the efficient dispersion of the internalized therapeutic payload, a full-length therapeutic anti-TNFα monoclonal antibody (Humira®, Abbvie) was conjugated to PEI-modified HSA via a PEG-based linker incorporating a disulfide bond. Similar to GFP, the antibody was modified with NHS-PEG4-SPDP. HSA modified with PEI (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 the HSA-PEI free thiols to create an antibody-HSA conjugate with labile disulfide bonds incorporated into the linker. As can be confirmed in Figure 12, the internalized monoclonal antibody was dispersed throughout the cytoplasm, further supporting the hypothesis that direct cationization hinders free dispersion in the cytoplasm and demonstrating the effectiveness of the solution that separates the payload from its cationic carrier.

[0266] This dispersibility can also be confirmed in Figures 5F - 5G and by comparing Figures 3 - 11. Payloads conjugated with linkers containing S-S cleavable bonds were more uniformly dispersed throughout the cell. However, this does not mean that constructs without cleavable linkers were not distributed at all or were non-functional. Figure 5G clearly shows the distribution of VHHs with non-cleavable linkers within the cell, and Figures 4A - D clearly show that conjugates with non-cleavable linkers can still bind to and function against the target, but it is noteworthy that constructs with cleavable linkers are superior in inducing apoptosis (Figures 5A - 5B). Thus, while dispersibility is beneficial and reaching / binding to the target can be somewhat challenging, non-cleavable linkers can still be fully functional.

[0267] Example 6: Cytosolic Stability Therapeutic agents not only need to enter the cytoplasm of target cells but also exhibit their biological activity. Molecules transported to other intracellular locations (such as the nucleus, ER, mitochondria, etc.) still pass through the cytoplasm. Many biological therapeutic agents are based on antibody scaffolds or their derivatives. In many cases, these therapeutic agents bind to specific targets and antagonize or act on those targets. The binding activity of these agents completely depends on their tertiary and quaternary structures. For antibody-based molecules, either full IgG or its truncated derivatives (such as Fab, scFv, etc.), these structures are based on and stabilized by intra-chain or inter-chain disulfide bonds. However, as mentioned above, the cytoplasm, as well as other intracellular organelles and compartments, is characterized by a highly reducing environment. The major reducing agent, glutathione, has a cytosolic concentration in the range of 1 - 11 mM. In contrast, its plasma levels are at low micromolar values. This characteristic of the cytoplasm has hindered the use of antibody-based agents as effective intracellular therapeutic routes, as well as other biological agents that utilize disulfide bonds in their structures.

[0268] Certain in vitro experiments have shown that exposure of antibodies to cytosolic levels of glutathione (GSH), as confirmed by the appearance of multiple bands on SDS-PAGE Western blots detected by anti-light chain antibodies, resulted in the reduction of disulfide bonds after just a few hours. Overnight exposure led to a lack of detection of antibodies in Western blots, presumably due to loss of 3D structure or further aggregation and precipitation. The latter is a known phenomenon for antibodies and their derivatives expressed intracellularly (Kabayama, H. et al., Nature Communications 2020, (11), 336, which is hereby incorporated by reference in its entirety). Any such effects on intracellular biological agents would be detrimental to their ability to bind biological targets and exert a therapeutic effect.

[0269] Considering these intracellular stability issues, single-domain antibodies were selected as the therapeutic payload. Such single-domain antibodies are single-chain protein-based molecules that have the ability to bind to other proteins. Since their structures lack any essential disulfide bonds, they are resistant to the reducing environment of the cytosol. Such single-domain binding proteins include truncated forms of heavy-chain antibodies (HcAbs) of camelid-derived variable heavy-chain homodimers (VHHs), also known as nanobodies, or shark-based immunoglobulin new antigen receptors (IgNARs). Other examples of such single-domain binding proteins include designed ankyrin repeat proteins (DARPins) and genetically engineered antibody mimetic proteins.

[0270] Single-domain binding proteins, especially VHHs, are very suitable as payloads. The absence of their structurally essential disulfide bonds makes them resistant to the cytoplasmic state. They are very small, approximately 15 KDa in the case of VHHs and 20 KDa in the case of DARPins, which aids their dispersibility into the cytosol. Their single-domain nature prevents them from causing any accidental intracellular cross-linking effects. They can be easily engineered to contain two or more parts with different architectures, allowing for more complex binding profiles. They are not considered immunogenic and have a good safety profile. A major feature is their suitability for site-selective conjugation to carriers. For example, in both VHHs and DARPins, their C-termini are located away from their antigen-binding regions (CDRs), allowing the use of this site for conjugation without affecting antigen binding. Furthermore, the C-terminus can be easily engineered to contain one cysteine amino acid with a free sulfhydryl group for conjugation. This group can be conjugated to carriers equipped with thiol-reactive groups. The free sulfhydryl at the C-terminus can be conjugated directly to the carrier or via a linker.

[0271] A commercially available anti-vimentin VHH (Q60c, QVQ) containing one cysteine amino acid at its C-terminus was conjugated to a PEI-modified HSA carrier further modified with a thiol-reactive group. HSA modified with PEI was linked to an NHS-activated PEG linker bearing a 2-pyridyldithio group (NHS-PEG n -SPDP, n = 4). SPDP readily reacts with the free thiol of VHH to generate the VHH-HSA moiety, where the connection between the VHH payload and the HSA carrier contains a disulfide bond. The general scheme of this construct can be seen in Figure 13A, where a conjugate containing two VHHs is shown to exemplify the fact that this conjugation methodology can create conjugates where the carrier is conjugated to multiple payloads. The bond between the carrier and the payload can be cleaved under reducing conditions, and the conjugation to VHH was similar regardless of the level of PEI. Furthermore, in all reactions, the payload was efficiently cleaved after treatment with 5 mM GSH, mimicking the reducing conditions of the cytosol.

[0272] The cellular internalization efficiency of the anti-vimentin PEI-modified HSA conjugate was evaluated by the disappearance of the conjugate from the culture medium during incubation of A375 cells with the conjugate. VHH was conjugated to HSA modified with PEI at two levels of an average of 3.5 PEI molecules and 8 PEI molecules per HSA. As already seen for PEI-modified IgG (Figure 6), the VHH-carrier conjugate was efficiently internalized by the cells (Figure 14), with nearly 80% of the conjugate being internalized during the first 24 hours of incubation. The level of the conjugate was measured using an in-house developed two-sided ELISA that measures only the VHH conjugated to HSA, utilizing an anti-VHH antibody as capture and an anti-HSA antibody as detection. Surprisingly, both PEI modification levels exhibited very similar internalization efficiencies and kinetics, with only a slight advantage for the 8-PEI modification. The levels of these VHH-HSA conjugates in cell-free cell culture medium were evaluated to confirm 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.

[0273] To further evaluate the efficiency of endosomal escape and the dispersibility of the payload VHH in the cytoplasm, anti-vimentin VHH conjugated to PEI-modified HSA (an average of 11 PEIs per HSA molecule) was incubated with A375 cells for 24 hours, and the cells were analyzed by confocal microscopy (Figures 15A - 15B). The presence of VHH inside the cells was shown in a slow-release profile using an anti-VHH antibody conjugated to Alexa Fluor 647 (Figure 15A). The cells were co-stained for vimentin using a standard fluorescently labeled anti-vimentin antibody (Figure 15B). A careful examination of the two images clearly shows that the profile obtained by anti-VHH staining is substantially identical to the vimentin staining, suggesting that anti-vimentin VHH is successfully delivered to the cytoplasm of the cells from which it was released from its carrier and can find and bind its target (Figure 15C). Further evidence of this binding can be confirmed in Figure 16A, where an enlarged view of one of the cells exposed to anti-vimentin VHH conjugated to PEI-modified HSA clearly shows the vimentin cytoskeleton pattern visualized by anti-VHH antibody staining. Figure 16B shows that cells exposed only to anti-vimentin VHH without carrier conjugation do not exhibit intracellular VHH staining.

[0274] The binding of the internalized anti-vimentin VHH to its vimentin target (Figures 15A, 16A) also demonstrates that the VHH agent maintains its structural stability inside the cytoplasm. The ability to bind to a target is highly dependent on its structure and the stability of that structure, as is the case for all binding biological agents. This data thus supports the selection of single-domain binding proteins as payload agents of the present invention.

[0275] Another important feature of the presented delivery system is the efficiency and uniformity of internalization in that all cells in the medium exhibit internalization. This can be seen throughout various microscopic images, particularly those including the images in Figures 15A - 15C. Many attempts in the literature regarding intracellular delivery of proteins have resulted in only some of the cells exhibiting protein internalization, suggesting very low efficiency.

[0276] Example 7: In vitro functional PoCs Further evaluation of the functionality of biologics delivered intracellularly was performed using VHHs against the E7 protein of human papillomavirus (HPV). Almost all cervical cancers are associated with human papillomavirus (HPV) infection, and two types, HPV16 and HPV18, account for 70% of cases. One of the major 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 unregulated cell growth stimulation. E7 can also interfere with the host histone deacetylation mediated by HDAC1 and HDAC2, causing transcriptional activation. Previous studies have suggested that inhibition of E7 function inhibits the growth of HPV-positive cervical cancer cells. Li et al (Molecular Immunology, 2019, 109, 12-19) showed that transfection of plasmids encoding VHHs against the E7 protein in HPV-positive cells (which was used due to the lack of an efficient intracellular delivery system for the protein itself) could interfere with E7 activity (interfere with the E7-RB1 interaction) and result in a decrease in the growth of HPV-positive cells. The inventors expressed and purified the same anti-E7 VHH with an added C-terminal cysteine and conjugated it to the intracellular carrier of the present invention.

[0277] To confirm the amplitude of the intracellular effects of E7 inhibition in HPV-positive cell lines, a live cell analysis system such as Incucyte® was used. Since E7 affects the control of the cell cycle, a specific HPV-positive HeLa cell line called FUCCI (Ubiquitination-based Cell Cycle Indicator) was used. The FUCCI reporter system enables the tracking of different cell cycle stages of cells. Cells in G1 emit red fluorescence, while cells in S, G2, or M emit green fluorescence. Cells were synchronized with thymidine for 24 hours before introducing various treatments into the cell medium. Cells were treated with anti-E7 VHH conjugated to PEI-modified HSA via a cleavable linker and the following controls: untreated, modified HSA, modified HSA conjugated to an irrelevant VHH (anti-vimentin), unmodified anti-E7 VHH, unmodified HSA, and anti-E7 HH conjugated to a cell cycle inhibitor (DP, a CDK4 / 6 inhibitor). As can be seen in Figure 17, all controls had no effect on the cell cycle, remaining the same as untreated cells, while anti-E7 VHH conjugated to PEI-modified HSA (3.5PEI) had a dramatic effect, causing a cell cycle arrest similar to that of direct cell cycle inhibitor use. Since there is no commercially available E7 inhibitor, palbociclib isethionate (PD0332991, Sigma, catalog number PZ0199), a CDK4 / 6 inhibitor known to cause cell cycle arrest, was used as a positive control. Anti-E7 VHH conjugated to modified HSA had a similar effect to the inhibitor, with slightly different kinetics. Furthermore, as can be seen in Figure 18, this arrest led to the death of HPV-positive cells.

[0278] Both anti-vimentin VHH and anti-E7 VHH show their durability in the reducing environment of the cytoplasm. To further exemplify the suitability of single-domain binding proteins that do not rely on disulfide bonds to stabilize and maintain their structures for the present invention, the inventors used a designed ankyrin repeat protein (DARPin) as a binder. DARPin is a genetically engineered antibody-mimicking protein that typically exhibits highly specific and high-affinity target protein binding. These are derived from natural ankyrin proteins, which are one of the most common binding proteins in nature and are responsible for various functions such as cell signaling, regulation, and the structural integrity of cells. DARPin consists of several repeating motifs, and their molecular weights are approximately 14 - 18 kDa (kilodaltons). They are also characterized, like VHH, by the fact that their structural integrity does not depend on disulfide bonds.

[0279] For this purpose, a DARPin against K-RAS was selected. The RAS protein plays an important role in signal transduction as a molecular switch. RAS is the most important target in cell transformation and is involved in cell growth and differentiation via the RAF-MEK-ERK cascade and cell survival via the activation of PI3K. Mutations in the RAS protein (K-RAS, H-RAS, or N-RAS) create a constitutively active GTP-bound form that promotes cell transformation in a signal-independent manner. Activating RAS gene mutations are found in 30% of human cancers and are most frequent in pancreatic cancer, colon cancer, and lung adenocarcinoma. Oncogenic RAS has been shown to be essential for early tumor onset and necessary for maintaining tumor survival. The most central positions of RAS mutations are in glycine 12, such as G12D and G12V.

[0280] Guillard et al. (Guillard, S. et al. Nat. Commun. 2017, 8, 16111, which is incorporated herein by reference in its entirety) generated an antibody mimetic, DARPin K27, that inhibits nucleotide exchange of Ras. K27 preferentially binds to the inactive RasGDP form with a Kd of 4 nM, and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 by transfection of a vector encoding the DARPin was shown to significantly reduce the amount of active Ras, inhibit downstream signaling, particularly the level of phosphorylated ERK, and slow the growth of HCT116 cells in soft agar. This group stated the following: "The barrier arises from the fact that Ras is intracellular. Since DARPin K27 does not have an inherent ability to enter cells, it cannot access Ras when added extracellularly. There are reports of delivery of DARPin to the cytoplasm of cells, but a substantial improvement in efficiency will be required to make this approach therapeutically feasible. Since the scaffold binds over a broad surface rather than defining a pocket, it may be difficult to develop small molecule inhibitors that bind at the same site as DARPin K27."

[0281] DARPin K27 was expressed based on the published sequence and conjugated to an HSA carrier modified with an average of 3.5 PEI molecules. Internalization and KRAS binding were evaluated by confocal microscopy (Figure 19). The K27 DARPin was not dispersed in the cytoplasm but rather was localized to the inner side of the plasma membrane, which is the major location of KRAS. Interestingly, some cells exhibited both a punctate profile and internal membrane localization with respect to the internalized DARPin. This punctation could be the result of the DARPin not yet having escaped from the endosome or having separated from the carrier and not yet freely found its target.

[0282] In further tests, pancreatic cancer cells (SU8686) were incubated with the anti-KRAS DARPin K27 conjugated to the HSA carrier. The carrier was modified with 8 or 3.5 PEI molecules. The apoptotic state of the cells was monitored using a classical annexin V assay and visualized using the Incucyte® continuous cell monitoring system. Cells exposed to the DARPin conjugated to the PEI-modified HSA via a cleavable linker showed a dramatic increase in apoptosis, particularly in the case of the DARPin conjugated to the carrier with a high level of PEI modification (Figure 20). This is probably due to enhanced internalization and, in particular, the rapid rate of the endosomal escape process. In contrast, untreated cells, cells treated with unmodified DARPin, or cells treated with PEI-modified HSA (8 molecules) alone showed baseline levels of apoptosis that underscored the intracellular effect of the DARPin.

[0283] Guillard et al. showed that the anti-KRAS DARPin K27 binds to native, non-mutated, inactive KRAS and to KRAS with different mutations mainly at the G12 position. Therefore, the anti-KRAS DARPin K27 was evaluated for its intracellular effects on the growth and apoptosis of the HeLa cell line. This cell line is characterized by constitutive expression of GFP in the nucleus, allowing these cells to be easily tracked using continuous image monitoring methods such as Incucyte®. Two different preparations of the anti-KRAS DARPin were conjugated to HSA carrying 8 PEI molecules, and their effects on HeLa-GFP cells were compared to the following control treatments: untreated as a negative control, treatment with a small molecule Pan-Ras inhibitor as a positive control, treatment with unmodified DARPin, treatment with DARPin conjugated to unmodified HSA, HSA carrier modified with 8 PEI molecules, and an irrelevant VHH, anti-vimentin VHH, conjugated to HSA modified with 8 PEI molecules. As can be seen in FIGS. 21A-21B, both preparations of the anti-KRAS DARPin conjugated to HSA carrying 8 PEI molecules dramatically affected both the growth (FIG. 21A) and apoptosis (FIG. 21B) of HeLa-GFP cells. The control treatments had no effect on these parameters. The effect on apoptosis can also be confirmed in FIG. 22, where the red staining of cells treated with anti-KRAS DARPin conjugated to PEI-modified HSA (apoptotic cells) and the loss of GFP associated with cell death are clearly observed.

[0284] Example 8: Pharmacokinetics and biodistribution (PK and BD) PEI modification imparts a strong positive charge concentrated on the carrier protein. In contrast, plasma components and cell membranes are generally negatively charged. Positively charged proteins are known to be "sticky" due to electrostatic binding to these negatively charged components. This "stickiness" can cause problems with short half-life and in vivo distribution. This phenomenon is known in proteins that are naturally positively charged and characterized by a somewhat basic isoelectric point (pI). The pharmacokinetic profile and in vivo distribution profile of PEI-modified proteins can be affected by their strong positive charge. Furthermore, such adhesion can also lead to "trapping" of the administered positively charged protein at the injection site. To avoid or at least minimize these effects, the effect of the level of PEI modification was investigated.

[0285] By identifying and using the minimum level of PEI modification that remains effective to enable passage through the cell membrane and endosomal escape, the PK and biodistribution of the proteins of the present invention can be improved. 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 23). These carriers were further conjugated to anti-vimentin VHH, and the internalization of these conjugates was evaluated using confocal microscopy (Figure 24). As can be confirmed by the results of confocal microscopy, even VHH conjugated to HSA with 2.0 and 3.6 PEI molecules exhibits a distinct presence inside the cell. The use of carriers modified with high levels of PEI appears to result in a clearly higher level of VHH inside the cell, but the profile of internalized VHH is more punctate and less dispersed in these cases, probably due to slower dissociation of VHH from the carrier, which is caused by a higher positive charge. The stronger staining of VHH inside the cell in the case of HSA modified with more than 3.6 PEI / HSA could be the result of stronger imaging due to a concentrated punctate profile and may not necessarily indicate an absolutely high level.

[0286] The results of confocal microscopy were also correlated with the internalization levels of these conjugates by measuring the residual levels of the conjugates in the cell culture medium using specific ELISA (Figure 14). Different from the results observed with IgG directly modified with PEI (Figure 6), the levels and kinetics of the HSA carriers (VHH conjugates) are not as much affected by the level of modification. Internalization is efficient with both 3.5 and 8 molecules of PEI, and the extent and rate are very similar.

[0287] These results suggest that HSA, when actually used as a carrier, can be modified with only 3.5 or fewer PEI molecules, which means reducing the adhesiveness of the HSA carrier in circulation. The effect of this reduction in PEI modification is investigated in in vivo PK and biodistribution studies. HSA modified with different levels of PEI in the range of 2 - 8 was further labeled with a near-infrared fluorescent dye, such as Vivo-Tag-750 (PerkinElmer). The labeled protein was administered IV to Balb-C mice. After administration, the injected animals were monitored using a live imaging system such as the IVIS in vivo imaging system (PerkinElmer). The distribution profiles of different PEI-modified HSA derivatives were monitored at various time points after administration. The animals were sacrificed at various time points, perfused, various organs were harvested, and the fluorescence levels in each organ were examined using the IVIS system. A similar protocol can be used in animals transplanted with tumors.

[0288] The PK and biodistribution of HSA modified with PEI at two different levels (3.5 or 8 PEI units) were investigated. PEI-modified HSA was injected into mice bearing B-16 melanoma tumors (SC tumors). The carrier was injected IV at a dose of 250 nmol / Kg. The PK and biodistribution of the tested carrier were evaluated using anti-HSA ELISA.

[0289] As can be confirmed in Figure 25A and Table 2, carriers with 8 PEI modifications exhibited very low AUC levels and high clearance, which are characteristic of highly cationized proteins. Carriers with 3.5 PEI modifications had somewhat better PK values with higher AUC and lower clearance values, presumably due to their lower positive charge. In contrast, when the VHH was directly conjugated to PEI1800 at its C-terminus, clearance from the blood was very rapid (Figure 25B, Table 2). Plasma exposure levels of both carriers can be confirmed in Figure 25C. As can be seen in Figure 25D, the high clearance and low AUC values of the carriers are not due to renal clearance, as can be seen from the very low levels of carriers seen in the urine of treated mice. This is one of the main reasons for generally using carriers, and especially HSA, to avoid such clearance due to their small size.

[0290]

Table 2

[0291] Example 9: Stability of Linker The design and precise structure of the linker are very important for the in vivo performance (PK and biodistribution) of the final conjugate product. The initial design of the linker was based on a PEG chain connected to the HSA carrier by a stable amide bond and to the payload via a disulfide bond. This disulfide bond was designed to be cleaved under the reducing environment of the cytoplasm. Figure 13A shows a schematic of the initial design of PEI-modified HSA linked to two VHHs. The VHHs can of course be replaced with any biologic. This design was used in a functional in vitro assay using anti-E7 VHH and anti-KRAS DARPin. In this design, HSA modified with PEI was conjugated to NHS-PEG n-Further modify with SPDP. The NHS group reacts with the primary amino groups on HSA or on its PEI modification to create stable amide bonds. The linker used contained a PEG4 chain. The reaction conditions were selected to obtain a modification level close to that of a single modification. In fact, most of the PEI-modified HSA had one modification, although some had two to three modifications. These moieties were further reacted with VHH or DARPin having a free cysteine at their C-terminus. In some cases, a pretreatment with a reducing agent such as TCEP was necessary to release the thiol of this cysteine from its dimer state or from its binding to other thiol-containing molecules. The final conjugate was characterized by having a labile disulfide bond near the active payload.

[0292] This initial design was shown to undergo cleavage under conditions mimicking the cytoplasm, i.e., in the presence of approximately 10 mM GSH, and was shown to be stable in cell culture medium, where it was used in functional in vitro tests.

[0293] According to the structure shown in Figure 13A, or having only a single VHH, the antivimentin VHH (QVQ, catalog number Q60C) conjugated to PEI-modified HSA (3.5 and 8 molecules) was evaluated for its PK and biodistribution in mice. Unmodified VHH at 10 mg / Kg (VHH weight basis), VHH conjugated to unmodified HSA, and VHH conjugated to PEI-modified HSA were all injected into the tail vein of mice, and plasma was collected 5 minutes and 24 hours after injection. Furthermore, various tissues were collected from the mice 24 hours after injection to evaluate the biodistribution. Tissues included the liver, spleen, kidney, lung, heart, and brain.

[0294] The initial analysis focused on the detection and measurement of the payload, i.e., the VHH. Samples were analyzed by ELISA used for the detection of VHH and by ELISA designed to measure intact carrier-VHH conjugates. Analysis of mouse plasma at 5 minutes post-injection using both methods revealed the presence of VHH in the plasma of all groups with a good correlation between VHH levels and levels of intact conjugate bodies (Figure 26). This suggests that the VHH is still conjugated to its carrier at this time point. Analysis of VHH levels in plasma samples taken 24 hours post-injection was unable to detect any VHH, regardless of whether it was unmodified or part of the conjugate body. Analysis of carrier (HSA) levels detected significant levels of unmodified carrier, regardless of whether it was conjugated to VHH (Figure 27). These results suggest that the VHH was cleaved from the carrier early. This hypothesis was further supported by the results of ELISA (Figure 28) and immunofluorescence analysis (Figure 29) of VHH in various tissues. These analyses showed that the levels and distribution profiles of VHH in the different organs analyzed were very similar, regardless of the level of PEI on the carrier or even if the VHH was not conjugated.

[0295] These results led to the hypothesis that the linker between the VHH payload and the HSA carrier was not sufficiently stable in mouse plasma. Therefore, ex vivo stability analysis of this linker was performed in mouse, monkey, and human plasma. The stability of VHH (without PEI modification) conjugated to HSA according to the general structure of Figure 13A was analyzed by incubating the conjugate in mouse plasma (Balb-C mice, Bioreclamation, K2EDTA, catalog number MSEO2PLK2YNN, lot number MSE347339), human (Bioreclamation, Na2EDTA, lot number HMN21123) and monkey serum (Bioreclamation, catalog number CYN262432). The levels of the conjugate were measured over time using an ELISA that measures only intact conjugate and normalized to the conjugate level when incubated in PBS. As can be seen in Figure 30, the conjugate was indeed unstable in mouse plasma and to a lesser extent in monkey serum. The decline in conjugate levels over time in human plasma was not as rapid as in mouse plasma, but the stability in human plasma was also not very ideal for efficient in vivo distribution. The stability of the conjugate was also evaluated in a 10 mM GSH solution that mimics the GSH level in the cytoplasm of cells. As expected and as designed, the disulfide bond that conjugates VHH to the carrier was unstable in the presence of GSH and substantially all of the conjugate was cleaved within 5 hours.

[0296] Understanding that the payload VHH was cleaved early in plasma, the levels of the carrier itself were analyzed in the various organs collected. ELISA directed only against the HSA protein was used to analyze extracts of brain, heart, spleen, lung, tail, and liver from the injected animals. Both unmodified HSA and HSA-PEIx3.5 were detected at very low levels in all organs (Figure 31). HSA-PEIx8, however, was detected at very high levels in all organs except the lung and tail (the injection site). These ELISA results were corroborated by immunofluorescence results showing high levels of HSA-PEIx8 protein in spleen frozen sections stained with anti-HSA-FITC antibody (Figure 32C). PEI-modified HSA was found at high levels in the spleen, while unmodified HSA was observed at much lower levels (Figure 32B), and in fact, at levels similar to the background signal observed after injection of unmodified VHH (Figure 32A). Both the ELISA and immunofluorescence results indicate that PEI modification can actually deliver the carrier and its cargo stably conjugated to cells in different organs after IV injection.

[0297] Example 10: New Conjugates and Linkers Based on these results, the conjugate was redesigned (Figure 33). This design aimed to create a conjugate in which one payload protein is conjugated to one HSA carrier. The previous method of using NHS-activated esters for linker conjugation to the carrier does not meet this requirement, so instead, the cysteine amino acid at position 34 of the HSA protein was utilized. This is the only free thiol present in HSA, and thus, conjugation of the payload to this position results in a 1:1 conjugate.

[0298] As can be confirmed in Figure 34, the biotin-PEG-HSA conjugate in which biotin is bound to HSA via a direct disulfide bond on Cys34 is very stable in both mouse plasma and human plasma.

[0299] However, an overly protected cleavage site will not be usable because cytoplasmic cleavage is beneficial for the dispersion of the payload within the cytoplasm. Thus, the stability of the biotin-PEG-HSA conjugate was evaluated under cytoplasmic-like conditions characterized by the presence of glutathione (10 mM). Surprisingly, biotin-PEG-HSA is highly cleavable under these conditions, and even at the first measurement time point designated as t = 0, approximately 50% of the conjugate is cleaved within the few minutes that elapse during the addition of the conjugate to the GSH solution and its freezing prior to sampling and analysis (Figure 35). By 6 hours, most of the conjugate is cleaved, suggesting that the Cys34 disulfide bond, while stable in plasma, is highly sensitive to reductive cleavage as seen in the cytoplasmic environment, making it ideal for use in the conjugates of the present invention.

[0300] Thus, different payload-carrier conjugates (C1 - C2, see Table 1) were designed and prepared using payload-carrier conjugation via a disulfide bond (e.g., between the linker and Cys34) or via click chemistry (e.g., Michael addition, or azide-DBCO addition cyclization, etc.) (Figures 13B - 13D and 36).

[0301] As described above, to conjugate the payload to the carrier in a site-selective manner that also does not interfere with the active domain, the preferred methodology involves using conjugation to a single free cysteine, preferably at the C-terminus of the payload. Conjugation of both the carrier and the payload to the linker occurs via a free thiol, which requires the linker to have two thiol-reactive groups at both ends, one based on SPDP that generates an unstable Cys34 disulfide bond and the other based on a thiol-reactive group that results in a stable bond near the VHH payload. To avoid reaction of the second site with the Cys34 thiol, a protected group that does not react with Cys34 and requires deprotection and / or activation prior to its reaction with a thiol without a payload is typically used.

[0302] Additional optimizations were evaluated for the linker presented. One such optimization involves the use of a linker containing a sterically protected disulfide bond on Cys34. This is achieved by using a linker in which the carbon at the alpha position of the activated thiol is substituted by one or two alkyl groups, preferably methyl groups.

[0303] Nevertheless, protecting groups on the free thiol can be used to bind the payload. For example, a commonly used thiol protecting group is the S-acetyl group, also known as SATA.

[0304] The deprotected thiol is then further activated to enable efficient reaction with the free thiol of Cys34 of HSA. Such activation can be achieved by reacting the deprotected free thiol with 2,2'-dipyridyldisulfide (aldrithiol) to generate an SPDP-activated thiol that has already been shown to react efficiently with Cys34 of HSA. An exemplary conjugation scheme is shown in Figure 36. Additional exemplary synthetic routes are shown in Table 3 below.

[0305] [Table 3] TIFF0007713030000021.tif216159TIFF0007713030000022.tif53160

[0306] In one design, HSA-PEG4-dimethyl (dm)-SS-VHH (S1) was prepared (note that the molar ratio of HSA to VHH may be greater than 1 because conjugation to HSA is not selective and thus multiple modifications can occur). For this purpose, HSA is modified with NHS-PEG4-dm-SPDP. The NHS group reacts with the primary amino groups on HSA to create stable amide bonds. The linker used contained a PEG4 chain. This reaction of dm-SPDP on modified HSA with VHH having a free cysteine at its C-terminus resulted in a low reaction yield. To improve the reaction yield, the dm-SPDP moiety on HSA was converted to dm-SPDP-NO2 to obtain a significantly more activated group. For this purpose, modified HSA having NHS-PEG4-SPDP was reduced with TCEP to receive HSA having a PEG4-dm-SH moiety, which was further activated with 2,2'-dithiobis(5-nitropyridine) to obtain the desired HSA modified with a PEG4-dm-SPDP-NO2 moiety. These moieties were further reacted with VHH or DARPin having a free cysteine at the C-terminus. In some cases, pretreatment with a reducing agent such as TCEP was necessary to liberate the thiol of this cysteine from the dimeric state or from binding with other thiol-containing molecules.

[0307] Another construct, HSA-SS-α-methyl-PEG 11 -triazole-PEG4-VHH (S2) was prepared. For this purpose, HSA was modified with N3-PEG 11 -α-methyl-SPDP-NO2 to obtain a mono-PEG modification on cysteine 34. In parallel, VHH was modified with MAL-PEG4-DBCO to obtain a mono-PEG modified on the VHH C-terminal cysteine. The two proteins were conjugated by a click reaction to obtain the desired HSA-S-S-α-methyl-PEG 11 -triazole-PEG4-VHH construct.

[0308] Peptide-based linker In this design, the VHH payload was expressed with an additional peptide linker at the C-terminus. The inventors synthesized various conjugate bodies having a peptide-based linker (S3A-B) with a disulfide bond unstable to the HSA cysteine at position 34. Briefly, the VHH expressed with a G4S-based peptide at its C-terminus and terminated with cysteine was activated by SPDP or nitro-SPDP and reacted with the C34 thiol group of HSA. S3A - B is schematically shown in Table 1.

[0309] To enhance the stability of the S - S bond, the inventors added leucine, a steric hindrance amino acid located at α or β with respect to the C-terminal cysteine, in the following sequences respectively; GGGGSGGGGSGGGGLC (SEQ ID NO: 4) (name "pep1") or GGGGSGGGGSGGGLGC (SEQ ID NO: 5) (name "pep2"). These conjugate bodies were prepared by activation of VHH on the C-terminal cysteine using aldritiol or 2,2'-dithiobis(5-nitropyridine) and subsequent conjugation to HSA. In some cases, pretreatment of the C-terminal VHH with a reducing agent such as TCEP was necessary to liberate the thiol of this cysteine from the dimeric state or from binding with other thiol-containing molecules.

[0310] To understand whether the leucine amino acid in the peptide contributes to the stability of these peptide linkers, an additional construct of VHHαE7-(G4S)3-C was expressed and conjugated to HSA to obtain an additional peptide linker-based conjugate, VHHαE7-(G4S)3-C-HSA (S3C).

[0311] Example 11: Stability of Novel Conjugate Bodies Containing Biodegradable (Reversible) Linkers and Non-Biodegradable (Irreversible) Linkers (S1 - S3C) The S1 conjugate was characterized by having a labile disulfide bond near the active payload. In vitro tests (Figure 37A) in the following media: PBS / human plasma / mouse plasma and GSH (10 mM) revealed that this conjugate was stable in PBS. In human plasma, the conjugate concentration did not decrease even after 96 hours. In GSH (10 mM), the conjugate concentration rapidly decreased over time as expected, and a slight decrease in the conjugate concentration was observed in mouse plasma (Figure 37A). Considering the similar behavior of the non-reversible linker (Figure 37B) and the in vivo PK results, this decrease in concentration over time is likely the result of a physical process, such as adsorption, rather than chemical decomposition of the disulfide bond (see Figures 38A - 38B).

[0312] In vitro stability analysis of S2 showed that the conjugate was very stable in PBS / mouse plasma and human plasma. In contrast to other HSA conjugates, this linker was also more stable under GSH (10 mM) conditions, and its reduction to separate VHH and HSA was not as rapid as that of the other linkers used. After 4 hours, the majority of the conjugate was still in the HSA - VHH conjugate form (more than 80%), while after 24 hours, only 20% of the sample was detected in the HSA - VHH form, and the reduction process continued over time (Figure 37C). These results correlate with the PK results, showing excellent stability for this conjugate compared to all other conjugates tested (see Figures 38A - 38B).

[0313] The in vitro stability of the peptide-based linkers was also tested. The results revealed that these conjugates were stable in PBS / human plasma and mouse plasma, as no decrease in conjugate concentration was observed during 96 h of incubation under either of these conditions. In the presence of GSH (10 mM), the conjugate concentration decreased rapidly with time as expected (Figs. 37D–37F). The stability of the conjugates in mouse plasma was further compared with that of the irreversible conjugate (VHH anti-E7-HSA), showing that the new constructs (S3A–B) were as stable as the irreversible construct in mouse plasma (Fig. 37F).

[0314] Conjugates C1 and C2, which have a labile disulfide bond at C34 cysteine of HSA, were evaluated in an in vivo PK study to determine their actual stability in mouse plasma (Fig. 38A). Table 4 below summarizes the kinetic parameters of different conjugates with different linkers, as well as their comparison with HSA alone and the irreversible conjugate (VHHαE7-PEG 11 -HSA, S4). As expected, these two have very similar PK profiles and parameters, including their half-lives and AUCs. Conjugates C1 and C2 had shorter half-lives than HSA and the irreversible conjugate, but the half-life corresponding to the stability of the disulfide bond was dramatically improved when compared with the first construct where the disulfide bond was at the C-terminus of the payload and there was no hindrance or natural protection provided by the C34 cavity in HSA. In the latter case, the conjugate decreased within minutes to hours and only a very small amount of the original injected dose could be detected in the blood of these animals 24 h after injection. This further indicates that the actual position of the disulfide bond at C34 provides protection from plasma reducing agents.

[0315] [Table 4]

[0316] To further evaluate the stability of different new linkers, impaired PEGs, and peptide linkers under actual mouse plasma conditions, PK studies were performed using the following conjugates: (1) S3A; (2) S3B; (3) VHHS2; (4) S1; (5) HSA. As can be seen in Figure 38B and the table below, the peptide linkers of S3A and S3B, and the dimethyl-impaired PEG linker of S1 gave very similar PK profiles and PK parameters. All of these linkers showed higher half-life values than those obtained for the simpler PEG linkers used in previous PK studies (Figure 38A), suggesting that these linkers result in better plasma stability of the labile disulfide bonds. The disulfide bond on C34 further impaired with a methyl group (S2) resulted in an even better stability profile, as evidenced by its longer half-life, closer to that of HSA. This improvement in stability was also evident in other PK parameters, particularly clearance and AUC (see Table 5 below).

[0317]

Table 5

[0318] The plasma half-life of the conjugate with the new linker (S1, S2, S3A-B) is slightly lower than that of HSA. To confirm whether the decrease in plasma conjugate concentration compared to that of HSA is actually the result of linker cleavage, additional ELISA was performed. In this ELISA, the concentration of the four new conjugates in plasma was evaluated by anti-HSA ELISA (which quantifies the amount of HSA even when the linker is cleaved). The concentration of the conjugate by HSA was compared with the concentration of the conjugate by sandwich ELISA (which quantifies only VHH αE7 bound to the HSA form). The PK profile of S3A measured by ELISA method compared to the PK profile of HSA is shown in Fig. 38C. The quantification of S3A by HSA ELISA gave conjugate concentrations very similar to those observed for HSA alone. This result indicates that the decrease in S3A concentration compared to HSA is actually the result of linker cleavage. Similar results were obtained for other conjugates with different linkers (data not shown).

[0319] Two constructs (VHHαE7-G4SG4SG4LC (SEQ ID NO: 4), S3A and VHHαE7-G4SG4S-GGGLG-C (SEQ ID NO: 5), S3B) with peptide linkers differing only in the position of leucine amino acids near the disulfide bond were evaluated for their in vitro and in vivo stability. The two conjugates exhibited similar stability to each other both in vitro and in vivo (PK), and also showed similar stability to the conjugate with the non-cleavable linker (S4). The stability of the new construct, VHH αE7-(G4S)3-C-HSA (S3C), was tested in vitro in human plasma and 10 mM GSH against S3A. As can be seen from Figure 39, both constructs exhibited very similar stability profiles in both human plasma and glutathione solution, suggesting that leucine amino acids do not contribute much to the stability of the conjugate. At 10 mM GSH, it should be noted that the linker containing leucine survived slightly better since about 8% of the initial level of the conjugate was still detectable after 24 hours while the residual levels of the conjugate without leucine were not detectable at this point. The conjugate with the leucine-containing linker was still detectable even after 48 hours (about 1%).

[0320] This suggests that leucine confers some advantage in terms of stability and that perhaps further testing is required to confirm this contribution. In any case, the inventors will continue to use linkers containing leucine near the disulfide bond with the carrier.

[0321] All four conjugate bodies (S1 - S3B) of VHHαE7 conjugated to HSA were further modified with PEI by conjugating VHHαE7 to HSA - PEIx3.5 using different linkers and chemical reactions, and their uptake capabilities were verified by confocal microscopy. Hela GFP cells were seeded, the test samples were incubated with the cells for 16 hours, and then, after the fixation and immunostaining processes, the uptake levels were evaluated with a confocal microscope. As can be confirmed in Figures 40A - 40F, untreated cells (Figure 40A) showed no presence of VHH, while treatment with VHH conjugated to HSA - PEIx3.5 via a new linker (S1 - PEI - S3B - PEI) showed a clear presence of VHH inside the cells (Figures 40B - 40F). Linker length (peptide linkers of different lengths; (G4S)n, n = 0, 1, 2, 3)

[0322] Different VHH and DARPin agents were expressed with a flexible peptide based on the G4S unit and subsequent terminal cysteine amino acids. The length of the peptide linker was determined by the number of (G4S)n units (n = 0, 1, 2, 3). The proteins were activated with 2,2’ - dithiobis(5 - nitropyridine) and conjugated to HSA.

[0323] When n = 1, different conjugation reaction yields were observed for different proteins. VHHαE7 - G4S - 1C resulted in an efficient conjugation reaction to the carrier, while K27 anti - KRAS DARPin gave a moderate reaction yield, and in the conjugation of VHH anti - BRAF to HSA / HSA - PEI, no conjugation product was observed. It should be noted that in the case of irreversible conjugation requiring the use of bis - MAL - PEGn that provides some elongation of the linker, the VHH anti - BRAF - G4SC construct can be used to successfully generate VHH anti - BRAF - G4SC - HSA - PEI (S5A).

[0324] VHH anti-BRAF (1C5) was expressed with different peptide linker lengths (shown in Table 6 below). Different 1C5 constructs with different linker lengths were treated with TCEP, and the free thiol of the resulting C-terminal cysteine was activated with 2,2'-dithiobis(5-nitropyridine) and then conjugated to the carrier HSA-PEI. All activated conjugate forms of 1C5 terminating with leucine-cysteine at the C-terminus showed efficient conjugation to HSA-PEIx4.5 regardless of the length of the peptide linker (n = 0, 1, 2, 3). These results are surprising considering the results obtained from the conjugation reaction of 1C5-G4SC (non-leucine-containing) with HSA-PEIx4.5, from which no conjugation product could be obtained.

[0325] The yields of the conjugation reactions of different payload constructs were evaluated by SDS gel, and the results are summarized in Table 6 below.

[0326]

Table 6

[0327] Without being bound to any particular theory, it is hypothesized that it may be beneficial to have a linker containing at least 4, at least 5, at least 6, at least 8 amino acids when the peptide linker is implemented for the covalent attachment of the protein carrier to the payload of the present invention. As shown in Table 6, the length of the linker required for efficient conjugation of the protein carrier to the payload of the present invention may vary depending on the particular size and / or sequence of the payload. VHH anti-BRAF (1C5) peptide linkers with different lengths and containing or not containing sterically hindered amino acids were expressed with the following C-terminal sequences:

[0328] Construct 1: anti-BRAF (1C5)-G4SC (SEQ ID NO: 9) (1C5) Construct 2: anti-BRAF (1C5)-GGGGSGGGGSGGGGLC (SEQ ID NO: 4) (1C5-flex-pep-LC) Construct 3: anti-BRAF (1C5)-AAASAEAAAKEAAAKEAAAKAAAGSGLC (SEQ ID NO: 10) (1C5-Hel20-LC)

[0329] Construct 2 is similar to the above constructs having a peptide linker with anti-E7 VHH (S3A). Construct 3 has a linker that is essentially an alpha helix, considered a rigid linker that is thought to avoid or minimize the interaction between the VHH payload and the carrier. The 1C5 VHH was observed to lose affinity for protein A-based resin when conjugated to HSA-PEI using a flexible G4S-based linker, presumably due to specific electrostatic interactions. The rigid linker actually restored this interaction with the protein A resin, suggesting that the rigid linker reduces this interaction with the carrier and maintains the exposed VHH. The rigid linker also terminates with leucine near the terminal cysteine used for conjugation of the carrier.

[0330] Construct 1 (1C5) was conjugated to HSA-PEI using an irreversible PEG linker (bis MAL-PEG 11) was conjugated to HSA-PEIx3.5 to obtain 1C5-HSA-PEIx3.5 (S5A). Both constructs 2 (1C5-flex-pep-LC) and 3 (1C5-Hel20-LC) were conjugated to HSA-PEIx3.5 to obtain reversible conjugates; 1C5-flex-pep-LC-HSA-PEIx3.5 (S5B) and 1C5-Hel20-LC-HSA-PEIx3.5 (S5C), respectively. 1C5-flex-pep-LC-HSA-PEIx3.5 (S5B) is a similar construct to E7-pep1-LC-HSA (S3A) that exhibited good stability in mouse plasma (in vivo Figure 38B and in vitro Figure 37F). S5C is a construct of an anti-BRAF VHH via a rigid peptide linker. This construct was tested for its in vitro stability (Figure 41), and it showed good stability under mouse plasma conditions.

[0331] Example 12: PEI Modification PEI modification imparts a very strong and concentrated positive charge to the carrier protein, responsible for the initial adhesion of the carrier to the cell membrane leading to cell membrane passage. The same PEI modification then is responsible for the efficient escape of the PEI-modified carrier and its payload from the endosomal pathway. Here, various carriers modified with different levels of PEI are shown. Particularly for HSA as a carrier, the inventors mainly focused on two levels of modification, namely a low level in the range of 3.5 - 4.5 PEI molecules per HSA molecule and a high level in the range of 7.5 - 9.5 PEI molecules (both the high and low PEI ranges enable efficient internalization and endosomal escape, and functionality in cells), to explain the internalization, functional, and in vivo results, and both the high and low levels enable efficient internalization and endosomal escape as well as functionality in cells. Both ranges have equivalent PK and biodistribution profiles, but the lower range was superior. The inventors further sought to establish how the effect of the lower level of PEI is, particularly with respect to the function of the payload-carrier conjugate in cells.

[0332] The anti-BRAF VHH with a rigid peptide linker, 1C5-Hel20-LC, was conjugated to HSA-PEIxn(S5C) (n = 4.5, 3, 2, 1). As described above, the level of PEI was controlled by the levels and ratios of the reagents used in the PEI modification reaction, and the final level was determined by analyzing the modified products for their intact masses by MALDI-ToF mass spectrometry. It should be noted and emphasized that the reported PEI level represents the average number of PEI molecules added to HSA. The level of PEI modification of the HSA protein in each reaction was evaluated by mass spectrometry, and various carriers were characterized for their zeta potential (Table 7).

[0333] Zeta potential measurements were performed using a Zeta Sizer Ultra (Malvern Instruments), and the sample buffer was exchanged to 1 mM NaCl with a protein concentration of 1 mg / mL. Twenty microliters from each sample was loaded into a zeta cell (DTS1070), and five repetitions of measurements were made for each sample to obtain the average zeta potential (mV).

[0334] [Table 7]

[0335] The 1C5-Hel20-LC-HSA-PEI(S5C)xn conjugate was prepared by reduction of 1C5-Hel20-LC and activation with 2,2'-dithiobis(5-nitropyridine), followed by conjugation to HSA-PEIxn. Intracellular BRAF inhibition by different constructs was evaluated by a functional assay in A375 cells. Different conjugates were incubated with A375 cells for 6 days in the presence of annexin V red reagent while monitoring fluorescence with IncuCyte® to identify apoptotic cells (Figures 42A, C). At the end of 6 days, a CellTiter-Glo assay was performed to evaluate the amount of live cells in each of the wells tested. After 6 days, the red areas at the same time point indicating the level of apoptosis can be normalized to the amount of live cells in each well tested. These normalized values were used to compare different test agents (Figures 42B, D).

[0336] Looking at the direct apoptosis evaluation (Figures 42A, C), at both concentration levels, S5C-PEIx4.5 provided significant inhibition of intracellular BRAF and resulted in a high apoptosis signal in the treated cells. However, when normalizing the apoptosis signal to the cell number, it is clear that there is a dose-dependence on the level of PEI (Figures 42B, D). S5C-PEIx3 also exhibited activity, but it was reduced, and furthermore, PEIx2 and PEIx1 showed limited effects, especially at the 3 μM concentration. Overall, the activity levels correlated well with the level of PEI.

[0337] As can be confirmed from these results, the level of PEI is important to obtain intracellular activity, and in the case of HSA, significant activity can be obtained when the carrier is modified with more than two PEI molecules, and even better activity seems to be obtained when the carrier is modified with more than three PEI molecules.

[0338] Furthermore, as shown in Figure 5A, the conjugate S5D (described in Example 1) exhibited effective BRAF inhibition that resulted in an obvious apoptosis signal. Therefore, conjugates with or without a biologically cleavable bond are hypothesized to be available for efficient cell delivery and payload function as described herein.

[0339] Example 13: Additional Carriers Four different proteins with different molecular weights were evaluated for their ability to act as carriers: K27 DARPin (20KDa; Guillard, S. et al. Nat. Commun. 8, 16111 doi:10.1038 / ncomms16111 (2017)), GFP (30KDa; biorbyt, catalog number orb84840), IgG (150KDa; monoclonal antibody, mouse anti-goat / sheep, clone GT-34, Sigma) and fibrinogen (300KDa; Sigma, catalog number 341578). The latter two proteins are endogenous proteins to the blood, similar to HSA which is used as the main carrier of the present invention. For this purpose, the different proteins were modified with PEI. The modification was carried out in MES buffer pH 5 in the presence of EDC and PEI. The proteins modified with PEI were purified from the excess reagent using size exclusion chromatography. The modification level was evaluated by mass spectrometry, and the modified carriers were further evaluated for their zeta potential. The level of PEI modification of each potential carrier can be optimized and controlled as can be seen in Table 8, but larger carrier proteins, namely IgG and fibrinogen, are more easily modified because they have more accessible PEI modification sites. In contrast, small carriers such as DARPin are modified with a lower level of PEI because they have fewer potential modification sites and these sites are more densely packed. GFP was modified at a relatively low level, but its size suggests that it can be further highly modified in the future.

[0340]

Table 8

[0341] The change in the charge of each carrier protein after modification with PEI was measured by zeta potential (Table 9). HSA containing 4.5 or 8 PEI modifications, which is a preferred carrier, is also shown for comparison.

[0342]

Table 9

[0343] Different carriers were tested for their ability to internalize into cells. Carriers modified with PEI were incubated with A375 cells for 24 hours, and their presence in the cells was evaluated by confocal microscopy using an anti-HSA-PEI antibody (produced in-house after immunization of animals with HSA-PEI, which has been shown to selectively identify HSA-PEI and other PEI-modified proteins).

[0344] Since attempts were not made to identify conjugation hotspots in these alternative carriers, different proteins modified with PEI were activated with NHS-PEG4-SPDP and then subjected to a conjugation reaction with the C-terminal cysteine of VHH anti-E7 to obtain VHH anti-E7 conjugated to the above alternative PEI-modified carriers for conjugating them to the payload. The anti-E7 VHH payload was conjugated using a reversible disulfide bond sensitive to the reducing environment of the cytoplasm. The ability of different carriers to deliver the payload into cells was evaluated by incubating the conjugate with A375 cells for 24 hours and then immunofluorescent staining with an anti-VHH antibody.

[0345] As can be confirmed in FIGS. 43A - 43L, all alternative carriers successfully delivered their VHH cargo to A375 cells. Also, as discussed above, carriers with higher levels of PEI modification, namely IgG, fibrinogen, and HSA, were able to deliver more VHH. Since the carriers here were conjugated via a reversible linker, the VHH was released in the cytoplasm and, as there was no target in the cytoplasm for the specific VHH used, the VHH diffused throughout the cytoplasm, thereby making its detection difficult as opposed to carrier detection (FIG. 44). This is more pronounced due to the phenomenon already discussed where it appears as a punctate profile due to electrostatic interactions with cytoplasmic proteins. Therefore, it is somewhat difficult to observe the VHH delivered by the DARPin carrier, which was able to somehow deliver a lower level of VHH with its lower level of PEI.

[0346] These results clearly show that other proteins, including other human blood endogenous proteins, can act as intracellular carriers, but it may be beneficial to use HSA as the protein carrier of the conjugates of the present invention, which is due to its abundant presence in blood, low immunogenicity, favorable physicochemical properties also involved in its favorable PK profile, and its unique conjugate site at C34 which can bring about high site selectivity and improved stability of the conjugate when a reversible bond is added to release the payload inside the cell.

[0347] Example 14: Functional assay Three constructs, S5A - C, were tested for their functional ability to inhibit intracellular BRAF and induce cell death in various cell lines: Hela - GFP, B16, Renca, and A375. As a positive control, PLX4032, vemurafenib was also tested. As a negative control, 2A1, an unrelated VHH, was conjugated to HSA - PEIx3.5 using an irreversible PEG linker. Cell death was monitored throughout the experiment (FIG. 45A) and quantified by cell titer at the end point of the experiment (140 hours) (FIG. 45B).

[0348] As can be confirmed in FIGS. 45A-45B, the anti-BRAF VHH conjugated via the novel peptide linker exhibited high activity in cells, suggesting that these linkers are compatible with the generation of conjugates that effectively enter cells and release the payload to intracellular targets. The anti-BRAF VHH (1C5) irreversibly conjugated to HSA-PEIx3.5(S5A) also induced apoptosis on the cells, but at a lower level than the reversibly conjugated construct, and this effect started at a later time point than the reversibly conjugated construct.

[0349] Although the invention has been described in conjunction with its specific embodiments, it will be apparent to those skilled in the art that many alternative, modification, and variation forms will be obvious. Therefore, it is intended to embrace all such alternative, modification, and variation forms that fall within the spirit and broad scope of the appended claims.

Claims

**Claim 1** a. A protein carrier covalently attached to a plurality of polyethyleneimine (PEI) moieties and characterized by a positive zeta potential of at least 8 mV; b. A payload that interacts with an intracellular target; c. A linker covalently attached to the protein carrier and the payload; A protein conjugate comprising: The protein conjugate is not a fusion protein, and the linker comprises a biocompatible polymer or is a peptide linker. **Claim 2** The protein conjugate according to claim 1, wherein the PEI is linear PEI or branched PEI having a molecular weight of less than 2000 Daltons, or the plurality of PEI moieties comprises 3 to 90 molecules, or both. **Claim 3** The protein conjugate according to claim 1, wherein the protein carrier is human serum albumin (HSA). **Claim 4** The protein conjugate according to any one of claims 1 to 3, wherein the payload is an antigen-binding molecule that binds to the intracellular target. **Claim 5** The protein conjugate according to claim 4, wherein the antigen-binding molecule is selected from single-chain antibodies, single-domain antibodies, variable heavy chain homodimers (VHHs), nanobodies, immunoglobulin new antigen receptors (IgNARs), designed ankyrin repeat proteins (DARPins), and antibody mimetic proteins. **Claim 6** The protein conjugate according to claim 5, wherein the antigen-binding molecule is selected from VHHs and DARPins. **Claim 7** The protein conjugate according to claim 1 or 2, wherein the protein carrier is an endogenous plasma protein. **Claim 8** The protein conjugate according to claim 7, wherein the protein carrier is at least 60 kDa in size. **Claim 9** The protein conjugate according to claim 1 or 2, wherein the protein carrier is selected from human serum albumin (HSA), fibrinogen, IgG, green fluorescent protein (GFP), and designed ankyrin repeat protein (DARPin). **Claim 10** The protein conjugate according to claim 9, wherein the protein carrier is HSA. **Claim 11** The protein conjugate according to claim 10, wherein the protein carrier is HSA and the protein carrier contains 3 to 10 PEI molecules.

12. The protein conjugate according to any one of claims 1 to 3, wherein the linker contains a biocompatible polymer.

13. The protein conjugate according to claim 12, wherein the biocompatible polymer contains polyethylene glycol (PEG).

14. The protein conjugate according to any one of claims 1 to 3, wherein the linker contains a biologically cleavable bond.

15. The protein conjugate according to claim 14, wherein the biologically cleavable bond contains a disulfide bond.

16. The protein conjugate according to any one of claims 1 to 3, wherein the linker is substantially stable in blood for at least 24 hours.

17. The protein conjugate according to claim 16, wherein the stability includes less than 25% cleavage in blood after 24 hours.

18. The protein conjugate according to claim 14, wherein the biologically cleavable bond is sterically hindered.

19. The protein conjugate according to any one of claims 1 to 3, wherein the protein carrier is HSA and the HSA contains SEQ ID NO: 1, or a fragment or homolog thereof containing cysteine 34 (C34).

20. The protein conjugate according to claim 19, wherein the linker is bound to the HSA via a disulfide bond.

21. The protein conjugate according to claim 19, wherein the linker is bound to the C34 of the HSA.

22. The protein conjugate according to claim 20, wherein the disulfide bond is proximal to the C34.

23. The protein conjugate according to claim 22, wherein the proximal is separated from the C34 in the range of 5 to 15 angstroms.

24. The protein conjugate according to any one of claims 1 to 3, wherein the linker is a peptide linker.

25. The protein conjugate according to claim 24, wherein the linker is selected from SEQ ID NOs: 4 to 15.

26. The protein conjugate according to any one of claims 1 to 3, wherein the protein carrier lacks DNA.

27. The protein conjugate according to any one of claims 1 to 3, wherein the biological payload does not bind to cell surface proteins.

28. The protein conjugate according to claim 6, wherein the antigen-binding molecule is a VHH.

29. a. HSA covalently bound to a plurality of polyethyleneimine (PEI) moieties and characterized by a positive zeta potential of at least 8 mV; b. a single-domain antibody; c. a linker covalently bound to the HSA and the single-domain antibody; comprising a protein conjugate, wherein the protein conjugate is not a fusion protein, and the linker comprises a biocompatible polymer or is a peptide linker.

30. The protein conjugate according to claim 29, wherein the HSA comprises 3 to 10 PEI molecules.

31. The protein conjugate according to claim 29 or 30, wherein the linker comprises a biocompatible polymer.

32. The protein conjugate according to claim 31, wherein the biocompatible polymer comprises polyethylene glycol (PEG).

33. The protein conjugate according to claim 29 or 30, wherein the linker comprises a biologically cleavable bond.

34. The protein conjugate according to claim 33, wherein the biologically cleavable bond comprises a disulfide bond.

35. The protein conjugate according to claim 29 or 30, wherein the linker is substantially stable in blood for at least 24 hours.

36. The protein conjugate according to claim 35, wherein stability comprises less than 25% cleavage in blood after 24 hours.

37. The protein conjugate according to claim 34, wherein the biologically cleavable bond is sterically hindered.

38. The protein conjugate according to claim 29 or 30, wherein the HSA comprises SEQ ID NO: 1, or a fragment or homolog thereof comprising cysteine 34 (C34).

39. The protein conjugate according to claim 38, wherein the linker is bound to the HSA via a disulfide bond.

40. The protein conjugate according to claim 39, wherein the linker is bound to C34 of HSA.

41. The protein conjugate according to claim 40, wherein the linker is a peptide linker.

42. The protein conjugate according to claim 41, wherein the linker is selected from SEQ ID NOs: 4 to 45.

43. The protein conjugate according to any one of claims 1 to 3 and 29 to 30, further comprising a detectable tag, and optionally the tag is conjugated to a biological payload.

44. The protein conjugate according to any one of claims 1 to 3 and 29 to 30, which is a blood-stable conjugate.

45. The protein conjugate according to any one of claims 1 to 3 and 29 to 30, which is a cell-permeable conjugate.

46. A method for producing a protein conjugate capable of binding to an intracellular target, comprising: a. providing a payload that binds to the intracellular target; b. providing a protein carrier covalently bound to a plurality of PEI moieties and characterized by a positive zeta potential of at least 8 mV; c. covalently linking a selected payload to a selected protein carrier via a linker to produce a protein conjugate, wherein the linker comprises a biocompatible polymer or is a peptide linker; d. determining the stability of the linker in human blood, plasma or serum; e. selecting a protein conjugate comprising a linker that is stable in the human blood, plasma or serum; thereby producing a protein conjugate capable of binding to an intracellular target. A method comprising the above steps.

47. The method according to claim 46, wherein the determining is performed before or after the formation of the protein conjugate.

48. The method according to claim 46 or 47, comprising confirming the binding of the biological payload to the intracellular target.

49. The method according to claim 46 or 47, wherein the payload is determined to lack a disulfide bond that reduces binding to the intracellular target when cleaved.

50. The method according to claim 46 or 47, wherein the protein carrier comprises HSA.

51. The method according to claim 46 or 47, wherein the protein carrier is covalently bound to at least 3 PEI molecules.

52. The method according to claim 46 or 47, further comprising contacting the selected protein conjugate with a cell and confirming that the biological payload enters the cytoplasm of the cell.

53. The method according to claim 46 or 47, wherein stable comprises less than 25% cleavage in blood after 24 hours and unstable comprises at least 50% cleavage in the cytoplasmic state after 24 hours.

54. The method according to claim 46 or 47, wherein the linker comprises a biocompatible polymer.

55. The method according to claim 46 or 47, wherein the covalent linking is via a click reaction.

56. The payload is covalently bound to a linker comprising a first reactive group; and the protein carrier is covalently bound to a linker comprising a second reactive group having reactivity towards the first reactive group, the method according to claim 46 or 47.

57. The method according to claim 56, wherein the covalent linking is by reacting the first reactive group with the second reactive group, thereby covalently linking the payload and the protein carrier.

58. The method according to claim 46 or 47, wherein the linker comprises a biologically cleavable bond.

59. The method according to claim 58, wherein the covalent linking comprises formation of a disulfide bond.

60. (i) the payload is covalently bound to a linker capable of forming a disulfide bond with a cysteine of the protein carrier; or (ii) the protein carrier is covalently bound to a linker capable of forming a disulfide bond with a cysteine of the payload, and the bond is optionally via a disulfide bond, the method according to claim 58.

61. The method according to claim 58, further comprising determining the stability of the linker in the cytoplasmic state and selecting a protein conjugate comprising a linker that is unstable in the cytoplasmic state.

62. A protein conjugate produced by the method according to claim 46 or 47.

63. An in vitro method of binding an intracellular target, comprising contacting a cell expressing the intracellular target with the protein conjugate according to any one of claims 1 to 3 and 29 to 30, wherein the biological payload binds to the intracellular target, thereby binding the intracellular target, an in vitro method.

64. An in vitro method of binding an intracellular target, comprising: a. producing a protein conjugate capable of binding the intracellular target by the method according to claim 46 or 47; b. contacting a cell expressing the intracellular target with the produced protein conjugate; thereby binding the intracellular target, an in vitro method.

65. The in vitro method according to claim 63, wherein the method is a method of detecting an intracellular target and the protein conjugate comprises a detectable tag, and further comprises detecting the detectable tag.

66. The in vitro method according to claim 65, wherein the method is a method of modulating the intracellular target and the payload is an agonist or antagonist of the intracellular target.

67. A pharmaceutical composition for use in a method of treating a condition in a subject in need thereof, comprising a protein conjugate according to any one of claims 1 to 3 and 29 to 30 and a pharmaceutically acceptable carrier, excipient or adjuvant, wherein the payload is an agonist or antagonist of an intracellular target and the condition is treatable by modulation of the intracellular target.

68. The composition according to claim 67, wherein the condition is cancer, the intracellular target is oncogenic and the biological payload is an antagonist.

69. The in vitro method according to claim 63, wherein the contacting is not in the presence of an agent other than the carrier protein designed to induce permeation of the protein conjugate into the cell.

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