Patent Application
20210284716
The content of the ASCII text file of the sequence listing named PAT.005234.US003_ST25, which is 99 KB in size was created on 15 May 2020 and electronically submitted via EFS-Web along with the present application. The sequence listing is incorporated by reference in its entirety.
The present disclosure relates to therapeutic and diagnostic compositions and methods, especially as they relate to hybrid constructs of angiotensin-converting enzyme 2 (ACE2) and an immunoglobulin “fragment crystallizable” (Fc) domain, useful for treatment and diagnosis of coronaviruses.
The background description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
ACE2 is a regulatory carboxypeptidase of the renin-angiotensin hormone system and functions as regulator of cardiovascular homeostasis. The ACE2 substrates are angiotensin I (which is converted by ACE2 to angiotensin 1-9) and angiotensin II (which is converted by ACE2 to angiotensin 1-7, a vasodilator). ACE2 also removes the C-terminal residue from three other vasoactive peptides, neurotensin, kinetensin, and des-Arg bradykinin, but is not active on bradykinin. Additional substrates for ACE2 include apelins, casomorphins, and dynorphin A. In addition, ACE2 C-terminus is homologous to collectrin and is responsible for regulating expression of the neutral amino acid transporter SL6A19 on the cell surface.
Despite its regulatory role in blood pressure, ACE2 is also expressed in numerous other tissues, including lung alveolar epithelial cells, enterocytes of the small intestine, Leydig cells and Sertoli cells, the renal proximal tubule, and testis. ACE2 is a receptor for human coronaviruses such as SARS-CoV and SARS-CoV-2, as well as human coronavirus NL63/HCoV-NL63.
Currently, there are no treatments that can reduce coronaviral pathogenic effects. While several antiviral and immunomodulatory drugs (e.g., remdesivir, hydroxychloroquine, azithromycin, immune suppressive steroids, etc.), have been proposed as COVID-19 treatments, most of these will not directly affect the virus or prevent its entry into the host cells.
A vaccine was prepared comprising a fusion protein with an IgG Fc portion and a viral protein of SARS-CoV as described in US 2010/0150923. However, the therapeutic effectiveness of such a construct was not reported.
In a further example, an ACE2/IgG Fc fusion was made as described in F1000Research 2020, 9:72 and in bioRxiv preprint at doi.org/10.1101/2020.02.01.929976. However, the extracellular ACE2 concentration achieved with such a fusion could have unknown effects on the body, particularly as the Fc domain prolongs serum half-life.
Thus, even though various compositions and methods of treatment for coronaviruses are known in the art, they suffer from several drawbacks. Therefore, there remains a need for improved compositions and methods for treatment of coronavirus infection.
Herein are disclosed various compositions of ACE2-IgA Fc fusion constructs and methods of using and uses of such constructs in the treatment of viral diseases, especially SARS-CoV-2 disease. Advantageously, these constructs will not only act as decoy receptors for coronaviruses by binding to the coronaviral Spike protein, but IgA-Fc domains will also localize them to mucous membranes. In certain embodiments, the ACE2 portion of the ACE2-Fc fusion construct comprises a mutation that inactivates the protein. Such constructs can advantageously be used as therapeutics to treat coronavirus.
In one aspect, a soluble recombinant ACE2-Fc hybrid construct comprises an ACE2 portion coupled to an IgA Fc portion.
In some embodiments, the ACE2 C-terminus is coupled to an IgA Fc N-terminus, optionally wherein the ACE2 portion has at least 85% sequence identity to SEQ ID NO:9 and/or wherein the IgA Fc portion has at least 85% sequence identity to SEQ ID NO:10 or 11. In certain embodiments, the hybrid construct will have at least 85%, for example 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:5. In certain embodiments, the hybrid construct will have at least 85%, for example 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:6. In further embodiments, the ACE2 portion has a mutation that renders the ACE2 portion catalytically inactive. The hybrid construct may also include a J-chain portion. For example, hybrid constructs may have an amino acid sequence with at least 85% sequence identity to SEQ ID NO:7 or 8. In certain embodiments, the hybrid construct 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:7. In certain embodiments, the hybrid construct 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:8. In still further embodiments, the hybrid construct may further comprise an affinity portion or a detectable label coupled to the hybrid construct. Moreover, the hybrid construct may be formulated in a pharmaceutically acceptable carrier (e.g., for inhalation, nasal administration, or injection).
In certain embodiments, the recombinant nucleic acid that encodes the ACE2-Fc hybrid construct as described herein may be an RNA or a DNA. Among other options, the recombinant nucleic acid may be an expression vector (e.g., having a nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4).
In another aspect, methods of treating a coronavirus infection are disclosed herein. These methods include administering to an individual in need thereof a therapeutically effective amount of an ACE2-Fc hybrid construct as described herein. Non-limiting examples of coronavirus infections that can be treated with these methods include those caused by SARS-CoV-2. Most typically, administration comprises nasal or pulmonary administration or intravenous injection.
In yet another aspect, methods of detecting a coronavirus are disclosed herein that include adding a test sample to a test surface to which ACE2-Fc is coupled to thereby bind Spike protein in the sample to the ACE2-Fc hybrid, contacting the bound Spike protein with a detectable binder, and detecting the detectable binder.
For example, the ACE2-Fc hybrid construct may be coupled to the test surface via a biotin group that is coupled to the ACE2-Fc hybrid construct, and/or detectable binder is an ACE2-Fc hybrid construct that is coupled to a detectable label. Among other options, the step of detecting the detectable binder may be performed using electrochemiluminescence.
Various objects, features, aspects and advantages of the compositions and methods disclosed herein will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.
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.
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.
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.
“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.
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.
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).
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.
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).
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.
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.
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).
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.
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).
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.
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.
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.
Selected exemplary constructs presented herein were expressed in CHO—S using Maxcyte electroporation. 3.2×108 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% CO2.
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.
Yield and relative purity for a small-scale Maxcyte production of IgG-Fc hybrid constructs were in a desirable range (
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 (
The purified ACE2-Fc hybrid constructs were then tested for binding capacity and avidity against 2019-n-CoV Spike protein.
Where modified ACE2-Fc hybrid constructs are used for diagnostic tests, multiple test formats can be chosen. One exemplary system is depicted in
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.
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.
This application claims priority to our co-pending U.S. 63/016,048, which was filed on 27 Apr. 2020, and which is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63022146 | May 2020 | US | |
| 63016048 | Apr 2020 | US | |
| 63016241 | Apr 2020 | US | |
| 63009960 | Apr 2020 | US | |
| 63010010 | Apr 2020 | US | |
| 62991504 | Mar 2020 | US | |
| 62988328 | Mar 2020 | US |
