ACE2-Fc Trap

Abstract

An ACE2-Fc hybrid construct is used as a therapeutic and/or analytic entity to treat an individual infected with a coronavirus or to detect a coronavirus in an analyte. In selected embodiments, the Fc portion of the hybrid construct is an IgA Fc portion, and in still further embodiments the ACE2 portion has a mutation that reduces or abolishes ACE2 catalytic activity.

Claims

1. A soluble ACE2-Fc hybrid construct, having an amino acid sequence at least 85% identical to SEQ ID NO:7 or SEQ ID NO:8.

2. The soluble hybrid construct of claim 1, wherein a C-terminus of the ACE2 portion is coupled to an N-terminus of the immunoglobulin IgA Fc portion, and wherein: a) the ACE2 portion has at least 85% sequence identity to SEQ ID NO:9; and/or b) the immunoglobulin IgA Fc portion has at least 85% sequence identity to SEQ ID NO:10 or 11.

3. The soluble hybrid construct of claim 1, wherein the ACE2 portion is catalytically inactive.

4. The soluble hybrid construct of claim 1, further comprising a J-chain portion.

5. The soluble hybrid construct of claim 1, having an amino acid sequence at least 95% identity to SEQ ID NO:7 or SEQ ID NO:8.

6. The soluble hybrid construct of claim 1, further comprising a detectable label coupled to the hybrid construct.

7. The soluble hybrid construct of claim 1, formulated in a pharmaceutically acceptable carrier.

8. The soluble hybrid construct of claim 7, wherein the pharmaceutically acceptable carrier is formulated for inhalation, nasal administration, or injection.

9. A recombinant nucleic acid encoding the ACE2-Fc hybrid construct of claim 1.

10. The recombinant nucleic acid of claim 9, wherein the nucleic acid is an RNA.

11. The recombinant nucleic acid of claim 9, wherein the nucleic acid is a DNA.

12. The recombinant nucleic acid of claim 11, wherein the recombinant nucleic acid is an expression vector.

13. The recombinant4 nucleic acid of claim 12, having a nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

14. A method of treating a coronavirus infection in an individual in need thereof, the method comprising: administering to the individual a therapeutically effective amount of an ACE2-Fc hybrid construct of claim 1.

15. The method of claim 14, wherein the coronavirus is SARS-CoV-2.

16. The method of claim 14, wherein the administering comprises nasal or pulmonary administration or intravenous injection.

17. A method of detecting a coronavirus, the method comprising: adding a test sample to a surface to which an ACE2-Fc hybrid construct is coupled, to thereby bind coronaviral Spike protein to the ACE2-Fc hybrid construct; contacting the Spike protein that is bound to the ACE2-Fc hybrid construct with a detectable binder; and detecting the detectable binder.

18. The method of claim 17, wherein the ACE2-Fc hybrid construct is coupled to the test surface via a biotin group that is coupled to the ACE2-Fc hybrid construct.

19. The method of claim 17 or claim 18, wherein the detectable binder is an ACE2-Fc hybrid construct that is coupled to a detectable label.

20. The method of any one of claims 17-19, wherein the detectable binder includes an electrochemiluminescent moiety.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS. 1A-1C are exemplary schematic illustrations of ACE2-IgG.sub.1Fc constructs.

[0021] FIGS. 2A-2B are exemplary schematic illustrations of ACE2-IgAFcJchain constructs.

[0022] FIG. 3 depicts exemplary results for ACE2-IgGiFc small scale production with Maxcyte.

[0023] FIG. 4 depicts exemplary results demonstrating that ACE2-IgAFc expression is as efficient as ACE2-IgG1Fc.

[0024] FIG. 5 is a schematic representation of an exemplary purification approach for ACE2-IgA constructs.

[0025] FIG. 6 depicts exemplary results for CaptureSelect IgA column purification.

[0026] FIG. 7 depicts exemplary results for anion exchange column purification.

[0027] FIGS. 8A-8C depict exemplary results demonstrating that Fc avidity improves binding affinity of ACE2 dimer against 2019-nCoV Spike.

[0028] FIG. 9 schematically depicts an exemplary Spike detection assay.

[0029] FIG. 10 is a graph showing exemplary results for the assay of FIG. 9.

DETAILED DESCRIPTION

[0030] Recited ranges of values herein are merely intended as a shorthand referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0031] As used herein, “administering” a pharmaceutical composition or drug refers to both direct and indirect administration of the pharmaceutical composition or drug. “Direct administration” is typically performed by a health care professional (e.g., physician, nurse, etc.). “Indirect administration” includes a step of providing or making available the pharmaceutical composition or drug to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.). “Prognosing” or “predicting” a condition, a susceptibility for development of a disease, or a response to an intended treatment covers the prediction (but not treatment or diagnosis of) the condition, susceptibility and/or response, including the rate of progression, improvement, and/or duration of the condition in a subject.

[0032] As used in the description herein and throughout the claims that follow, “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Also, “in” includes “in” and “on” unless the context clearly dictates otherwise. As also used herein, and unless the context dictates otherwise, “coupled to” includes both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, “coupled to” and “coupled with” are synonymous.

[0033] “Comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification or claims refer to at least one of something selected from the group consisting of A, B, C, . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

[0034] Various ACE2-Fc hybrid constructs can be prepared and employed therapeutically and/or diagnostically. In some embodiments, soluble ACE2-Fc hybrid constructs as presented herein trap coronavirus when the ACE2 portion binds to the coronavirus Spike protein. When used in conjunction with an ACE2-Fc hybrid construct, “soluble” means that the ACE2-Fc hybrid construct is not bound to a cell membrane via a transmembrane domain. Soluble ACE2-Fc hybrid constructs can be administered by injection or inhalation without being bound to a cell membrane. In further embodiments, the constructs as described herein act as decoy receptors to reduce or even eliminate viral entry.

[0035] In other embodiments, the ACE2-Fc hybrid constructs as described herein can be modified with an affinity portion and/or a detectable portion to facilitate use in assays to detect or quantify SARS-CoV-2 in a biological fluid (e.g., in a sandwich ELISA).

[0036] Most typically, the ACE2-Fc hybrid constructs comprise an ACE2 portion coupled to an immunoglobulin Fc portion in a single polypeptide chain. As detailed below, the coupling is preferably covalent, such that the ACE2 C-terminus joins to an immunoglobulin Fc N-terminus, optionally via a flexible linker. The ACE2 portion and the immunoglobulin Fc portion may be of any origin. However, especially preferred ACE2 portions and immunoglobulin Fc portions will be human portions—i.e., sequences having at least 85% sequence identity to any one of SEQ ID NOs:9-11. In certain embodiments the ACE2 portion will have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to SEQ ID NO:9. Additionally or alternatively, in certain embodiments the Fc portion will have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to SEQ ID NO:10, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity to SEQ ID NO:11.

[0037] The ACE2 portion may also be catalytically active. Alternatively, the ACE2 portion may carry one or more mutations to reduce or even entirely abolish ACE2 catalytic activity. It is generally preferred that such mutants not affect ACE2 binding to the coronaviral Spike protein. Non-limiting examples of coronaviruses include MERS-CoV, SARS-CoV, SARS-CoV-2, and NL63/HCoV-NL63. Suitable ACE2 portions include those from human, mouse, rat, bovine, or yeast (S. cerevisiae) described. For example, contemplated ACE2 portions are found under UniProtKB identifier Q9BYF1 (human), Q8R0I0 (mouse), Q5EGZ1 (rat), Q58DD0 (bovine), A0A2J8KU96 (chimpanzee), P21192 (S. cerevisiae), Q56H28 (cat), etc. Most preferably, however, the ACE2 sequence will be a human sequence in any isoform (e.g., isoform 1 or 2).

[0038] Additionally or alternatively, the ACE2 portion may be full length portion or truncated. Where a truncated portion is used, the ACE2 sequence is preferably truncated on the C-terminus. The modified forms will be changed to preserve at least three domains known to interact with the SARS-CoV Spike glycoprotein. Likewise, ACE2 portions preferably include a leader peptide to allow secretion from a recombinant cell producing ACE2-Fc constructs. Thus, where a truncation is present on the C terminus, the truncation may remove the sequence motif needed for cleavage by ADAM17 and/or the sequence motif needed for cleavage by TMPRSS11D and/or TMPRSS2.

[0039] In further preferred aspects, the ACE2 portion will have one or more inactivating mutations that reduce or abolish ACE2 proteolytic activity while maintaining binding to Spike. Various mutations are known in the art that reduce or abolish ACE2 activity, and all of these are suitable. For example, suitable mutations reduce ACE2 catalytic activity by at least 20%, or by at least 30%, or by at least 40%, or by at least 50%, or by at least 60%, or by at least 70%, or by at least 80%, or by at least 90%, or by at least 90%, or even more. In one preferred embodiment, the ACE2 mutation is R273Q, which substantially reduces catalytic activity but retained binding capacity to Spike.

[0040] The immunoglobulin Fc portion is an IgG or IgA Fc portion, preferably with cysteine amino acids in place to dimerize in vivo and in vitro. An IgA Fc portion may further comprise a J-chain portion coupling it to rest of the hybrid construct. An IgG Fc portion enhances serum half-life and, in some cases, even binding avidity. On the other hand, an IgA Fc will preferentially collocate with mucous membrane. As such, the ACE2-Fc hybrid construct may be administered via injection/infusion, or inhaled (e.g., as nasal spray or inhaled composition).

[0041] All known IgG and IgA sequences are suitable. Particularly preferred IgG and IgA Fc sequences are human, or mammalian (e.g., SEQ ID NOs:10 or 11). Most typically the sequences will retain amino acids required for N-glycosylation in a host cell (e.g., CHO cell EC7 cell, etc.). Therefore, suitable Fc portions include at least the second and third constant portions of the heavy chain. Where desired, the IgA Fc portion may further include a J-chain portion to associate two IgA Fcs. Most typically, the J chain will be derived from human or other mammal. An exemplary J chain sequence can be found at UniProtKB entry P01591.

[0042] Additionally, the ACE2 portion and the IgA or IgG Fc portions may be directly coupled to each other. Alternatively, they may be coupled via a flexible peptide linker. Typically, such linkers will have between 5 and 25 amino acids and all known flexible linkers are deemed appropriate for use herein. Particularly suitable linkers include a run of glycines interspersed with serines (e.g., GGGS or SEQ ID NO:12).

[0043] In yet further embodiments, the ACE2-Fc hybrid construct is immobilized on a carrier. All manners of modifications to permit immobilization are suitable, e.g., biotinylation, cellulose binding domain, etc. A detectable label may also be added to the ACE2-Fc hybrid construct to enable in situ detection and/or quantification in a quantitative assay. Suitable labels include luminescent labels, radioisotope labels, enzymatic labels, etc.

[0044] ACE2-Fc hybrid constructs can be prepared in numerous manners. Sequences for the ACE2 portion and the Fc portions are well known in the art, so the hybrid constructs can be expressed from recombinant nucleic acids. Most typically, such recombinant nucleic acids may be mRNA for transfection into producer cells or may be DNA expression vectors (typically mammalian expression vector). Among other suitable configurations, expression vectors include those having SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. Recombinant cells may comprise the recombinant nucleic acids or expression vectors described herein encoding ACE2-Fc hybrid constructs.

[0045] Once prepared, therapeutically effective amounts of the ACE2-Fc hybrid construct may be administered to an individual in need thereof via inhalation or injection, or other suitable route, alone or in combination with other therapeutic agents. Alternatively, modified ACE2-Fc hybrid constructs may also be used in a diagnostic qualitative or quantitative assay as described in more detail below.

Examples

[0046] Selected exemplary constructs presented herein were expressed in CHO—S using Maxcyte electroporation. 3.2×10.sup.8 cells were electroporated in Maxcyte electroporation buffer and cultured in CD Opti CHO media with CD CHO efficient feed for 14 days at 32° C. and 3% CO.sub.2. FIGS. 1A-1B depict exemplary IgG1 containing constructs. FIG. 1A shows an ACE2-IgGiFc construct encoded by expression plasmid pWH184Lig93 (SEQ ID NO:1). FIG. 1B shows an ACE2.sub.R273Q-IgG.sub.1Fc construct (with an ACE2 inactivating mutation), encoded by expression plasmid pWH185Lig94 (SEQ ID NO:2). FIG. 1C depicts a biotinylated version of FIG. 1A for diagnostic tests. Similarly, FIG. 2A shows an ACE2IgAFc construct with a J-chain, encoded by expression plasmid pWH190 2B-2 (SEQ ID NO:3). FIG. 2B shows an ACE2.sub.R273Q-IgAFc construct with a J-chain encoded by expression plasmid pWH1913B-1 (SEQ ID NO:4).

[0047] ACE2-Fc hybrid constructs were isolated following known isolation procedures. The ACE2-Fc IgG Hybrid Protein had an amino acid sequence of SEQ ID NO:5, while the ACE2-Fc IgG R273Q Hybrid Protein (lacking ACE2 activity) had an amino acid sequence SEQ ID NO:6. The ACE2-Fc IgA Hybrid Protein had an amino acid sequence of SEQ ID NO:7, while the ACE2-Fc IgA R273Q Hybrid Protein (lacking ACE2 activity) had an amino acid sequence SEQ ID NO:8.

[0048] Yield and relative purity for a small-scale Maxcyte production of IgG-Fc hybrid constructs were in a desirable range (FIG. 3). Table 1 provides numerical results for the size exclusion chromatography.

TABLE-US-00001 TABLE 1 Production Culture % Main on ID Volume (mL) Yield SEC-HPLC ACE2-IgG1Fc 30 5.2 90.9 ACE2(R273Q)-IgG1Fc 30 8.4 91.7 ACE2-IgG1Fc-Avi-tag 40 6.2 94.5

[0049] ACE2-IgAFc expression was as efficient as the expression of ACE2-IgGiFc, with no apparent differences in the mutated form (R273Q) versus non-mutated form (FIG. 4). Traditional purification processes for IgG are often not effective for IgA, so the hybrid constructs were isolated using ion exchange chromatographic media and affinity media selective for IgA. FIG. 5 depicts schematically various purification options for the recombinant polypeptides. FIG. 6 shows exemplary results for the CaptureSelect IgA column purification, while FIG. 7 shows exemplary results for anion exchange column purification. FIGS. 6 & 7 show that the Fc IgA hybrid constructs could be isolated in a meaningful quantity and at reasonable purity.

[0050] The purified ACE2-Fc hybrid constructs were then tested for binding capacity and avidity against 2019-n-CoV Spike protein. FIGS. 8A-8C depict selected results for binding. SPR demonstrated improved binding affinity of ACE2 dimer against 2019-nCoV Spike. Table 2 lists exemplary test results in numerical format.

TABLE-US-00002 TABLE 2 KD Method Ligand Analyte k.sub.on (1/Ms) k.sub.off (1/s) (nM) Octet-SA sensor Spike-RBD ACE2-IgG1Fc 1.03E+3005 7.89E-05 0.76 Octet-SA sensor Spike-RBD ACE2 (R273Q)-IgG1Fc 1.04E+3005 9.86E-05 0.95 Octet-SA sensor Spike-RBD rACE2 (1-740aa) dimer 5.37E+3004 6.63E-05 1.2 SPR 2019-nCoV Spike ACE2 (1-615aa) 1.88E+3005* 2.76E-03* 14.7* monomer

[0051] Where modified ACE2-Fc hybrid constructs are used for diagnostic tests, multiple test formats can be chosen. One exemplary system is depicted in FIG. 9 using a biotin tagged ACE2-Fc hybrid construct immobilized to MSD streptavidin plate and another modified ACE2-Fc hybrid construct for detection (sulfo-tag MSD label). Results using such test system are shown in FIG. 10. As shown, test systems with ACE2-Fc hybrid constructs have high sensitivity and specificity.

[0052] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0053] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest manner consistent with the context.