Engineered CD47 extracellular domain for bioconjugation

Abstract

Compositions and methods related to engineered CD47 extracellular domain (ECD) proteins are provided.

Description

[Background technology]

[0001] Nanoparticle (NP)-based targeted delivery is a promising approach for drug delivery, including improved cancer treatment and diagnosis. However, effective delivery of cargo presents challenges that must be overcome. In addition to particle stability and specific targeting, evasion of the immune system is a key challenge. Only a small fraction of the NP dose reaches the target tissue, as the majority of administered NPs are cleared by the mononuclear phagocyte system. The liver and spleen are involved, and for smaller NPs, the renal system also participates in clearance.

[0002] Coating nanoparticles with polyethylene glycol (PEG) may evade phagocytes, including Kupffer cells in the liver, and extend blood circulation time by creating a "stealth" brush (see Hong et al. Clin. Cancer Res. 5, 3645-52 (1999), and Armstrong et al. Cancer 110, 103-11 (2007)). However, PEGylation can reduce uptake of NPs by target cells and may be immunogenic. Such NPs may also be opsonized over time by serum proteins such as immunoglobulin G (IgG), increasing clearance by phagocytes (Rodriguez et al. Science 339, 971-5 (2013)).

[0003] CD47 is a widely expressed transmembrane glycoprotein with a single Ig-like extracellular domain and five transmembrane regions. It functions as a cellular ligand for SIRPα with binding via its NH2-terminal V-like domain. SIRPα is mainly expressed on myeloid cells, including macrophages, granulocytes, myeloid dendritic cells (DCs), mast cells, monocytes, and their precursor cells, including hematopoietic stem cells. The structural determinants of SIRPα that mediate CD47 binding are discussed in Lee et al. (2007) J. Immunol. 179:7741-7750, Hatherley et al. (2007) JBC 282:14567-75, and the role of SIRPα cis-dimerization in CD47 binding is discussed in Lee et al. (2010) JBC 285:37953-63 and reviewed in Barclay and van den Berg Annu. Rev. Immunol. 32, 25-50 (2014).

[0004] The extracellular domain (ECD) of CD47 has been proposed as a means of avoiding phagocytosis. When CD47-ECD interacts with SIRPα ECD displayed on the surface of phagocytes, it generates a "don't eat me" signal to inhibit phagocytosis. Foreign bodies such as bacteria, viruses, and NPs are phagocytes, in part due to the absence of surface CD47. Polymer-based NPs and P22 virus-like particles (VLPs) displaying CD47-ECD or CD47 "self" peptides on their surface have been reported to undergo less phagocytic clearance (see Qie et al. Sci. Rep. 6, 26269 (2016) and Schwarz et al. ACS Nano 9, 9134-47 (2015)).

[0005] Improved methods for reducing undesirable phagocytic clearance are of great interest for the development of implantable devices and drug delivery modalities and are addressed herein. Summary of the Invention

[0006] Compositions and methods related to engineered CD47 extracellular domain (ECD) proteins are provided. CD47-ECD is modified by specific amino acid alterations to provide benefits in surface conjugation, where CD47-ECD is modified to properly orient on the surface and engage its counter-receptor, SIRPα. Engagement with SIRPα is required for the biological activity of CD47-ECD in reducing phagocytic clearance. Modifications for these purposes include, but are not limited to, (i) the addition of a cleavable N-terminal extension to generate a pyroglutamic acid N-terminus, and (ii) substitution of residues to allow for the introduction of non-natural amino acids (nnAA) at the desired attachment site to the surface of CD47-ECD. CD47-ECD can be generated using cell-free protein synthesis (CFPS). Sometimes reference is made to human CD47-ECD, but similar alterations have been made to murine CD47-ECD.

[0007] In some embodiments, the cleavable extension is provided at the N-terminus of the ECD, where the cleavage site for the extension is immediately adjacent to the N-terminal glutamine of the mature CD47-ECD. When the extension is cleaved, this glutamine is exposed at the N-terminus. In some embodiments, the exposed glutamine is converted to pyroglutamic acid with glutaminyl cyclase. In some embodiments, the extension includes a recognition site for enterokinase (e.g., DDDDK) and is cleaved by enterokinase. In some embodiments, the extension includes a recognition site for factor Xa cleavage (e.g., IEGR) and is cleaved by factor Xa. The extension optionally includes a purification tag (e.g., a histidine tag, etc.) and may include a short linker between the tag and the cleavage site.

[0008] An nnAA is introduced at the desired site for attaching CD47-ECD to a surface. The site for introducing the nnAA can be the naturally occurring double bond site, C15 and V116 (relative to the reference human CD47-ECD of SEQ ID NO: 1). The unnatural amino acid for this purpose is selected to provide a reactive group for click chemistry or other bioorthogonal reactions. The nnAA can, for example, contain an alkyne or azide functional group (e.g., homopropargylglycine (HPG) or azidohomoalanine (AHA), respectively). Conveniently, this is achieved by global methionine substitution, although other methods of introducing the nnAA can alternatively be used. In an embodiment in which the nnAA is introduced by methionine substitution, both the C15 and V116 codons of the coding sequence are changed to ATG.

[0009] In those embodiments utilizing methionine substitutions, the CD47-ECD may also be engineered to replace naturally occurring methionines at sites that are undesirable for attachment, i.e., M28 and M82 (numbering relative to the human reference protein). In some embodiments, M28 and M82 are replaced with amino acids other than methionine. In some embodiments, the replaced amino acids are conservative variants (e.g., hydrophobic amino acids such as L, I, V, F). In some embodiments, the particular amino acid substitutions are M28V and M82L / I.

[0010] Optionally, CD47-ECD is further engineered to improve the solubility of the protein. Or, for example, hydrophobic residues on the surface of CD47-ECD that faces the cell membrane in the native protein can be replaced with hydrophilic residues. In some embodiments, residues F14 and V115 are replaced with hydrophilic uncharged amino acids. In some embodiments, the amino acid replacement is one or both of F14N and V115N.

[0011] In one embodiment, an engineered CD47-ECD is provided that includes (i) the addition of a cleavable N-terminal extension to generate a proglutamic acid N-terminus, and (ii) changes in residue substitutions compared to the native protein to allow for the introduction of non-natural amino acids (nnAAs) at desired attachment sites to the surface of CD47-ECD. In some embodiments, nnAAs are introduced at positions C15 and V116. In some embodiments, nnAAs include alkyne or azide functional groups. In some embodiments, the engineered protein further includes (iii) altered substitutions of naturally occurring methionines at M28 and M82. In some embodiments, the substituted amino acids are conservative variants. In some embodiments, the engineered CD47-ECD further includes (iv) substitutions of hydrophobic residues on the membrane-facing surface. In some embodiments, residues F14 and V115 are substituted with hydrophilic uncharged amino acids.

[0012] Methods are provided for using CD47-ECD engineered as described herein to coat the surfaces of articles intended for internal use or administration that are exposed to phagocytic cells, typically articles such as particles, microparticles, inserts for internal delivery of therapeutics such as nanoparticles, sustained release implants that may be biodegradable, osmotic pumps, implantable devices and prostheses, etc. In some embodiments, coating with CD47-ECD reduces phagocytic clearance of the article.

[0013] The coating method utilizes reactive groups in the nnAA to provide a bond (e.g., a covalent bond) between CD47-ECD and reactive groups provided on the surface. Surface reactive groups provide spacing of reactants as an array or as pairs. Preferred pairs of reactants are about 5 to about 15 Å, about 7 to about 13 Å apart, and may be about 10 Å apart. Alternatively, an array of reactants is provided on the surface providing multiple reactants, or on an array that displays reactive groups on a linker that is fixed to the surface at inconsistent spacing, but allows the reactive groups to move freely to a spacing that allows two-point attachment. After CD47-ECD is attached, unreacted groups may be blocked.

[0014] In one such embodiment, the nnAA at positions 15 and 116 of CD47-ECD provide either an alkyne or an azide functional group. The surface provides reactants of the nnAA, azide to alkyne or alkyne to azide. Conjugation is achieved by copper(I)-catalyzed azide-alkyne cycloaddition ("click" reaction). Cleavage of the N-terminal extension and conversion of glutamine to pyroglutamic acid can be performed before or after the click reaction.

[0015] In some embodiments, the article is a nanoparticle. In some embodiments, the article, including but not limited to a nanoparticle, comprises a protein that provides a reactive group for binding to CD47-ECD. In some embodiments, the reactive group on the protein is nnAA. In some embodiments, the nanoparticle is a virus-like particle and the reactant protein is a viral core protein. In some embodiments, the protein is a Hepatitis B core protein.

[0016] In other embodiments, articles are provided that include engineered CD47-ECD on a surface as described herein. Articles include particles, microparticles, inserts, slow release implants that may be biodegradable, osmotic pumps, implantable devices, and prostheses, for example, for internal delivery of therapeutic agents such as nanoparticles. In some embodiments, coating with CD47-ECD reduces phagocytic clearance of the article relative to the article in the absence of engineered CD47-ECD. In such embodiments, the optimal number and spacing of attached engineered CD47-ECD is determined by experimentation known to those skilled in the art. In some embodiments, CD47-ECD is covalently attached to a protein present on the article, including, but not limited to, a protein present on a virus-like particle. The coated article may be purified and formulated in a pharmacologically acceptable vehicle for administration to a patient. In some embodiments, the article is a VLP covalently attached to engineered CD47-ECD for formulation. In some embodiments, the VLP contains a protein or drug for delivery.

[0017] Engineered CD47-ECD can be made by generating a nucleic acid construct encoding the engineered protein and producing the polypeptide by cell-free synthesis, which may include coupled transcription and translation reactions. CFPS provides convenient methods for introducing nnAAs during synthesis (e.g., using orthogonal tRNAs, global methionine substitution, etc.). Vectors and polynucleotides encoding the engineered proteins are also provided.

[0018] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Conversely, dimensions of the various features have been arbitrarily expanded or reduced for clarity. The drawings include the following figures: [Brief description of the drawings]

[0019] [Figure 1] Upon interacting with SIRPα on phagocytes, CD47 transmits a "don't eat-me" survival signal. [Diagram 2] The structure of human CD47-ECD and its binding partner, human SIRPα domain 1. The FG loop region (amino acid positions 97-106, shown in pink) and the N-terminal (NT) pyroglutamic acid (shown in light green) of CD47-ECD are important in binding to SIRPα. The ECD is anchored to the surface of the cell by the C-terminal (CT) polypeptide and a disulfide bond link at the C15 position. [Diagram 3] An N-terminal extension was introduced into human CD47-ECD to expose a pyroglutamic acid on the N-terminus after enzymatic reaction. The fusion protein was first digested with enterokinase to remove the N-terminal tag. Then, the exposed N-terminal glutamine was converted to proglutamic acid using glutaminyl cyclase. [Figure 4]Mutations are introduced into the CD47-ECD for improved conjugation to the surface of VLPs and production. A shows the sites where mutations are introduced into human CD47-ECD for nnAA incorporation by global methionine substitution. B shows the creation of a double bond between CD47-ECD and the HepBc dimer to mimic the native anchoring of CD47-ECD to the retina. C shows the substitution of a hydrophobic amino acid to improve protein solubility. The original methionine positions that are substituted to avoid nnAA incorporation at these sites are shown in green, and the nnAA incorporation sites for VLP attachment are shown in yellow. The sites of hydrophilic mutations are shown in blue. [Diagram 5] CD47-ECD amino acid sequence conservation is shown. [Figure 6A] The total cell-free accumulation levels (total product and total soluble product accumulation) of human CD47-ECD variants are shown. [Figure 6B] Soluble accumulation of human CD47-ECD mutants. Protein solubility of human CD47-ECD mutants was expressed with methionine, HPG, or AHA. WTC15G has only the C15G mutant. The nnAA(L / I) mutant has C15M, M28V, M82Lorl, and V116M. The nnAA(L / I)NN mutant has the nnAA(L / I) mutant plus F14N and V115N. In this notation, C15M and V116M refer to modifications in the coding region that change the native codon to ATG. [Figure 7A] Purification and purification of human CD47-ECD with N-terminal pyroglutamic acid. SDS-PAGE gel of human CD47-ECD nnAA(I)NN samples at each purification step: 1st lane: protein ladder (Mark12), 2nd lane: after buffer exchange with CFPS, 3rd lane: after aggregate removal, 4th lane: Ni-NTA elution, 5th lane: after enterokinase reaction, 6th lane: purified (1Q) hCD47-ECD nnAA(I)NN. [Figure 7B]Detection of proglutamate formation by QC as indicated by NADH consumption. GLDH=glutamate dehydrogenase, QC=glutaminyl cyclase. [Figure 8] 1 shows a comparison of human and mouse CD47-ECD mutants, with the amino acid sequences showing the incorporation of methionine in place of nnAA. [Figure 9A] Figure 1 shows total cell-free and soluble protein accumulation of mouse CD47-ECD mutants, and cell-free protein expression levels of mouse CD47-ECD nnAA(I). [Figure 9B] Indicates the solubility of the protein. [Figure 10A] Shows mouse CD47-ECD attachment to HepBc VLPs. Reduced SDS-PAGE and autoradiography of VLPs with mouse CD47 (mCD47)-ECD attached or disassembled. (Only the HepBc protein is radioactive.) 79nnAA and 80nnAA refer to VLPs with nnAA incorporated at these sites at the tips of the surface spikes of the VLPs. [Figure 10B] The mCD47-ECD numbers attached to the surface of the VLPs are indicated. [Figure 10C] The surface attachment sites of the HepBc dimer are shown. (For clarity, only the sites on either monomer are shown. Sites on the back of the molecule are shown in brackets.) [Figure 11] Fluorescence images of RAW264.7 cell internalization of BDFL-loaded VLPs are shown. The BDFL dye of HepBc VLPs is shown in green, and cell nuclei are shown in magenta. Uptake of BDFL-loaded VLPs by RAW264.7 cells was detected by BDFL fluorescence (middle). In comparison, uptake of mouse CD47-ECD-functionalized BDFL-VLPs was significantly reduced (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Compositions and methods related to engineered CD47 extracellular domain (ECD) proteins are provided. CD47-ECD is modified by specific amino acid alterations to provide benefits in surface conjugation, where CD47-ECD is modified to properly orient on the surface and engage with its counter-receptor, SIRPα. Engagement with SIRPα is required for the biological activity of CD47-ECD in reducing phagocyte clearance. Modifications for these purposes include, but are not limited to, (i) the addition of a cleavable N-terminal extension to generate a pyroglutamic acid N-terminus, and (ii) the substitution of residues to allow the introduction of non-natural amino acids (nnAA) at the desired attachment site to the surface of CD47-ECD.

[0021] In some embodiments, what is provided is the use of an engineered protein disclosed herein or an article comprising an engineered protein, e.g., in the manufacture of a pharmaceutical product. In one embodiment, what is provided is the use of an engineered protein or an article comprising an engineered protein disclosed herein for drug delivery in an article to a tissue.

[0022] CD47 is a 50 kDa membrane receptor with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. There are four alternatively spliced ​​isoforms of CD47 that differ only in the length of their cytoplasmic portion. It binds to signal inhibitory protein alpha (SIRPα). CD47 / SIRPα interaction triggers bidirectional signaling, resulting in different cell-cell responses including inhibition of phagocytosis, stimulation of cell-cell fusion, activation of T cells, and its activity as a don't eat me signal for phagocytes of the immune system. For example, red blood cells lacking CD47 are rapidly cleared from the bloodstream by macrophages, a process mediated by its interaction with SIRPα.

[0023] The sequences of human and mouse CD47 are publicly available, e.g., at GenBank The human protein reference sequence is NP_034711.1 and the mouse reference protein sequence is NP For convenience, numbering of amino acid residues can be made herein with reference to SEQ ID NO: 1, for human CD47-ECD QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVS and for mouse CD47-ECD SEQ ID NO: 2 QLLFSNVNSIEFTSCNETVVIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTTTDQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSREGKTVIELKNRTS.

[0024] For physiologically relevant purposes, binding of SIRPα to CD47 is typically an event between SIRPα on phagocytes and their precursor cells (e.g., microphages and monocytes), and CD47 on articles, particularly particulate matter such as nanoparticles, microparticles, etc., may be targets for phagocytosis.

[0025] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring and non-naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid.

[0026] The term "amino acid" refers to naturally occurring and artificial amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are subsequently modified (e.g., hydroxyproline, gamma-carboxyglutamate, O-phosphoserine). Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., alpha, a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0027] The engineered polypeptides disclosed herein can include at least one unnatural amino acid at a given site, and can include or contain 1, 2, 3, 4, 5, or more unnatural amino acids. When present at two or more sites in a polypeptide, the unnatural amino acids can be the same or different. When the unnatural amino acids are different, an orthogonal tRNA and cognate tRNA synthetase are present for each unnatural amino acid. In some embodiments, a single unnatural amino acid is present at residue 80.

[0028] Examples of unnatural amino acids that can be used include: unnatural analogs of tyrosine amino acids, unnatural analogs of glutamine amino acids, unnatural analogs of phenylalanine amino acids, unnatural analogs of methionine amino acids, unnatural analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronic acid, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, or any combination thereof; amino acids with photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; amino acids with novel functional groups; alternative functional groups; These include amino acids that interact covalently or non-covalently with molecules; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; biotin or biotin analog-containing amino acids; glycosylated or carbohydrate-modified amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyethers; heavy-atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with elongated side chains; amino acids containing toxic groups; sugar-substituted amino acids (e.g., sugar-substituted serine, etc.); carbon-linked sugar-containing amino acids; redox-active amino acids; alpha-hydroxy-containing acids; aminothioacid-containing amino acids; alpha,alpha disubstituted amino acids; beta-amino acids; cyclic amino acids other than proline, etc.

[0029] Unnatural amino acids of interest include, but are not limited to, amino acids that provide reactive groups for "click" chemistry reactions (see Click Chemistry: Diverse Chemical Function from a Few Good Reactions Hartmuth C. Kolb, MG Finn, K. Barry Sharpless Angewandte Chemie International Edition Volume 40, 2001, P. 2004, specifically incorporated herein by reference). For example, the amino acids azidohomoalanine, homopropargylglycine, p-acetyl-L-phenylalanine and p-azido-L-phenylalanine are of interest.

[0030] In some embodiments, the unnatural amino acid is introduced by global replacement of methionine on the protein, for example, methionine can be released from the cell-free reaction mixture and replaced with 0.25-2.5 mM azidohomoalanine (AHA). In such embodiments, it is preferred to replace the natural methionine with a different amino acid, while an ATG codon is introduced into the coding sequence at the desired site for the introduction of the unnatural amino acid.

[0031] Alternatively, the unnatural amino acid is introduced by an orthogonal component. The orthogonal component includes a tRNA that is aminoacylated with an unnatural amino acid, where the orthogonal tRNA base pairs with a codon that is not normally associated with an amino acid, e.g., a stop codon, a 4 bp codon, etc. The reaction mixture can further include a tRNA synthetase that can aminoacylate (with an unnatural amino acid) the cognate orthogonal tRNA. Such components are known in the art, e.g., as described in U.S. Patent No. 7,045,337, issued May 16, 2006. The orthogonal tRNA recognizes a selector codon, which can be a nonsense codon, e.g., a stop codon (e.g., amber, ochre, and opal codons, four or more base codons, codons derived from natural or unnatural base pairs, etc.). The orthogonal tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates the unnatural amino acid at this site into the polypeptide.

[0032] Orthogonal tRNA synthetases can be exogenously synthesized, purified, and added to reaction mixtures of the invention, typically in defined amounts of at least about 10 μg / ml, at least about 20 μg / ml, at least about 30 μg / ml, and up to about 200 μg / ml. Proteins can be synthesized in bacteria or eukaryotic cells and purified, for example, by affinity chromatography, PAGE, gel exclusion chromatography, reverse phase chromatography, and the like, as known in the art.

[0033] The term "conjugation partner" may be used to refer to any moiety, such as a peptide or protein, a nucleic acid, a polysaccharide, a label, etc., that is conjugated to an engineered CD47-ECD. The conjugation partner may be present on or form the surface of an article. The conjugation partner may contain complementary active groups for "click" chemistry conjugation. For example, the conjugation partner may be synthesized with one or more non-natural amino acids that allow for conjugation to non-natural amino acids present in the CD47-ECD protein. Those skilled in the art will appreciate that chemistry for conjugation is well known and can be readily applied to a variety of groups, such as, for example, CpG DNA sequences, detectable labels, antigens, polypeptides, etc.

[0034] In some embodiments, the conjugation partner is a structural protein, such as collagen, keratin, actin, myosin, elastin, fibrillin, lamin, etc. In some embodiments, the conjugation partner is a virus-like particle protein, including but not limited to, Hepatitis B core protein HBc. In some embodiments, the conjugation partner is a polymer, such as PEG, that has been modified to provide a reactive group.

[0035] The term "HBc" refers to the amino acid peptide sequence of Hepatitis B core protein, or a truncated version thereof, or an equivalent protein, e.g., a protein modified with one or more disulfide bonds, modified to provide sites for the introduction of unnatural amino acids, including such end modifications as described in U.S. Patent No. 9,896,483, specifically incorporated herein by reference. In some embodiments, HBc is the conjugation partner of an engineered CD47-ECD.

[0036] Various groups can be introduced into the peptide during synthesis or expression and can be attached to a molecule or surface, such as CD47-ECD. Additionally, such surfaces, when modified to display reactive species, can be modified by first attaching an article, such as a VLP, to the surface, followed by attachment of an engineered CD47-ECD to the article. For example, a surface can display an alkyne first to allow attachment of an azide-displaying VLP, followed by attachment of an engineered CD47-ECD displaying an alkyne. The introduced groups need not be included in the HBc domain itself, but can be introduced as tags or fusions to the C- or N-terminus of the HBc domain. Thus, cysteines can be used to create metal ion complexes, carboxyl groups to form amides or esters, thioethers to attach to amino groups to form amides, polyhistidines, etc. Insertion of three amino acids (ASV) after the initiator formyl methionine to remove translation initiation non-natural methionine analogs by methionyl aminopeptidase can be included to avoid surface conjugation at undesired positions.

[0037] In some embodiments, the unnatural amino acid is included at one or more defined sites of the protein. The HBc polypeptide of the present invention can include an unnatural amino acid to control direct attachment to the conjugation partner CD47-ECD. The nnAA on the conjugation partner is different and reactive to the nnAA present on the HBc polypeptide.

[0038] Those skilled in the art will appreciate that minor amino acid changes can be made in the sequence without altering the function of the protein, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 amino acid changes, and the full-length protein can be substituted for the truncated versions exemplified herein. HBc is functionally capable of self-assembly to form icosahedral virus like particles. The HBc polypeptides of the present invention can also include a cargo loading domain.

[0039] As used herein, the term "virus-like particle" refers to a stable macromolecular assembly of one or more viral proteins, usually viral coat proteins. The number of separate protein chains in a VLP is usually at least about 60 proteins, about 80 proteins, at least about 120 proteins, or more, depending on the specific viral shape. In the method of the present invention, the VLP comprises HBc conjugated to an engineered CD47-ECD. The method of the present invention provides for the synthesis of coat proteins in the absence of a viral polynucleotide genome, and thus the capsid may be empty or may contain non-viral components, such as mRNA fragments, drug cargo, etc.

[0040] Stable VLPs maintain the association of proteins in the capsid structure under physiological conditions for extended periods, e.g., at least about 24 hours, at least about one week, at least about one month or more. Once assembled, VLPs can have a stability commensurate with that of native virus particles, e.g., when exposed to pH changes, heat, freezing, ionic changes, etc. Additional components of the VLP can be included within or disposed on the VLP, as known in the art. VLPs do not contain intact viral nucleic acid and are non-infectious. In some embodiments, there are sufficient viral surface envelope glycoproteins and / or adjuvant molecules on the surface of the VLPs such that when the VLP preparation is formulated into an immunogenic composition and administered to an animal or human, an immune response (cell-mediated or humoral) is raised.

[0041] As used herein, the terms "purified" and "isolated" when used in the context of a polypeptide are substantially free of contaminants from the material from which the cellular material is obtained, such as, for example, but not limited to, cell debris, cell wall material, membranes, organelles, the bulk of nucleic acids, carbohydrates, proteins, and / or lipids present in the cell. Thus, an isolated polypeptide includes preparations of a polypeptide having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellular material and / or contaminants. As used herein, the terms "purified" and "isolated" when used in the context of a chemically synthesized polypeptide refer to a polypeptide that is substantially free of chemical precursors or other chemicals involved in the synthesis of the polypeptide.

[0042] Polypeptides may be isolated and purified according to conventional methods of recombinant synthesis or cell-free protein synthesis. Separation procedures of interest include affinity chromatography. Affinity chromatography uses highly specific binding sites, usually present in biological macromolecules, to separate molecules according to their ability to bind to specific ligands. Covalent binding binds the ligand to an insoluble porous support medium in a manner that clearly presents the ligand to the protein sample, thereby using the natural biospecific binding of one molecular species to separate and purify a second species from a mixture. Antibodies are commonly used in affinity chromatography. Preferably, microparticles or matrices are used as supports for affinity chromatography. Such supports are known in the art and commercially available, including activated supports that can be coupled to linker molecules. For example, Affi-Gel supports based on agarose or polyacrylamide are low-pressure gels suitable for most laboratory-scale purifications by peristaltic pumps or gravity flow elution. Affi-Prep supports are based on pressure-stable macroporous polymers and are suitable for formulation and process-scale applications.

[0043] Proteins may be separated by ion exchange chromatography, and / or concentration, filtration, dialysis, etc., using methods known in the art. The methods of the invention provide proteins containing unnatural amino acids with biological activity comparable to the natural proteins. The specific activity of the proteins in the composition may be determined by determining the activity level in functional assays, non-functional assays, such as immunostaining, ELISA, quantification on Coomassie or silver stained gels, as well as determining the ratio of biologically active protein to total protein. Generally, the specific activity so defined is at least about 5% of the natural protein, usually at least about 10% of the natural protein, and may be about 25%, about 50%, about 90% or more.

[0044] Exemplary coding sequences are provided, but one of skill in the art can easily design a suitable coding sequence based on the amino acid sequence provided. Methods well known to those of skill in the art can be used to construct expression vectors containing the coding sequence and appropriate transcriptional / translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. Alternatively, RNA capable of encoding a polypeptide of interest can be chemically synthesized. One of skill in the art can easily utilize well-known codon usage tables and synthetic methods to provide a suitable coding sequence for any of the polypeptides of the invention. The nucleic acids are isolated and obtained in substantial purity. Usually, the nucleic acids, either as DNA or RNA, are obtained substantially free of other naturally occurring nucleic acid sequences, generally at least about 50%, usually at least about 90% pure, and typically are "recombinant", e.g., flanked by one or more nucleotides that are not normally associated on naturally occurring chromosomes. The nucleic acids of the invention can be provided as linear or circular molecules, and can be provided in autonomously replicating molecules (vectors) or in molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or other regulatory sequences known in the art. The nucleic acids of the invention can be introduced into suitable host cells using a variety of techniques available in the art.

[0045] The term "diagnosis" is used herein to refer to the identification of a molecular or pathological state, disease or condition. The term "prognosis" is used herein to indicate a prediction of the likelihood of death or progression due to cancer, including recurrence, metastatic spread, and drug resistance. The term "prediction" is used herein to refer to the act of foreseeing or inferring based on observation, experience, or scientific reasoning. In one example, a physician may predict a patient's chances of survival after treatment.

[0046] As used herein, the terms "treatment," "treating," and the like refer to administering an agent or performing a procedure for the purpose of obtaining an effect. The effect may be completely or partially preventive of a disease or its symptoms, or may be therapeutic in that it results in a partial or complete cure of the disease itself and / or the symptoms of the disease.

[0047] Treating refers to indications of success in treating, improving, or preventing disease, including objective or subjective parameters such as reduction, remission, relief of symptoms, or making the condition tolerable to the patient, slowing the rate of degeneration or decline, or making the end point of degeneration less debilitating. Treating or improving symptoms can be based on objective or subjective parameters, including the results of a physician's examination. Thus, the term "treating" includes administration of the compounds or agents of the present invention to prevent or delay, alleviate, or inhibit or inhibit the onset of symptoms or conditions associated with cancer or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of disease, symptoms of disease, or side effects of disease in a subject.

[0048] "In combination," "combination therapy," and "combination product" refer, in certain embodiments, to the simultaneous administration of a first therapeutic agent and a compound used herein to a patient. When administered in combination, each component can be administered simultaneously at different times or sequentially in any order. Thus, each component can be administered separately but sufficiently close in time to provide the desired therapeutic effect.

[0049] An "effective amount" or "sufficient amount" of a substance is an amount sufficient to cause a desired biological effect, such as a beneficial outcome, including a clinical outcome, and thus, an "effective amount" depends on the context in which it is applied. An effective amount can be administered in one or more administrations. For example, an effective amount or density of engineered CD47-ECD within a particle can be an amount or density that reduces phagocytic clearance by at least 10%, at least 20%, at least 50%, at least 75%, at least 90%, at least 95% or more compared to particles lacking CD47-ECD.

[0050] Cargo. The VLP may encapsulate cargo (e.g., a molecule that is released when the VLP is intracellular). Encapsulated cargo is protected within the VLP and is typically not displayed on the surface of the VLP. Stably loaded cargo is retained within the VLP after washing.

[0051] Many molecules are suitable as cargo, including but not limited to RNA, such as guide RNA, siRNA, antisense RNA, etc.; DNA, such as double-stranded or single-stranded DNA, including but not limited to plasmids, coding sequences, etc.; proteins, such as toxin proteins, including diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, auristatin-E, etc.; genetically modified proteins, including but not limited to CRISPR; binding proteins, such as antibodies or fragments derived therefrom, etc. Cytotoxic agents are numerous and varied. One exemplary class of cytotoxic agents is chemotherapeutic agents. Exemplary chemotherapeutic agents include aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ibuprofen, rifametidine ... These include, but are not limited to, phosphamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, mertansine, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel, pilocarpine, prochlorperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, vinorelbine tartrate, and the like.

[0052] In some embodiments, the cargo is modified to enhance encapsulation by VLP, for example, by adding a free sulfhydryl group for conjugation to HBc protein. Alternatively, a cargo agent that includes a free sulfhydryl group, such as mertansine, can be selected. Polar cargo, for example, nucleic acid such as ssDNA, RNA, can be conjugated to a hydrophobic group, for example, cholesterol, to enhance loading. Alternatively, one or more polar or charged amino acids can be conjugated to the cargo.

[0053] Cell-free protein synthesis, as used herein, refers to cell-free synthesis of polypeptides in a reaction mixture that contains biological extracts and / or defined reagents. The reaction mixture includes templates for the production of macromolecules, such as DNA, mRNA, etc., monomers for the synthesized macromolecules, such as amino acids, nucleotides, etc., as well as cofactors, enzymes, and other reagents required for synthesis, such as ribosomes, tRNA, polymerase, transcription factors, etc. Such synthesis reaction systems are well known in the art and described in the literature. Cell-free synthesis reactions can be carried out as batch, continuous flow, or semi-continuous flow, as known in the art.

[0054] CFPS and other subsequent steps may be performed under reducing conditions, e.g., in the presence of 1 mM DTT or equivalent. After assembly of the VLPs, the conditions may be changed to an oxidizing environment, e.g., optionally by dialysis to remove the reducing agent in the presence of salt (e.g., up to about 1 M salt, up to about 1.5 M salt, up to about 2.5 M salt, such as NaCl), and then oxidation to form disulfide bonds by adding 5-10 mM HO, 5-20 mM diamide, or equivalent.

[0055] In some embodiments of the present invention, cell-free synthesis is carried out in a reaction in which oxidative phosphorylation is activated, for example, in the CYTOMIM™ system. Activation of the respiratory chain and oxidative phosphorylation are demonstrated by an increase in polypeptide synthesis in the presence of O2. In a reaction in which oxidative phosphorylation is activated, the overall polypeptide synthesis in the presence of O2 is reduced by at least about 40% in the presence of certain electron transport chain inhibitors such as HQNO or in the absence of O2. The reaction chemistry may be as described in International Patent Application No. WO2004 / 016778, which is incorporated herein by reference.

[0056] The CYTOMIM™ environment for synthesis utilizes a cell extract from bacterial cells grown in a medium containing glucose and phosphate, with glucose initially present at a concentration of at least about 0.25% (weight / volume), more usually at least about 1%, and usually not more than about 4%, more usually not more than about 2%. An example of such a medium is 2YTPG medium, but those skilled in the art will appreciate that many culture media can be adapted for this purpose, as there are many published media suitable for the growth of bacteria such as E. coli, using both defined and undefined sources of nutrients (for examples of glucose-containing media, see Sambrook, J., EF Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor University Press, Cold Spring Harbor, NY). Alternatively, the culture may be grown using a protocol that continuously supplies glucose as needed to maintain high growth rates in either a defined or complex growth medium. The reaction mixture may be supplemented by including vesicles (e.g., an inner membrane vesicle solution). When provided, such vesicles may contain from about 0 to about 0.5 volumes, usually from about 0.1 to about 0.4 volumes.

[0057] In some embodiments, PEG is present in trace amounts or less, for example, less than 0.1%, and may be less than 0.01%. A reaction that is substantially free of PEG contains a sufficiently low level of PEG that, for example, oxidative phosphorylation is not inhibited by PEG. Spermidine molecules and putrescine can be used instead of PEG. Spermine or spermidine is present at a concentration of at least about 0.5 mM, usually at least about 1 mM, preferably about 1.5 mM, and not more than about 2.5 mM. Putrescine is present at a concentration of at least about 0.5 mM, preferably at least about 1 mM, preferably about 1.5 mM, and not more than about 2.5 mM. Spermidine and / or putrescine can be present in the initial cell extract or can be added separately.

[0058] The concentration of magnesium in the reaction mixture affects the overall synthesis. Magnesium is often present in cell extracts and may be adjusted with additional magnesium to optimize the concentration. Sources of magnesium salts useful for such methods are known in the art. In one embodiment of the present invention, the source of magnesium is magnesium glutamate. The preferred magnesium concentration is at least about 5 mM, usually at least about 10 mM, and preferably at least about 12 mM, and at a concentration of about 25 mM or less, usually about 20 mM or less. Other changes that may enhance synthesis or reduce costs include omitting HEPES buffer and phosphoenolpyruvate from the reaction mixture.

[0059] The system can be run under aerobic and anaerobic conditions. Oxygen can be supplied to increase synthesis yields, especially for reactions larger than 15 μl. The head space of the reaction chamber can be filled with oxygen, oxygen can be pumped into the reaction mixture, etc. Oxygen can be supplied continuously, or the head space of the reaction chamber can be refilled during the course of protein expression for longer reaction times. Other electron acceptors, such as nitrate, sulfate, or fumarate, may also be supplied in conjunction with the formulation of cell extracts, such that the necessary enzymes are active in the cell extract.

[0060] No exogenous cofactors are required to activate oxidative phosphorylation. + Compounds such as acetylcholinesterase (Asp), acetylcholinesterase (AAS), or acetyl-coenzyme A can be used to supplement protein synthesis yields, but are not required. The addition of oxalic acid, a metabolic inhibitor of phosphoenolpyruvate synthase (Pps), can be beneficial in increasing protein yields, but is not required.

[0061] The template for cell-free protein synthesis can be either mRNA or DNA, preferably the combined system continuously produces mRNA from a DNA template with a recognizable promoter. Either an endogenous RNA polymerase is used or an exogenous phage RNA polymerase (usually T7 or SP6) is added directly to the reaction mixture. Alternatively, the mRNA can be continuously amplified by inserting the message into a template for QB replicase, an RNA-dependent RNA polymerase. The purified mRNA is generally stabilized by chemical modification before being added to the reaction mixture. Nucleases can be removed from the extract to help stabilize the mRNA levels. The template can code for any particular gene of interest.

[0062] Other salts may also be added, particularly biologically relevant salts such as manganese. Potassium is generally present at a concentration of at least about 50 mM, and not more than about 250 mM. Ammonium may usually be present at a concentration of not more than 200 mM, more usually not more than about 100 mM. Typically, the reaction is maintained at a pH range of about 5-10 and at a temperature of about 20° C.-50° C., more typically at a pH range of about 6-9 and at a temperature of about 25° C.-40° C., although these ranges may be extended for particular conditions of interest.

[0063] Metabolic inhibitors for undesired enzyme activity may be added to the reaction mixture, alternatively, enzymes or factors causing undesired activity may be directly removed from the extract, or genes encoding undesired enzymes may be inactivated or deleted from the chromosome.

[0064] As used herein, the terms "subject" or "patient" are used interchangeably to refer to animals (e.g., birds, reptiles, and mammals). In some embodiments, the subject is a non-primate (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., monkey, chimpanzee, and human). In certain embodiments, the subject is a non-human animal. In some embodiments, the subject is a livestock or pet. In other embodiments, the subject is a human (e.g., an infant, including a premature baby, a child, an adult, and / or an elderly person).

[0065] "Fused" or "operably linked" as used herein means that two or more polypeptides are linked together to form a continuous polypeptide chain. A fusion polypeptide (or a fusion polynucleotide encoding a fusion polypeptide) may contain additional components, including multiple peptides in multiple loops, fusion partners, etc. The exact location where the fusion is made is not critical, and the specific site is known and may be selected to optimize the biological activity, secretion or binding properties of the binding partner. The optimal location is determined by routine experimentation.

[0066] As used herein, the terms "correlated" or "related to each other," and similar terms, refer to a statistical association between instances of two events, where events include numbers, data sets, and the like. For example, where events include quantities, a positive correlation (also referred to herein as a "direct correlation") means that as one increases, the other also increases. A negative correlation (also referred to herein as an "inverse correlation") means that as one increases, the other decreases.

[0067] "Dosage unit" refers to a physically discrete unit suited as a single dosage for a particular individual to be treated. Each unit may contain a predetermined quantity of active compound(s) calculated to produce a desired therapeutic effect in association with the required medical carrier. The specifications for the dosage unit forms may be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technology of compounding such active compounds.

[0068] By "pharmaceutically acceptable excipient" is meant an excipient that is generally safe, non-toxic and useful for formulating a desired medical composition, and includes excipients that are acceptable for veterinary and human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

[0069] "Pharmaceutically acceptable salts and esters" refers to salts and esters that are pharma-ceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds can react with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as, for example, sodium, potassium, magnesium, calcium, aluminum, etc. Suitable organic salts include those formed with organic bases, such as amine bases (e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and equivalents). Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkanesulfonic and arenesulfonic acids, such as methasulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, such as, for example, C 1-6Alkyl esters are included. When two acidic groups are present, the pharma- ceutically acceptable salts or esters may be mono-acid-mono-salts or esters, or di-salts or esters; similarly, when two or more acidic groups are present, some or all of such groups may be salified or esterified. The compounds named in this invention may exist in unsalted or unesterified form, or salified and / or esterified form, and the naming of such compounds is meant to include both the parent (unsalted and unesterified) compound and its pharma- ceutically acceptable salts and esters. Also, the compounds named in this invention may exist in multiple stereoisomeric forms, and the naming of such compounds is intended to include all single stereoisomers and all mixtures of such stereoisomers (whether racemic or not).

[0070] The terms "pharmacologically acceptable," "biologically acceptable," and grammatical variations thereof are used interchangeably as they refer to compositions, carriers, diluents, and materials, and indicate that the material can be administered to a human without producing undesirable physiological effects to the extent that would prohibit administration of the composition.

[0071] protein Engineered CD47 extracellular domain (ECD) proteins (e.g., human CD47-ECD protein) are modified by specific amino acid changes to provide utility for conjugation to surfaces, where CD47-ECD is properly oriented on the surface and modified to bind its counter-receptor, SIRPα. Such modifications include, but are not limited to, (i) the addition of a cleavable N-terminal extension to generate a pyroglutamic acid N-terminus, and (ii) the substitution of residues to allow the introduction of non-natural amino acids (nnAA) at the desired attachment site to the surface of CD47-ECD. In certain embodiments, the sequences include any of SEQ ID NOs: 3, 5, 7, 9, 11, or 13, or sequences substantially similar (e.g., greater than 99% sequence identity, greater than 95% sequence identity, greater than 90% sequence identity) to any of SEQ ID NOs: 3, 5, 7, 9, 11, or 13.

[0072] The cleavable extension is provided at the N-terminus of the ECD, with a specific cleavage recognition site in the extension being in direct proximity to the N-terminal glutamine of the mature CD47-ECD. When the extension is cleaved, this glutamine is exposed at the N-terminus. The exposed glutamine is converted to proglutamic acid, for example with glutaminyl cyclase. The extension can be of any length, usually at least three, at least four, at least five, at least six or more residues, and can be considerably longer depending on the desired function, so long as it contains a specific cleavage recognition site. Conveniently, the extension is about 50 residues or less, 40 residues or less, 30 residues or less, 20 residues or less in length. The cleavage recognition site can be a recognition site for enterokinase (DDDDK) for factor Xa cleavage (e.g. IEGR), and the cognate enzyme is used to cleave the extension without the CD47-ECD.

[0073] The extension optionally includes a protein tag for purification or other purposes. The tag may provide for purification. Many such tags are known and used in the art, including histidine tags, polyglutamic acid tags, chitin-binding protein (CBP), maltose-binding protein (MBP), E-tag, FLAG-tag, S-tag, SBP-tag, Strep-tag, calmodulin tag, glutathione-S-transferase (GST), and the like. The tag may be an epitope tag, such as V5-tag, Myc-tag, HA-tag, Spot-tag, NE-tag, Strep-tag, TC-tag, Ty-tag, V5-tag, VSV-tag, Xpress-tag, and the like. AviTag provides for biotinylation by the enzyme BirA.

[0074] An nnAA is introduced at a desired site for attaching CD47-ECD to a surface. The site for introducing the nnAA can be C15 and V116 (relative to the reference human CD47-ECD of SEQ ID NO: 1), which are naturally occurring double bond sites. The unnatural amino acid for this purpose is selected to provide a reactive group for click chemistry or other bioorthogonal reactions. The nnAA can, for example, contain an alkyne or azide functional group (e.g., homopropargylglycine (HPG) or azidohomoalanine (AHA), respectively). Conveniently, this is achieved by global methionine substitution, although orthogonal methods of introducing nnAA can be used instead. In an embodiment where the nnAA is introduced by methionine substitution, the amino acid substitution can be C15nnAA and V116nnAA.

[0075] In those embodiments utilizing methionine substitutions, the CD47-ECD may also be engineered to replace naturally occurring methionines at sites that are undesirable for attachment, i.e., M28 and M82 (numbering relative to the human reference protein). In some embodiments, M28 and M82 are replaced with amino acids other than methionine. In some embodiments, the replaced amino acids are conservative variants (e.g., hydrophobic amino acids such as L, I, V, F). In some embodiments, the particular amino acid substitutions are M28V and M82L / I.

[0076] Optionally, CD47-ECD is further engineered to improve the solubility of the protein. Or, for example, hydrophobic residues on the surface of CD47-ECD that faces the cell membrane in the native protein can be replaced with hydrophilic residues. In some embodiments, residues F14 and V115 are replaced with hydrophilic uncharged amino acids. In some embodiments, the amino acid replacement is one or both of F14N and V115N.

[0077] A murine version of CD47-ECD is also provided, in which, for example, the naturally occurring methionines at residues M36, M82, and M88 are replaced with conservative substitutions. Cleavable extensions are provided. nnAA are introduced at C15 and V116.

[0078] Engineered CD47-ECD can be made by generating a nucleic acid construct encoding the engineered protein and producing the polypeptide by cell-free synthesis, which may include coupled transcription and translation reactions. CFPS provides convenient methods for introducing nnAAs during synthesis (e.g., using orthogonal tRNAs, global methionine substitution, etc.). Vectors and polynucleotides encoding the engineered proteins are also provided.

[0079] Optionally, various groups may be introduced into the peptide during synthesis or expression, which allows for linkage to other molecules or surfaces. Thus, cysteines can be used to create thioethers, histidines for linking to metal ion complexes, carboxyl groups to form amides or esters, amino groups to form amides, etc. In some embodiments, unnatural amino acids are included at one or more defined sites on the protein, particularly at the tips of protruding surface spikes on the VLP to provide steric availability.

[0080] The present invention further provides the nucleic acid that codes for the polypeptide.As those skilled in the art will understand, due to the degradation of genetic code, a huge number of nucleic acids can be made, all of which code for the fusion protein of the present invention.Therefore, those skilled in the art who identify a specific amino acid sequence can simply modify one or more codon sequences in a way that does not change the amino acid sequence of the protein to make any number of different nucleic acids.

[0081] Nucleic acids of the present invention that code for proteins can be used to create various expression constructs. Expression constructs can be self-replicating extrachromosomal vectors or vectors that are integrated into host genomes. Alternatively, for the purpose of cell-free expression, constructs can include those elements that are necessary for the transcription or translation of desired polypeptides, but may not include elements such as origin of replication, selection markers, etc. Cell-free constructs can be replicated in vitro, for example by PCR, and can include optimized terminal sequences for amplification reactions.

[0082] Generally, an expression construct comprises transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the fusion protein. The term "control sequence" refers to a DNA sequence required for the expression of an operably linked coding sequence in a particular expression system, such as mammalian cells, bacterial cells, cell-free synthesis, etc. Control sequences suitable for prokaryotic systems include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic systems may utilize promoters, polyadenylation signals, and enhancers.

[0083] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate the initiation of translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation or by an amplification reaction. Synthetic oligonucleotide adaptors or linkers can be used to link sequences according to conventional practices.

[0084] In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

[0085] The promoter sequence encodes either a constitutive promoter or an inducible promoter. The promoter can be either a naturally occurring promoter or a hybrid promoter. Hybrid promoters that combine elements of multiple promoters are also known in the art and are useful in the present invention. In a preferred embodiment, the promoter is a strong promoter, allowing high expression in in vitro expression systems, such as the T7 promoter.

[0086] In addition, expression constructs may contain additional elements. For example, expression vectors may have one or two replication systems, thus allowing them to be maintained in organisms, such as mammalian or insect cells for expression, and prokaryotic hosts for cloning and amplification. In addition, expression constructs may contain selection marker genes to allow the selection of transformed host cells. Selection genes are well known in the art and vary with the host cells used.

[0087] Formulation and Use Methods are provided for using CD47-ECD engineered as described herein to coat the surfaces of articles intended for internal use or administration that are exposed to phagocytic cells, such as, for example, particles, microparticles, inserts for internal delivery of therapeutic agents, such as nanoparticles, sustained release implants that may be biodegradable, osmotic pumps, implantable devices and prosthetics. In some embodiments, coating with CD47-ECD reduces phagocytic clearance of the article.

[0088] The method of coating utilizes reactive groups in the nnAA to provide a bond (e.g., a covalent bond) between CD47-ECD and reactive groups provided on the surface. Surface reactive groups provide spacing of reactants as an array or as pairs. Preferred pairs of reactants are about 5 to about 15 Å, about 7 to about 13 Å apart, and may be about 10 Å apart. Alternatively, an array of reactants is provided on the surface providing multiple reactants, or on an array displaying reactive groups on a linker that is fixed to the surface at inconsistent spacing, but allows the reactive groups to move freely to spacings that allow two-point attachment. After CD47-ECD is attached, unreacted groups may be blocked.

[0089] In one such embodiment, the nnAA at positions 15 and 116 of CD47-ECD provide either an alkyne or an azide functional group. The surface provides reactants of the nnAA, azide to alkyne or alkyne to azide. Conjugation is achieved by copper(I)-catalyzed azide-alkyne cycloaddition ("click" reaction). Cleavage of the N-terminal extension and conversion of glutamine to pyroglutamic acid can be performed before or after the click reaction.

[0090] In some embodiments, the article is a nanoparticle. In some embodiments, the article, including but not limited to a nanoparticle, comprises a protein that provides a reactive group for binding to CD47-ECD. In some embodiments, the reactive group on the protein is nnAA. In some embodiments, the nanoparticle is a virus-like particle and the reactant protein is a viral core protein. In some embodiments, the protein is a Hepatitis B core protein.

[0091] In other embodiments, articles are provided that include engineered CD47-ECD on a surface as described herein. Articles include particles, microparticles, inserts, slow release implants that may be biodegradable, osmotic pumps, implantable devices, and prostheses, for example, for internal delivery of therapeutic agents such as nanoparticles. In some embodiments, coating with CD47-ECD reduces phagocytic clearance of the article relative to the article in the absence of engineered CD47-ECD. In such embodiments, the optimal number and spacing of attached engineered CD47-ECD is determined by experimentation known to those skilled in the art. In some embodiments, CD47-ECD is covalently attached to a protein present on the article, including, but not limited to, a protein present on a virus-like particle. The coated article may be purified and formulated in a pharmacologically acceptable vehicle for administration to a patient. In some embodiments, the article is a VLP covalently attached to engineered CD47-ECD for formulation. In some embodiments, the VLP contains a protein or drug for delivery.

[0092] The compositions of the invention can be formulated as CD47-ECD suitable for conjugation, e.g., in kit form, reacted with a desired surface. Formulations of articles (e.g., nanoparticles, etc.) containing engineered CD47-ECD are also provided. Such formulations can be used therapeutically or prophylactically, e.g., as drug delivery. The toxicity and therapeutic efficacy of such compounds can be determined, e.g., by LD 50 (lethal dose to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of the population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 Compounds that exhibit large therapeutic indices are preferred.

[0093] The data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. Such compounds can be administered at doses that are consistent with the ED5001 with little or no toxicity. 50 It is desirable for the circulating concentration of the compound to be within a range including the range of 100-150 mg / kg / day. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.

[0094] For all such treatments above, the exact formulation, route of administration, and dosage can be selected by the individual physician in view of the patient's condition (see, for example, Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics"). It should be added that the attending physician will already know how and when to terminate, interrupt, or adjust administration due to toxicity, or organ dysfunction. Conversely, the attending physician will also already know to adjust treatment to higher levels if the clinical response is not adequate (precluding toxicity). The dosage of administration in the management of a subject's viral infection will vary with the severity of the condition being treated and with the route of administration. Dosage will also vary with the age, weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0095] The pharmacologically active compounds can be processed in accordance with conventional pharmaceutical methods to formulate medicinal agents for administration to patients, such as mammals, including humans.

[0096] Generally, the composition of the present invention preferably also includes a pharma- ceutically acceptable excipient and can be in various formulations.As is well known in the art, a pharma- ceutically acceptable excipient is a relatively inert substance that stabilizes and facilitates the administration of a pharmacologically effective substance.For example, an excipient can provide form or consistency, or act as a diluent.Suitable excipients include, but are not limited to, stabilizing agents, wetting agents and emulsifying agents, salts for various osmolarity, encapsulating agents, buffers, and skin penetration enhancers.Excipients and formulations for parenteral and non-parenteral drug delivery are described in Remington's Pharmaceutical Sciences 19th Ed.Mack Publishing (1995).

[0097] Generally, these compositions are formulated for administration by injection, e.g., intraperitoneally, intravenously, subcutaneously, intradermally, intramuscularly, or by inhalation. Thus, these compositions are combined with a pharma- ceutically acceptable vehicle, such as saline, Ringer's solution, dextrose solution, etc. The particular dosing regimen, i.e., dosage, timing, and repetition, will depend on the particular individual and that individual's medical history.

[0098] It is to be understood that the invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein.

[0100] All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing, for example, reagents, cells, constructs, and methodologies described therein that may be used in connection with the present invention. The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

[0101] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what is considered to be the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, concentrations, etc.), but some experimental error and deviation should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0102] experiment Engineering the CD47 extracellular domain for bioconjugation to the surface of hepatitis B core virus-like particles to avoid phagocyte phagocytosis Although CD47-ECD has the potential to be widely used to evade phagocytes, efficient production and purification of functional CD47-ECD using E. coli is limited. Due to disulfide bonds and natural hydrophobicity within the ECD structure, E. coli expression of CD47-ECD required the use of bulky fusion tags such as GST and extensive processing to unfold and refold the protein (see Lin et al. Protein Expr. Purif. 85, 109-116 (2012), and Han et al. J. Biol. Chem. 275, 37984-92 (2000)).

[0103] Furthermore, pyroglutamate formation at the N-terminus, which is important for binding to SIRPα, is a challenge as the N-terminal polypeptide needs to be removed to expose the glutamine residue at the N-terminus, which must be converted to pyroglutamate by glutaminyl cyclase (Figure 2, see Hatherley et al. Mol. Cell 31, 266-77 (2008)). There are several examples where CD47-ECD has been expressed and purified in various types of cells, including bacterial, mammalian (CHO) and insect (High Five) cells, but all of them had extensions at either the N- or C-terminus (see Ho et al. J. Biol. Chem. 290, 12650-63 (2015)).

[0104] Another challenge is to mimic the native CD47-ECD orientation on the cell surface to achieve ideal and authentic phagocyte evasion. As shown in "Error! Reference source not found", CD47-ECD is anchored to the cell membrane by double bonds (polypeptide and disulfide bonds) between the ECD and the cell surface.

[0105] Here, we developed a method to generate CD47-ECD variants that attach to the surface of HepBc VLPs to avoid immune clearance using E. coli-based cell-free protein synthesis (CFPS). First, an N-terminal extension was introduced to aid in the generation of pyroglutamic acid N-termini by an enzymatic reaction. Second, two unnatural amino acid (nnAA) incorporation sites were introduced so that attachment of CD47-ECD to the surface of VLPs could replicate the native double bonds between the ECD and the cell membrane. Using copper(I)-catalyzed azide-alkyne cycloaddition ("click" reaction), the two nnAAs on CD47-ECD could form two covalent bonds with two other nnAAs exposed at the tips of the surface spikes of HepBc VLPs. Next, CD47-ECD was further engineered to increase its solubility without being affected in binding to SIRPα. This engineered CD47-ECD was expressed and purified, and then attached to the surface of HepBc VLPs. CD47-ECD attachment then successfully inhibited VLP E. coli by phagocytes. Combined with recent efforts to engineer HepBc for targeted delivery of therapeutic cargo (U.S. Patent Application No. 62 / 785,866), murine CD47-ECD-functionalized VLPs significantly improve delivery efficiency and therapeutic efficacy.

[0106] material and method Construction of expression plasmids. The original sequence encoding human CD47-ECD (UniProt accession number Q08722, mature protein residues 1-117 (original amino acid positions 19-135)) was codon-optimized for E. coli expression and synthesized as "gBlocks" from IDT. The gene was cloned into the minimal vector pY71 with the restriction enzyme sites Ndel and Sall using Gibson assembly. Based on this, N-terminal extensions (His8-EKseq), mutants of unnatural amino acid incorporation (C15M, M28V, M82L / I, V116M), and mutants of improved protein solubility (F14N and V115N) were introduced using QuikChange mutagenesis and primers from IDT. The 5' untranslated region of the mRNA transcribed from the plasmid was analyzed and optimized to reduce the formulation of RNA hairpins using MFold.

[0107] The sequence encoding mouse CD47-ECD (UniProt accession number Q61735, mature protein residues 1-115 (amino acid positions 19-133)) with N-terminal extension (His8-EKseq) and mutants with unnatural amino acid incorporation (C15M, M36V, M82I, M88V, V114M) was optimized for E. coli expression and directly synthesized as "gBlocks" from IDT. The gene was cloned into the pY71 vector using the same method as above.

[0108] Cell-free protein synthesis (CFPS). CD47-ECD variants were expressed using the PANОx-SP (PEP, amino acids, nicotinamide adenine dinucleotide (NAD), oxalate, spermidine, and putrescine) CFPS system to generate disulfide-linked proteins as previously described (Lu et al. Proc. Natl. Acad. Sci. 201510533 (2015)) with some modifications. First, cell extracts were pretreated with 0.5 mM iodoacetamide for 30 min at room temperature. Then, the CFPS reaction mixture was supplemented with 4 mM oxidized glutathione (GSSG, AppliChem) and 1 mM reduced glutathione (GSH) to stabilize the thiol / disulfide redox potential. Also, a disulfide bond isomerase, E. coli DsbC, was added. Finally, to improve protein solubility, the potassium glutamate concentration was reduced to 50 mM from the original 175 mM. The final reaction mixture contained 10 mM magnesium glutamate, 10 mM ammonium glutamate, 50 mM potassium glutamate, 4 mM GSSG, 1 mM GSH, 100 μg / mL DsbC, 1.25 mM ATP, 1 mM GTP, 1 mM UTP, 1 mM CTP, 34 μg / mL folinic acid, 170.6 μg / mL tRNA (Roche Molecular Biochemicals), 2 mM each of the 18 amino acids (all except Met and Glu), 33.3 mM phosphoenolpyruvate (PEP, Roche Molecular Biochemicals), and 1 mM phosphate buffer (PBS). The incubation time consisted of 10 min at 37 °C for 24 h at 4 °C (Biochemicals), 0.33 mM NAD, 0.27 mM coenzyme A (CoA), 2.7 mM potassium oxalate, 1 mM putrescine, 1.5 mM spermidine, 2 mM methionine, 6 µM plasmid DNA encoding the protein of interest under a T7 promoter, approximately 100-300 µg / mL purified T7 RNA polymerase, and 0.25 volumes of pretreated KC6 S30 extract (prepared from A19met+, ΔtоnA, ΔtnaA, ΔspeA, ΔendA, ΔsdaA, ΔsdaB, ΔgshA E. coli cells). All reagents were obtained from Sigma-Aldrich unless otherwise stated.To quantify protein production, 2–10 μM L-[U- 14 For global methionine replacement to incorporate nnAAs, methionine was released from the CFPS reactant mixture and 2 mM azidohomoalanine (AHA, Medchem Source LLP) or 2 mM homopropargylglycine (HPG, Chiralix) was added instead.

[0109] Reactions were performed at multiple scales: 15-50 μL volumes in 2 mL Eppendorf tubes, 500 μL volumes in sealed 6-well culture plates, or 5 mL volumes in sealed 10 cm Petri dishes. Reactions were incubated at 30 °C or room temperature for 16 h.

[0110] Analysis of the concentration and solubility of the synthesized proteins. Protein concentration was determined by measuring the incorporated radioactivity using a Beckman LS6000 liquid scintillation counter. Total expressed protein concentration was measured directly, and soluble protein concentration was measured after removing the insoluble fraction using centrifugation at 10,000 × g for 15 min. Unincorporated proteins were removed by permeabilization three times with 5% trichloroacetic acid (TCA). 14 Both samples (3–5 μL) were spotted and precipitated onto Whatman filter paper before removal of C-leucine.

[0111] The soluble protein produced may precipitate from the supernatant after centrifugation. 14 C - defined as the percentage of the total amount of labeled protein (soluble protein).

[0112]

number

[0113] Purification of CD47-ECD with N-terminal extension using immobilized metal affinity chromatography (IMAC). CD47-ECD fusion protein (His8-EKseq-CD47-ECD) was expressed using CFPS as described above. The purification method first used a Sephadex G-25 PD-10 desalting column (GE Healthcare Life Science) to exchange the CFPS-reacted polymer into 50 mM Tris-HCI pH 7.4, 25 mM imidazole, 0.01% Tween-20. Next, centrifugation at 10,000 × g for 15 min was used to remove insoluble material before the insoluble fraction (supernatant) was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen), equilibrated with 50 mM Tris-HCI pH 7.4, 25 mM imidazole, 0.01% Tween-20. The sample was washed with 7 column volumes (CV) of the same buffer and eluted with 5 CV of 50 mM Tris-HCI pH 7.4, 250 mM imidazole, 0.01% Tween-20, collecting 0.5 CV fractions. Fractions containing the CD47-ECD fusion protein were then pooled and concentrated using Amicon® Ultra Centrifugal Filters (10 kDa molecular weight cutoff) (Millipore).

[0114] Preparation of CD47-ECD containing pyroglutamic acid at the N-terminus. The purified CD47-ECD fusion protein (His8-EKseq-CD47-ECD) was first loaded onto a Sephadex G-25 PD-10 desalting column (GE Healthcare Life Science) to exchange the protein into 50 mM Tris-HCI pH 7.4, 50 mM NaCI, 1 mM CaCI2, 0.01% Tween-20. The protein was then digested using human His-tagged enterokinase cleavage enzyme (Applied Biological Materials) for 20–23 h at 4 °C. The uncleaved fusion protein (His8-EKseq-CD47-ECD), the cleaved N-terminal tag (His8-EKseq), and the His-tagged enterokinase were then removed by Ni-NTA IMAC as described above. Mature CD47-ECD protein that did not bind to Ni-NTA resin (Qiagen) was collected from the flow-through and wash fractions and loaded onto a Sephadex G-25 PD-10 desalting column (GE Healthcare Life Science) for exchange into 50 mM Tris-HCl, 0.01% Tween-20. Finally, protein samples were concentrated using Amicon® Ultra Centrifugal Filters (10 kDa molecular weight cut-off) (Millipore) and treated with glutaminyl cyclase (QC, Novus biologicals) for 1–3 h at room temperature to convert N-terminal glutamine residues to proglutamic acid.

[0115] Detection of pyroglutamate formation. The conversion efficiency of the N-terminal glutamine residue of CD47-ECD to proglutamate was examined using a spectrophotometric continuous assay established by Schilling et al. Briefly, 50 μM (1Q)CD47-ECD protein was mixed with 150 μM NADH (Sigma), 7 mM α-ketoglutarate (Sigma), 15 U / mL glutamate dehydrogenase (GLDH, Sigma), and QC enzyme in 50 mM Tris-HCI pH 7.4 buffer. The decrease in NADH concentration, reflecting pyroglutamate formation, was continuously detected by measuring the absorbance at 340 nm using a SpectraMax® iD3 multimode microplate reader (Molecular Devices).

[0116] Generation of HepBc VLPs. Previously developed HepBc variants for loading small molecules (HepBc HP 2ASVins SS1 SS8 79M(IG)2IC-His6 and HepBc HP 2ASVins SS1 SS8 80M (IG)2IC-His6) were used in this study. HepBc protein containing AHA instead of methionine was expressed using CFPS, and VLPs displaying azide functional groups were generated as previously described (US Patent Application 62 / 788,558). Empty VLPs were used to assess CD47-ECD attachment on the surface of the VLPs, and VLPs loaded with Bodipy FL cysteine ​​(BDFL) were used for macrophage evasion assays.

[0117] Copper(I)-catalyzed azide-alkyne cycloaddition ("click" chemistry). For the "click" reaction attachment, the CD47-ECD protein was expressed with HPG (containing an alkyne functional group) and the protein of the HepBc mutant was expressed with AHA (containing an azide functional group) instead of methionine, using CFPS. The CD47-ECD protein was conjugated to the surface of HepBc VLPs based on a previously developed protocol (Patel & Swartz Biocojug Chem. 22, 376-387 (2011)). The reaction was carried out in an anaerobic glovebox (Coy Laboratories) to maintain reducing conditions for the tetrakis(acetonitrile)copper(I) hexafluorophosphate catalyst (Sigma Aldrich). 41.67 nM HepBc VLPs (monomer protein concentration was 10 μM) were combined with 1.5–20 μM CD47-ECD, 0.5 mM tris(triazolylmethyl)amine (TTMA), 0.01% Tween-20, and 2 mM tetrakis catalyst in 50 mM phosphate buffer pH 8 and reacted anaerobically for 16 h.

[0118] Analysis of "click" chemistry bioconjugation using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Nonradioactive CD47-ECD and radiolabeled HepBc VLPs were used for bioconjugation analysis, and the size of the bioconjugate products was analyzed by SDS-PAGE and autoradiography. NuPAGE Novex precast gels and reagents were purchased from Invitrоgen. To reduce SDS-PAGE, samples were mixed with LDS running buffer and 50 mM dithiothreitol (DTT) and denatured at 75 °C for 10 min. Samples were loaded onto a 12% (w / v) Bis-Tris precast gel with a separate lane for Mark12 molecular weight protein standards (Thermo Fisher Scientific) and electrophoresed in MES / SDS running buffer. SimplyBlue SafeStain (Thermo Fisher Scientific) was used to stain and fix the gels according to the manufacturer's recommendations. The gel was dried using a gel dryer model 583 (Bio-Rad) before being exposed to a storage phosphor screen (Molecular Dynamics) and then scanned for autoradiography using a Typhoon Scanner (GE Healthcare). The degraded and reduced HepBc monomer protein bands and the conjugated product bands (HepBc monomer + CD47-ECD and two HepBc monomer + CD47-ECD) were visible in the autoradiography. Based on the density of the control and conjugated bands, the conjugation efficiency was estimated by densitometry using ImageJ software.

[0119] CD47-ECD-functionalized VLP purification using size-exclusion chromatography (SEC). After the "click" reaction, the copper(I) catalyst and unconjugated ligand were removed by SEC. CD47-ECD-functionalized HepBc VLPs were purified by applying 200 μL of the reaction solution to 2.2 mL of Sepharose6 Fast Flоw resin (GE Healthcare) and eluting 24 × 150 μL fractions. PBS containing 0.01% Tween-20 was used to equilibrate the SEC column and elute the VLPs. The fractions of the VLP peak (usually between fractions 5 and 8) were detected by measuring the fluorescence intensity of the loaded fluorescent dye BDFL. Finally, the VLP fractions were pooled and concentrated using Amicon® Ultra Centrifugal Filters (30 kDa molecular weight cutoff) (Millipore).

[0120] Macrophage evasion assay. The ability of the indicated engineered mouse CD47-ECD mutants to evade VLP phagocytosis was examined using mouse macrophage-like RAW264.7 cells constitutively expressing H2B fused with miRFP670 (obtained from Markus Covert Lab) to provide nuclear labeling. Two days prior to imaging, these RAW264.7 cells were seeded at 7,500 cells / well in glass-bottom 96-well tissue culture plates precoated with 10 μg / mL human fibronectin (FC010, Millpore). Then, one day before imaging, cells were stimulated for 20 h with 100 ng / mL lipopolysaccharide (LPS, Escherichia coli, serotype EH100 (Ra), Enzo Life Sciences) and 20 ng / mL mouse interferon gamma (IFNγ, PeproTech) in 100 μL DMEM (Life Technologies) supplemented with 10% FBS (Omega Scientific), 2 mM L-glutamine (Life Technologies), and 1x penicillin / streptomycin (P / S, Life Technologies) to allow them to differentiate for enhanced phagocytosis. On the day of imaging, cells were incubated for 2 h at 37 °C with 0.05 nM (final concentration) BDFL-loaded VLPs to allow phagocytosis, followed by washing cells three times with PBS containing 0.1% Tween-20 to remove free VLPs. Finally, the medium was changed to FluоrоBrite DMEM (Life Technologies) supplemented with 1% FBS and 2 mM L-glutamine for imaging. A Nikon Eclipse Ti-E inverted microscope equipped with an Andor Neo 5.5s CMOS camera was used for fluorescence imaging to detect phagocytosed VLPs and BDFL dye molecules in RAW264.7 cells. The far-red channel (645 / 30 nm excitation filter and 705 / 72 nm emission filter) and the FITC channel (490 / 20 nm excitation filter and 525 / 36 nm emission filter) were used to detect cell nuclei and BDFL dye, respectively.

[0121] Results and Discussion Cell-free expression of N-terminally extended human CD47 extracellular domain. First, we engineered an N-terminally extended human CD47-ECD that allows the generation of CD47-ECD with a glutamine N-terminus that can be converted to an authentic pyroglutamic acid terminus, which is important for SIRPα recognition. At the N-terminus, an octahistidine tag (His×8) for purification was introduced, followed by a short Gly Ser Ala linker and an enterokinase recognition sequence (Asp Asp Asp Asp Lys) (Figure 3). After purification of this fusion protein (His8-EKseq-hCD47-ECD), the extension is removed immediately after the recognition sequence using enterokinase. This exposes an N-terminal glutamine residue that can be converted to pyroglutamic acid using glutaminyl cyclase.

[0122] We then introduced several mutations into human CD47-ECD to enable nnAA incorporation using CFPS and global methionine substitution. Using endogenous methionyl-tRNA synthase, methionine analogs are incorporated, which allows for "click" conjugation to the surface of VLPs. First, we replaced two natural methionine residues (M28 and M82) that have no recognition interface between CD47 and SIRPα (Figure 4A). Based on a CD47 conservation sequence search, M28 was replaced with valine (Figure 4C). Meanwhile, M82 was conserved in most of the available CD47 amino acid sequences, so several different mutants were created (e.g., M82L and M82I). Next, we introduced two nnAA incorporation sites (ATG codons) at C15 and V116, which are the dual binding sites that anchor the ECD to the cell membrane (Figure 4A). We hypothesized that using "click" conjugation, the double bond could incorporate two other nnAAs at the protruding spike tip of the HepBc dimeric subunit and replicate on the surface of the HepBc VLP to achieve authentic CD47-SIRPα binding (Figure 4B). In addition to these mutants, two hydrophobic residues on the surface of CD47-ECD (or the surface of the VLP) facing the cell membrane were replaced with asparagine residues, the most hydrophilic uncharged amino acid (F14N and V115N), to improve the solubility of the protein (Figure 4C).

[0123] We then used these mutants to test the cell-free protein expression levels and solubility using CFPS with nnAAs containing methionine, alkyne functional groups (homopropargylglycine, HPG), or azide functional groups (azidohomoalanine, AHA) (Figure 6A, 6B). Compared with the mutant with only the C15G variant (WT G15G), which was introduced to avoid protein aggregation due to incorrect disulfide bond formation, the accumulation level of soluble protein was reduced in the mutants designed to avoid undesired nnAA engagement (nnAA(L) and nnAA(I)). However, in the case of engineered CD47-ECD with hydrophilic asparagine mutations (F14N+V115N, mutants were designated as nnAA(L)NN and nnAA(I)NN), the accumulation of soluble protein was restored to the level of WTC15G, and the relative solubility increased from approximately 70% to >90% (Figure 6B). Those hydrophilic mutants were used for further studies.

[0124] Purification of human CD47-ECD. The engineered human CD47-ECD with the N-terminal extensions and mutants described above was then used to purify and cleave the initial expression product to generate human CD47-ECD with an N-terminal glutamine. Samples at each stage of purification were evaluated by SDS-PAGE (Figure 7A). The fusion protein was expressed using CFPS, purified using Ni-NTA IMAC, and cleaved with enterokinase. During this protease reaction, several non-specific cleavage products were observed that were presumably cleaved near 83D and 84K due to sequence similarity to the enterokinase recognition sequence (DDDDK). The level of this undesired cleavage varied based on the mutant at position 82. The nnAA(I)NN mutant underwent less non-specific cleavage than the nnAA(L)NN mutant, and therefore the nnAA(I)NN mutant was selected for further study. Using Ni-NTA IMAC again, enterokinase, the cleaved N-terminal tag (His8-EKseq), and the uncleaved fusion protein (His8-EKseq-hCD47-ECD) were all removed, leaving human CD47-ECD nnAA(I)NN with an N-terminal glutamine ((1Q)hCD47-ECD). We then tested the conversion of glutamine to pyroglutamate by QC. Using the spectrophotometric continuous assay established by Schilling et al., we showed up to 80% pyroglutamate formation (Figure 7B).

[0125] Generation of mouse CD47-ECD. Next, based on our experience with human CD47-ECD, we generated a mouse version of CD47-ECD with the same mutations for future mouse studies, taking into account the fact that CD47-SIRPα interaction is species-restricted. As shown in the figure, the original three methionine residues were replaced based on sequence conservation (M36V, M82I, M88V) (Figure 8). The hydrophilic mutations used in human CD47-ECD (F14N and V115N) were not introduced into mouse CD47-ECD because there were no hydrophobic residues at these positions (the corresponding amino acids are S14 and T113). We then tested protein expression using CFPS, and the N-terminally extended mouse CD47-ECD accumulated to the same concentration as the human version with approximately 80% of the protein solubility of CD47-ECD incorporating met, HPG, and AHA (Figure 9A, 9B). Mouse CD47-ECD with an N-terminal pyroglutamic acid was purified and produced using the same methods described above for the human version.

[0126] Evaluation of mouse CD47-ECD attachment to the surface of HepBc VLPs. We next tested the bioconjugation of purified mouse CD47-ECD to the surface of HepBc VLPs using "click" chemistry. An nnAA displaying an alkyne functional group (HPG) and an nnAA displaying an azide functional group (AHA) were introduced into HepBc and mouse CD47-ECD, respectively. Several nnAA incorporation sites at the tip of the HepBc spike region shown in Figure 10C were tested. For mouse CD47-ECD attachment, positions 79 and 80 were tested, D78 was avoided to stabilize the HepBc helical structure as a capping residue, and L76 was not selected because it was not expected to achieve the designed double bond with CD47-ECD due to the relatively large distance between the two L76 residues in each dimer. Bioconjugation was performed using various concentrations of mouse CD47-ECD with radiolabeled HepBc VLPs (79nnAA VLPs and 80nnAA VLPs) in the presence of Cu(I) and then analyzed by reducing SDS-PAGE and autoradiography (Figure 10A). For both types of VLPs, in addition to the HepBc monomer band, two bands were observed indicating conjugation products; one for CD47-ECD conjugated to one HepBc monomer (single conjugation) and another for CD47-ECD conjugated to two HepBc monomers (one dimer) (double conjugation). Figure B shows the number of attached CD47-ECD per VLP based on gel densitometry. Because the incompletely reduced HepBc dimer band and the single linkage band were partially merged, the HepBc dimer band density was subtracted from the merged band density to calculate the single linkage band density. Although there were some single linkage conjugates, these results suggested that more than 10 mouse CD47-ECDs were bound to the surface of HepBc VLPs by double bonds, similar to bona fide anchoring to the cell membrane.Because the 80nnAA VLP had a slightly higher ratio of double linkages over single linkages compared to the 79nnAA VLP, the 80nnAA VLP was used for further studies.

[0127] Surface modification with CD47-ECD reduces VLP uptake by macrophage-like cells. Based on the bioconjugation analysis, the majority of attached mouse CD47-ECD was expected to display the SIRPα-binding side of the VLP in the authentic orientation. Therefore, we used mouse macrophage-like RAW264.7 cells to test their function for evading phagocytosis. RAW264.7 cells were first stimulated with LPS and IFNγ to allow differentiation for more active phagocytosis, and then incubated for 2 h at 37 °C with fluorescent dye BDFL-loaded VLPs previously generated with or without mouse CD47-ECD on the surface. The medium was then removed and the cells were washed three times before being imaged under a fluorescent microscope. As shown in the figure, the uptake of mouse CD47-ECD-functionalized and BDFL-loaded VLPs by stimulated RAW264.7 cells was significantly reduced compared to the same VLPs without CD47-EDC. This indicates that the engineered CD47-ECD retains the function of inhibiting phagocytosis after attachment to VLPs.

[0128] In this work, we successfully engineered CD47-ECD for bioconjugation to HepBc VLPs and demonstrated its ability to circumvent phagocytosis by macrophage-like cells. Notably, our design to replicate the dual bond that endogenously orients CD47-ECD relative to the cell membrane surface can provide an authentic interaction with SIRPα and avoid immunogenicity. CD47-ECD may also benefit other types of NPs that display pairs of reactive functional groups on their surface in suitable proximity for "click" bioconjugation of CD47-ECD.

[0129] The CD47-ECD function can be combined with specific targeting functions for effective targeted delivery using NPs. Examples of targeting ligands are single chain variable fragments (scFv) and DNA aptamers that recognize specific cell surface markers of target cells. The one-pot "click" conjugation method is beneficial for easily creating NPs with different densities of each ligand by changing the relative ligand concentrations in the reaction.

[0130] The benefit is that even when the number of CD47-ECDs attached to the surface of the NPs is relatively low (Figure 10B), there remain approximately 100 surface spikes available for subsequent or simultaneous attachment of cell-targeting agents, thus greatly reducing phagocytic clearance but not NP uptake by target cells.

[0131] The table below shows the protein and nucleotide sequences used in the examples. Sites of incorporation of unnatural amino acids (nnAA) are shown as "Z" in the primary sequence of the protein, mutation sites substituting the original methionine residues are shown, asparagine hydrophilic mutation sites. Regions with a significant effect on SIRPα binding are underlined, and N-terminal extensions are double underlined.

[0132] [Table 1-1]

[0133] [Table 1-2]

[0134] [Table 1-3]

[0135] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 813,510, filed March 4, 2019, which is incorporated herein by reference in its entirety.

Claims

1. 1. An engineered CD47 extracellular domain polypeptide (ECD), comprising: (i) an N-terminal extension that includes a cleavage recognition site immediately adjacent to a position corresponding to residue Q1 in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein the N-terminal extension is cleaved, thereby exposing an N-terminal glutamine that is converted to pyroglutamic acid; and (ii) a pair of unnatural amino acids (nnAA) that provide a reactant for a bioorthogonal reaction at a site on CD47-ECD for attachment to a surface via a bioorthogonal reaction, optionally produced by cell-free protein synthesis, where one of the nnAAs is present at a position corresponding to residue C15 in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2; An engineered CD47-ECD comprising:

2. The engineered CD47-ECD of claim 1, wherein the nnAA provides a reactant for azide present on the surface of the article.

3. 3. The engineered CD47-ECD of claim 1 or 2, wherein the N-terminal extension comprises a cleavage recognition sequence for enterokinase.

4. 3. The engineered CD47-ECD of claim 1 or 2, wherein the N-terminal extension comprises a cleavage recognition sequence for Factor Xa.

5. 3. The engineered CD47-ECD of claim 1 or 2, wherein the N-terminal extension comprises a polypeptide tag for purification.

6. 6. The engineered CD47-ECD of any one of claims 1 to 5, comprising at least one amino acid substitution to replace a hydrophobic residue on the surface of CD47-ECD that faces the cell membrane in the native protein with a hydrophilic residue.

7. The engineered CD47-ECD of claim 6, wherein residues at positions corresponding to residues F14 and V115 in the amino acid sequence of SEQ ID NO:1 have been replaced with hydrophilic, uncharged amino acids.

8. 8. The engineered CD47-ECD of claim 7, which comprises one or both of the amino acid substitutions F14N and V115N.

9. 9. The engineered CD47-ECD of any one of claims 1 to 8, produced by cell-free protein synthesis.

10. An article having an engineered CD47-ECD of any one of claims 1 to 9 bound to its surface through reaction of an azide with a nnAA.

11. The article of claim 10 , wherein the article is a nanoparticle.

12. The article of claim 11 , wherein the nanoparticle is a virus-like particle.

13. The article of claim 12, wherein the virus-like particle comprises Hepatitis B core protein conjugated to CD47-ECD.

14. The article of claim 13 comprising a therapeutic agent.

15. The article of any one of claims 11 to 14, wherein phagocytic clearance of the article is reduced by at least 10% compared to the article in the absence of CD47-ECD.

16. A method of coating an article with an engineered CD47-ECD according to any one of claims 1 to 9, comprising the steps of: reacting an azide group on the surface of the article with a nnAA present on CD47-ECD; A method in which an array of reactive azide groups is provided on a surface, nanoparticles displaying conjugated azide groups are attached first, and engineered CD47-ECD is attached to the nanoparticles.

17. The method of claim 16, wherein the reactive alkyne or azide groups are about 5 to about 15 Å apart.

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