Patent Application
20230203466
Coronaviruses (CoVs) are widely distributed in nature and pose a serious threat to humans and a range of mammalian hosts, causing respiratory, gastrointestinal, and central nervous system diseases (Li, 2016). CoVs are enveloped non-segmented positive-sense single stranded RNA viruses and are classified into α-, β-, γ-, and δ-CoVs (Li, 2016). While α- and β-CoVs infect mammals, the γ- and δ-CoVs generally infect birds (Li, 2016). Previously, α-CoVs HCoV-229E and HCoV-NL63, and β-CoVs HCoV-HKU1 and HCoV-OC43 have been found to infect humans leading to mild symptoms (Graham and Baric, 2010; Li, 2016). More recently, three β-CoVs: severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 (Holmes, 2003; Li, 2016), Middle-East respiratory syndrome coronavirus in 2012 (MERS-CoV) (Li, 2016; Zaki et al., 2012), and more recently SARS-CoV-2 in 2019 (Chan et al., 2020a; Huang et al., 2020; Zhu et al., 2020) have crossed the species barrier to infect humans resulting in respiratory illnesses including pneumonia that can be fatal.
SARS-CoV-2 is a novel coronavirus (2019-nCoV) first reported in December 2019 and is the cause of an ongoing global pandemic (Chan et al., 2020a; Huang et al., 2020; Zhu et al., 2020). It has infected over 39 million people in 181 countries leading to over 1.2 million deaths as of Oct. 19, 2020 (JHU, 2020). Sequence analysis of the SARS-CoV-2 genome revealed that it is closer to the bat CoV RaTG13 (96.2% identical) than to SARS-CoV (79.5% identical) that was responsible for the 2003 epidemic, suggesting that this novel virus originated in bats independently before jumping to humans either directly or through a yet to be determined intermediary host (Guo et al., 2020).
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease (COVID-19) that has resulted in a global pandemic. It is a highly contagious positive strand RNA virus and its clinical presentation includes severe to critical respiratory disease that appears to be fatal in ˜3-5% of the cases. The viral spike (S) coat protein engages the human angiotensin-converting enzyme 2 (ACE2) cell surface protein to invade the host cell. The SARS-CoV-2 S-protein has acquired mutations that increase its affinity to human ACE2 by ˜10-15-fold compared to SARS-CoV S-protein, making it highly infectious. In this study, we assessed if ACE2 polymorphisms might alter host susceptibility to SARS-CoV-2 by affecting the ACE2 S-protein interaction. Our comprehensive analysis of several large genomic datasets that included over 290,000 samples representing >400 population groups identified multiple ACE2 protein-altering variants, some of which mapped to the S-protein-interacting surface. Using recently reported structural data and a recent S-protein-interacting synthetic mutant map of ACE2, we have identified natural ACE2 variants that are predicted to alter the virus-host interaction and thereby potentially alter host susceptibility. In particular, human ACE2 variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R are predicted to increase susceptibility. The T92I variant, part of a consensus NxT/S N-glycosylation motif, confirmed the role of N90 glycosylation in providing some protection against non-human CoVs. Other ACE2 variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y are putative protective variants predicted to show decreased binding to SARS-CoV-2 S-protein.
Using biochemical assays, we found that while K31R and E37K had a decreased affinity, K26R and T92I variants had an increased affinity for SARS-CoV-2 S-protein when compared to wildtype ACE2, confirming our structural predictions. Consistent with this, soluble ACE2 K26R and T92I were more effective in blocking entry of S-protein pseudotyped virus. These data suggest that ACE2 variants can modulate the susceptibility to SARS-CoV-2.
As with SARS-CoV and a related alphacoronaviruses NL63 (HCoV-NL63), the SARS-CoV-2 employs the human angiotensin-converting enzyme 2 (ACE2) cell surface protein as a receptor to gain entry into the cells (Hoffmann et al., 2020; Letko et al., 2020; Lin et al., 2008; Wan et al., 2020; Zhou et al., 2020). The virus surface spike glycoprotein (S-protein) constitutes a key determinant of viral host range and contains two domains, S1 and S2, which are separated by a protease cleavage site (Li, 2016). A successful host cell invasion by the virus involves direct binding of the virus S1 receptor binding domain (RBD) to the host ACE2 peptidase extracellular domain (PD), exposing the S1-S2 inter-domain protease site that upon cleavage by host proteases, leads to S2-mediated virus-host cell membrane fusion (Belouzard et al., 2009; Hoffmann et al., 2020; Li, 2016; Li et al., 2005a; Simmons et al., 2005).
The receptor binding domain (RBD) within S1 binds directly to the peptidase domain (PD) of ACE2, while S2 mediates membrane fusion (Li, 2016; Li et al., 2005a; Simmons et al., 2005). As the S1 subunit binds the host ACE2, an exposed protease site on S2 is cleaved by host proteases facilitating membrane fusion and viral infection (Belouzard et al., 2009; Simmons et al., 2005).
The SARS-CoV-2 S-protein is 98% identical to the bat CoV RaTG13 S-protein, with the exception of an insertion that is also absent in the SARS-CoV S-protein in the S1/S2 inter-domain protease cleavage site. This difference has been proposed to alter SARS-CoV-2 tropism and enhance its transmissibility (Walls et al., 2020).
Several structural studies involving the SARS-CoV-2 S-protein RBD and ACE2 peptidase domain (PD) have identified the key residues involved in their interaction (Shang et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020). The S-protein RBD was reported to bind ACE2 PD with ˜10- to 20-fold higher affinity (˜15 nM) when compared to the SARS-CoV S-protein RBD (Shang et al., 2020; Wrapp et al., 2020), potentially contributing to the high rate of SARS-CoV-2 infection.
As the interactions between the ACE2 receptor and S-protein RBD interface are critical for the cellular entry of the virus, we wanted to ascertain if there were natural ACE2 variations that would decrease or increase its affinity to the S-protein RBD and may thus protect or render individuals more susceptible to the virus. Consistent with this possibility, a saturation mutagenesis screen of select ACE2 PD residues identified variants that showed enhanced or decreased binding to S-protein (Chan et al., 2020b).
Since COVID-19 poses a serious threat to animals and humans, it is important to be able to identify it accurately and quickly to reduce COVID-19's deleterious health and economic impact. We have analyzed the ACE2 protein altering variants in a large number of data set populations and identified polymorphisms that will likely either protect or render them more susceptible to the virus.
We have addressed this need by discovering rationally designed, catalytically inactive, human ACE2 that carries one or more of the natural variants to obtain improved binding to SARS viral S-protein RBD that can be developed as a soluble protein with or without an Fc domain for treatment of COVID-19. Such a recombinant ACE2 protein can be engineered to create a pan-CoV neutralizing drug that is broad and can neutralize CoVs that may emerge during future epidemics.
In this study, we have analyzed ACE2 protein-altering variants in a large cohort of human population groups and identified polymorphisms that either likely protect or render individuals more susceptible to the virus. Understanding these changes at the molecular level, combined with the genotype and epidemiological data will allow the elucidation of population risk profiles and also help advance therapeutics such as a rationally designed soluble ACE2 decoy-receptor for treatment of COVID-19.
Isolated SARS-CoV-2 binding protein complexes comprising ACE2 receptor variations and variants which may predict resistance and sensitivity to a SARS coronavirus, COVID-19 are provided, which proteins comprise sequence modification that enhance the stability and/or utility of the protein. Human ACE2 receptor variations and variants are preferred. The ACE2 receptor variants may be used for diagnosis and treatment of COVID-19.
The invention also provides methods for monitoring the course of SARS-CoV-2 infection in a subject. In one embodiment, the method comprises obtaining a sample from the subject, determining amino acid sequence of ACE2 of the subject, comparing identity of amino acid so determined to reference amino acids known to affect SARS-CoV-2 interaction with ACE2, wherein finding an amino acid change favoring interaction with surface spike glycoprotein, S protein, of SARS-CoV-2 are any of S19P, I21T/V, E23K, A25T, K26E or K26R, T27A, F40L, Q60R, N64K, W69C, T92I, Q102P, Q325R, M366T, D367V, H374R, H378R, M383T, E398D, E398K, T445M, I446M, and Y510H, and wherein an amino acid change resulting in less favorable interaction with S protein of SARS-CoV-2 are any of K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51D or N51S, M62I or M62V, A65S, K68E, F72H, M82I, Y83H, P84T, V93G, N290H, G326E, E329G, P346S, G352V, D355N, T371K, Q388L, P389H, F504I or F504L, H505R, D509Y, S511P, R514G, Y515C and R518T and predicting a subject to have a more severe course of infection for the subject with an amino acid change favoring interaction with S protein of SARS-CoV-2 or a milder course of infection for the subject with an amino acid change resulting in less favorable interaction with S protein of SARS-CoV-2.
The invention also provides methods for assessing risk of being infected by SARS-CoV-2 virus in a subject. In one embodiment, the method comprises obtaining a sample from the subject, determining amino acid sequence of ACE2 of the subject, comparing identity of amino acid so determined to reference amino acids known to affect SARS-CoV-2 interaction with ACE2, wherein finding an amino acid change resulting in increased risk of being infected are any of S19P, I2T/V, E23K, A25T, K26E or K26R, T27A, F40L, Q60R, N64K, W69C, T92I, Q102P, Q325R, M366T, D367V, H374R, H378R, M383T, E398D, E398K, T445M, 1446M, and Y510H, and wherein an amino acid change resulting in decreased risk of being infect are any of K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51D or N51S, M62I or M62V, A65S, K68E, F72H, M82I, Y83H, P84T, V93G, N290H, G326E, E329G, P346S, G352V, D355N, T371K, Q388L, P389H, F504I or F504L, H505R, D509Y, S511P, R514G, Y515C and R518T, and predicting a subject to have an increased or decreased risk based on finding a match falling into the two groups.
The invention also provides kits for assessing risk or course of a SARS-CoV-2. In one embodiment, the kit comprises oligonucleotide or nucleic acid fragment for assessing polymorphism of ACE2 gene and instruction for use. In a further embodiment, the polymorphism is directed to the coding region of the ACE2 gene. In another embodiment, the polymorphism is directed to the SARS-CoV-2 S protein interaction site on ACE2 protein as provided in
The invention also provides kits for detecting COVID-19 comprising an ACE2 variant from any of the Tables herein and an informational insert.
It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, 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 present invention, which will be limited only by the appended claims.
As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the culture” includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
As used herein, the terms “purified” and “isolated” when used in the context of a polypeptide that is substantially free of contaminating materials from the material from which it was obtained, e.g. cellular materials, such as but not limited to cell debris, cell wall materials, membranes, organelles, the bulk of the nucleic acids, carbohydrates, proteins, and/or lipids present in cells. Thus, a polypeptide that is isolated includes preparations of a polypeptide having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellular materials and/or contaminating materials. As used herein, the terms “purified” and “isolated” when used in the context of a polypeptide that is chemically synthesized refers to a polypeptide which is substantially free of chemical precursors or other chemicals which are involved in the syntheses of the polypeptide.
The term “polypeptide,” “peptide,” “oligopeptide,” and “protein,” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically, or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
The polypeptides may be isolated and purified in accordance with conventional methods of recombinant synthesis. Exemplary coding sequences are provided, however one of skill in the art can readily design a suitable coding sequence based on the provided amino acid sequences. Methods which are well known to those skilled in the art can be used to construct expression vectors containing coding sequences 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 the polypeptides of interest may be chemically synthesized. One of skill in the art can readily 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 may be isolated and obtained in substantial purity. Usually, the nucleic acids, either as DNA or RNA, will be obtained substantially free of other naturally-occurring nucleic acid sequences, generally being at least about 50%, usually at least about 90% pure and are typically “recombinant,” e.g., flanked by one or more nucleotides with which it is not normally associated on a naturally occurring chromosome. The nucleic acids of the invention can be provided as a linear molecule or within a circular molecule, and can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences. Expression of the nucleic acids can be regulated by their own or by 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.
An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to cause a desired biological effect, such as beneficial results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. In the context of this invention, an example of an effective amount of a vaccine is an amount sufficient to induce an immune response (e.g., antibody production) in an individual. An effective amount can be administered in one or more administrations.
Folding, as used herein, refers to the process of forming the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure. Non-covalent interactions are important in determining structure, and the effect of membrane contacts with the protein may be important for the correct structure. For naturally occurring proteins and polypeptides or derivatives and variants thereof, the result of proper folding is typically the arrangement that results in optimal biological activity, and can conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc.
In some instances, for example where the desired product is of synthetic origin, assays based on biological activity will be less meaningful. The proper folding of such molecules may be determined on the basis of physical properties, energetic considerations, modeling studies, and the like.
Separation procedures of interest include affinity chromatography. Affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. Covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using 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 a microsphere or matrix is used as the support for affinity chromatography. Such supports are known in the art and are commercially available, and include activated supports that can be combined to the linker molecules. For example, Affi-Gel supports, based on agarose or polyacrylamide are low pressure gels suitable for most laboratory-scale purifications with a peristaltic pump or gravity flow elution. Affi-Prep supports, based on a pressure-stable macroporous polymer, are suitable for preparative and process scale applications.
Proteins may also be separated by ion exchange chromatography, and/or concentrated, filtered, dialyzed, etc., using methods known in the art. The methods of the present invention provide for proteins containing unnatural amino acids that have biological activity comparable to the native protein. One may determine the specific activity of a protein in a composition by determining the level of activity in a functional assay, quantitating the amount of protein present in a non-functional assay, e.g. immunostaining, ELISA, quantitation on coomassie or silver stained gel, etc., and determining the ratio of biologically active protein to total protein. Generally, the specific activity as thus defined will be at least about 5% that of the native protein, usually at least about 10% that of the native protein, and may be about 25%, about 50%, about 90% or greater.
The invention provides SARS-CoV-2 binding protein complexes comprising ACE2 receptor variations and variants which may predict resistance and sensitivity to a SARS coronavirus, COVID-19. Human ACE2 receptor variations and variants are preferred. The ACE2 receptor variants may be used for diagnosis and treatment of COVID-19.
The invention also provides isolated SARS-CoV-2 binding protein complexes. As used herein, examples of a complex includes conjugates and fusion proteins. In one embodiment, the SARS-CoV-2 binding protein complex comprises an extracellular domain or fragment thereof of an angiotensin converting enzyme 2 (ACE2) protein or its variant joined to a non-ACE2 molecule or compound.
In accordance with the practice of the invention, the non-ACE2 compound may be a biological entity. Examples of suitable biological entities include, but are not limited to, proteins, polypeptide, peptides and albumin. The proteins may be serum proteins. The serum proteins may comprises any of antibody, serum albumin, beta-1-B-glycoprotein or Hemopexin (Hpx).
The protein may be an immunoglobulin molecule or antibody molecule or variant or fragment thereof. The antibody fragment may be a Fc. Examples of suitable antibody fragment include, but are not limited to, Fab, Fab′, F(ab)′, scFv, and F(ab)′2. In a preferred embodiment, the antibody recognizes and binds a SARS-CoV-2. SARS-CoV-2 antibodies are known (ter Meulen J, van den Brink E N, Poon L L M, Marissen W E, Leung C S W, et al. (2006) Human monoclonal antibody combination against SARS coronavirus: Synergy and coverage of escape mutants. PLoS Med 3(7): e237. DOI: 10.1371/jourmal.pmed.0030237; Meng Yuan et al., Science 3 Apr. 2020: eabb7269, DOI: 10.1126/science.abb7269; Author links open overlay panel; ShiboJiang et al., Trends in Immunology, Volume 41, Issue 5, May 2020, Pages 355-359; Catalan-Dibene, J. Human antibodies can neutralize SARS-CoV-2. Nat Rev Immunol (2020). https://doi.org/10.1038/s41577-020-0313-6; Bin Ju, et al. Potent human neutralizing antibodies elicited by SARS-CoV-2 infection, bioRxiv 2020.03.21.990770; doi: https://doi.org/10.1101/2020.03.21.990770).
In another embodiment of the invention the non-ACE2 compound may be a chemical entity. Examples of suitable chemical entity include, but are not limited to, poly(ethylene glycol) (“PEG”). The PEG may be linear or branched. In one embodiment, the PEG has a molecular weight of from about 5,000 Daltons (5 kDa) to about 100,000 Daltons (100 kDa). In another embodiment, the PEG has a molecular weight of from about 10 kDa to about 60 kDa.
In one embodiment of the isolated SARS-CoV-2 binding protein complex, the ACE2 protein is derived from a mammal. Examples of mammals include, but are not limited to, mouse, rat, dog, cat, civet, pangolin, bat, pig, guinea pig, goat, sheep, donkey, horse, camel, chimpanzee, monkey, gorilla, cattle, and human. In a preferred embodiment of the invention, the mammal is human.
In one embodiment, the ACE2 protein may be a full length human ACE2 protein as shown in
In one embodiment, the extracellular domain of the ACE2 protein comprises or consists of the amino acid sequences between a signal sequence and a transmembrane domain of the ACE2 protein but lacks a signal sequence, transmembrane domain and cytosolic domain.
In one embodiment, the extracellular domain of the ACE2 protein consists of or comprises a peptidase domain and collectrin domain. In a further embodiment, the extracellular domain encompasses amino acid residues 18 to 740 of sequence provided in
In another embodiment, the extracellular domain is about 723 amino acids in length.
In accordance with the practice of the invention, in one embodiment, the ACE2 variant has at least one amino acid change from a reference full length ACE2 protein as provided in
In one embodiment, the amino acid change increases binding or binding affinity of the extracellular domain or fragment thereof for a SARS-CoV-2 virus or a SARS-CoV-2 spike glycoprotein (S-protein) as shown in
In one embodiment, the amino acid change prevents glycosylation at amino acid N90. In a further embodiment, the amino acid change which prevents glycosylation at amino acid N90 is substituting asparagine at amino acid residue 90 with another amino acid. In another embodiment, another amino acid is substituted for asparagine. Examples of the amino acid being substituted include, but are not limited to, alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
In one embodiment, the amino acid change which prevents glycosylation is a change at amino acid residue 91. The leucine at position 91 is substituted with a proline (L91P) or a change at amino acid residue 92, wherein threonine is substituted with another amino acid other than a serine. Examples of the amino acid being substituted for threonine include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine and valine.
In another embodiment of the isolated SARS-CoV-2 binding protein complex of the invention, the isolated SARS-CoV-2 binding protein complex further comprises a signal sequence located at an amino terminus of the protein.
Examples of the signal sequence include, but are not limited to, SEQ ID NO: 2A-2L as shown below.
In one embodiment, the extracellular domain of the ACE2 protein is a variant or allelic variant of amino acid 18-740 of SEQ ID NO: 1 (UniProtKB ID: Q9BYF1-1). In another embodiment, the extracellular domain or fragment thereof comprises a functional peptidase. The functional peptidase may be a carboxypeptidase. The carboxypeptidase may be a metallocarboxypeptidase.
In another embodiment, the extracellular domain or fragment thereof of the ACE2 protein variant comprises a HEXXH zinc-binding motif at amino acids 374 to 378 of
In another embodiment, the ACE2 extracellular domain or fragment thereof lacks a functional peptidase activity. For example, the functional peptidase activity so lacking may be a carboxypeptidase activity.
In one embodiment, the extracellular domain or fragment thereof of ACE2 protein or ACE2 protein variant comprises an alteration at HEXXH zinc-binding motif corresponding to amino acids 374 to 378 of
In another embodiment, the variant comprises an amino acid change at any of S19, E22, E23, Q24, A25, K26, T27, L29, D30, K31, N33, H34, E35, L39, F40, Y41, Q42, A65, W69, F72, E75, Q76, L79, A80, M82, Q89, N90, L91, T92, V93, T324, Q325, N330, L351, G352, D382, A386, P389, R393, S511 and R518 or a combination thereof. Examples of the amino acid change include, but are not limited to, S19V, S19W, S19Y, S19F, S19P, E22T, E23M, E23T, E23Q, E23F, E23C, Q24T, A251, A25V, A25T, A25F, K26I, K26V, K26A, K26D, K26R, T27M, T27L, T27A, T27D, T27K, T27H, 127W, T27Y, T27F, T27C, L29F, D301, D30V, D30E, K31W, K31Y, N33D, N33C, N33I, H34V, H34A, H34S, H34P, E35M, E35V, E35D, E35C, L391, L39V, L39K, L39R, Y41R, Q42M, Q42L, Q42T, Q42V, Q42K, Q42H, Q42C, A65W, W69L, W691, W69V, W69T, W69K, W69C, F72W, F72Y, E75A, E75S, E75T, E75Q, E75K, E75R, E75H, E75W, E75G, Q76M, Q76I, Q76V, Q76T, Q76R, Q76Y, L791, L79V, L79T, L79W, L79Y, L79F, L79P, A80G, M82C, Q89I, Q89D, Q89P, N90M, N90L, N90I, N90V, N90A, N90S, N90T, N90Q, N90D, N90E, N90K, N90R, N90H, N90W, N90Y, N90F, N90P, N900, N90C, L91P, T92M, T92L, T92I, T92V. T92A, T92N, T92Q, T92D, T92E, T92K, T92R, T92H, T92W, T92Y, T92F, T92P, T92G, T92C, V93P, T324A, T324E, T324P, Q325P, N330L, N330H, N330W, N330Y, N330F, L351F, A386L, A386I, P389D, R393K, S511D and R518G or a combination thereof.
In yet another embodiment, the variant comprises an amino acid change at any of S19, E23, A25, K26, T27, D30, K31, N33, H34, L39, Y41, Q42, W69, F72, E75, Q76, L79, A80, Q89, N90, L91, T92, T324, N330, A386 and R393 or a combination thereof. Examples of the amino acid change include, but are not limited to, S19P, E23F, A25V, K26I, K26D, T27M, T27L, T27A, T27D, T27H, T27W, T27Y, T27F, T27C, D30E, K31W, N33D, N33I, H34V, H34A, H34P, L39K, L39R, Y41R, Q42M, Q42L, Q42C, W691, W69V, W69T, W69K, F72Y, E75K, E75R, Q76I, Q76V, Q76T, 1,791, L79V, L79T, L79W, L79Y, L79F, A80G, Q89P, N90M, N90L, N90I, N90V, N90A, N90S, N90T, N90Q, N90D, N90E, N90K, N90R, N90H, N90W, N90Y, N90F, N90P, N90G, N90C, L91P, T92M, T92L, T92I, T92V, T92A, T92N, T92Q, T92D, T92E, T92K, T92R, T92H, T92W, T92Y, T92F, T92P, T92G, T92C, T324E, T324P, N330L, N330H, N330W, N330Y, N330F, A386L and R393K or a combination thereof.
In another embodiment, the variant comprises an amino acid change at any of S19, E22, E23, Q24, A25, K26. T27, L29, D30, K31, N33, H34, E35, L39, Q42, A65, W69, F72, F75, Q76, L79, A80, M82, Q89, T92, V93, T324, Q325, L351, A386, P389, S511 and R518 or a combination thereof. Examples of the amino acid change include, but are not limited to, S19V, S19W, S19Y, S19F, E22T, E23M, E23T, E23Q, E23C, Q24T, A251, A25T, A25F, K26V, K26A, K26R, T27K, L29F, D301, D30V, K31Y, N33C, N33I, H34S, E35M, E35V, E35D, E35C, L391, L39V, Q421, Q42V, Q42K, Q42H, A65W, W69L, W69C, F72W, E75A, E75S, E75T, E75Q, E75H, E75W, E75G, Q76M, Q76R, Q76Y, L79P, A80G, M82C, Q89I, Q89D, T92I, V93P, T324A, Q325P, L351F, A386I, P389D, S511D and R518G or a combination thereof.
In another embodiment, the variant comprises an amino acid change at any of S19, I21, E23, K26, T27, N33, F40, Q60, N64, A80, N90, T92, Q102, H378, M383, T445 and Y510 or a combination thereof. Examples of the amino acid change include, but are not limited to, S19P, I21V, E23K, K26E, K26R, T27A, F40L, Q60R, N64K, N90I, N90T, T92I, Q102P, H378R, M383T, T445M and Y510H or a combination thereof.
In yet another embodiment, the allelic variant comprises an amino acid change at any of 519, 121, E23, K26, T27, N33, F40, Q60, N64, A80, T92, Q102, H378, M383, T445 and Y510 or a combination thereof. Examples of the amino acid change include, but are not limited to, S19P, 21V, E23K, K26E, K26R, T27A, N33I, F40L, Q60R, N64K, A80G, T92I, Q102P, H378R, M383T, T445M and Y510H or a combination thereof.
In another embodiment, the allelic variant comprises an amino acid change at any of S19, T27, N33I, A80G and T92 or a combination thereof. Examples of the amino acid change include, but are not limited to, S19P, T27A, N33I, A80G and T92I and a combination thereof.
In another embodiment, the allelic variant comprises an amino acid change at any of I21, K26, N64, Q102 and H378 or a combination thereof. Examples of the amino acid change include, but are not limited to, I21V, K26R, N64K, Q102P and H378R or a combination thereof.
In another embodiment, the variant comprises an amino acid change at any of E23, K26, F40, Q60, M383, T445 and Y510 or a combination thereof. Examples of the amino acid change include, but are not limited to, E23K, K26E, F40L, Q60R, M383T, T445M and Y510H or a combination thereof.
In yet another embodiment, the variant comprises an amino acid change at any of S19, 121, E23, K26, T27, F40, N64, N90, T92, Q102, H378, M383 and T445 or a combination thereof. Examples of the amino acid change include, but are not limited to, S19P, I21V, E23K, K26E, K26R, T27A, F40L, N64K, N90I, N90T, T92I, Q102P, H378R, M383T and T445M or a combination thereof.
In another embodiment, the variant comprises amino acid changes at amino acid S19, 121, E23, K26, T27, F40, N64, N90, T92, Q102, H378, M383 and T445. In a further embodiment, the variant comprises amino acid changes S19P, 21V, E23K, K26E, T27A, F40L, N64K, N90I, N90T, T92I, Q102P, H378R, M383T and T445M. In another embodiment, the variant comprises amino acid changes S19P, I21V, E23K, K26R, T27A, F40L, N64K, N90L N90T, T92I, Q102P, H378R, M383T and T445M.
In one embodiment, the fragment of ACE2 extracellular domain consists of peptidase or carboxypeptidase domain.
In another embodiment, the fragment of ACE2 extracellular domain lacks a signal peptide or sequence, collectrin domain, transmembrane domain and cytosolic domain. In a further embodiment, the peptidase or carboxypeptidase domain consists of or comprises amino acid residues 18-615 as provided in
or a variant thereof.
In one embodiment, the fragment of ACE2 extracellular domain consists of or comprises about 598 amino acids. In another embodiment, the fragment of ACE2 extracellular domain is greater than about 5 amino acids. In another embodiment, the fragment of ACE2 extracellular domain is less than about 723 amino acids. In yet another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 10 and 723 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 601 and 700 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 501 and 600 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 401 and 500 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 301 and 400 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 201 and 300 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 101 and 200 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 50 and 100 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 25 and 65 amino acids. In another embodiment, the fragment of ACE2 extracellular domain consists or comprises between about 9 and 35 amino acids.
In one embodiment, the extracellular domain fragment consists of amino acid residues 18-393 as provided in
or a variant thereof or a portion thereof, wherein the portion is 35 or more amino acids.
In one embodiment, the fragment of ACE2 extracellular domain consists of or comprises about 376 amino acids.
In another embodiment, the extracellular domain fragment consists of or comprises amino terminus of ACE2 extracellular domain. In another embodiment, the amino terminus of ACE2 extracellular domain consists or comprises amino acid residues 18-48 as provided in
or a variant thereof.
In another embodiment, the fragment of ACE2 extracellular domain consists of or comprises about 31 amino acids.
In one embodiment of the isolated SARS-CoV-2 binding protein complex of the invention, the complex further comprises at least one additional extracellular domain fragment such that two or more extracellular domain fragments are functionally linked so as to permit binding to SARS-CoV-2 virus or SARS-CoV-2 spike glycoprotein (S-protein), wherein each extracellular domain fragment consists of or comprises a polypeptide secondary structural element. In a further embodiment, a polypeptide secondary structural element is any of helix, alpha helix, 310 helix, π helix, β-turn, hydrogen bonded turn, extended strand in parallel and/or antiparallel β-sheet conformation, residue in isolated β-bridge, bend and coil.
Examples of the extracellular domain fragment include, but are not limited to, a helix forming peptide, TEENVQNMNNAGDKWSAFLKEQSTLAQMY (SEQ ID NO: 6), corresponding to amino acid residue 55-83 as provided in
In one embodiment, the fragment of a helix forming peptide of SEQ ID NO: 6 is: AGDKWSAFLKEQSTLAQMY (SEQ ID NO: 9), corresponding to amino acid residue 65-83 as provided in
In another embodiment, the fragment of a helix forming peptide of SEQ ID NO: 7 is: EEQAKTFLDKFNHEAEDLFYQSS (SEQ ID NO: 10), corresponding to amino acid residue 22-44 as provided in
In another embodiment, the fragment of a β-turn peptide of SEQ ID NO: 8 is: DLGKGDFR (SEQ ID NO: 11), corresponding to amino acid residue 350-357 as provided in
In another embodiment, two or more extracellular domain fragments are ordered and covalently linked to form a polypeptide chain. In another embodiment, the extracellular domain fragments are in the same order or form overlapping fragments having an order as present in the primary amino acid sequence of ACE2 protein. In a further embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 7] or [helix forming peptide with SEQ ID NO: 10] followed by [helix forming peptide with SEQ ID NO: 6] or [helix forming peptide with SEQ ID NO: 9] and lastly followed by [β-turn peptide of SEQ ID NO: 8] or [β-turn peptide of SEQ ID NO: 11].
In another embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 7] or [helix forming peptide with SEQ ID NO: 10] followed by [helix forming peptide with SEQ ID NO: 6] or [helix forming peptide with SEQ ID NO: 9].
In another embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 6] or [helix forming peptide with SEQ ID NO: 9] and lastly followed by [β-turn peptide of SEQ ID NO: 8] or [β-turn peptide of SEQ ID NO: 11].
In yet another embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 7] or [helix forming peptide with SEQ ID NO: 10] followed by [β-turn peptide of SEQ ID NO: 8] or [β-turn peptide of SEQ ID NO: 11].
In another embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 7] followed by [helix forming peptide with SEQ ID NO: 6] and lastly followed by [β-turn peptide of SEQ ID NO: 8].
In an additional embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 10] followed by [helix forming peptide with SEQ ID NO: 9] and lastly followed by [0-turn peptide of SEQ ID NO: 11].
In yet another embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 7] followed by [β-turn peptide of SEQ ID NO: 11].
Further still, in one embodiment, the order from amino-to-carboxyl direction is: [helix forming peptide with SEQ ID NO: 10] followed by [β-turn peptide of SEQ ID NO: 8].
In one embodiment, the extracellular domain fragments are ordered such that at least one fragment is not in the same order as present in the primary amino acid sequence of ACE2 protein. In a further embodiment, the at least one fragment that is not in the same order has the following from amino-to-carboxyl direction: [helix forming peptide with SEQ ID NO: 6] or [helix forming peptide with SEQ ID NO: 9] followed by [helix forming peptide with SEQ ID NO: 7] or: [helix forming peptide with SEQ ID NO: 10] and lastly by [β-turn peptide of SEQ ID NO: 8] or [β-turn peptide of SEQ ID NO: 11]. In another embodiment, the at least one fragment that is not in the same order has the following from amino-to-carboxyl direction: [helix forming peptide with SEQ ID NO: 6]-[helix forming peptide with SEQ ID NO: 7]-[β-turn peptide of SEQ ID NO: 8]. In yet another embodiment, the at least one fragment that is not in the same order has the following from amino-to-carboxyl direction: [helix forming peptide with SEQ ID NO: 9]-[helix forming peptide with SEQ ID NO: 10]-[β-turn peptide of SEQ ID NO: 11].
In one embodiment of the invention, the fragments are separated by a peptide linker. The peptide linker may be between one to ten amino acids. The peptide linker may be glycine and/or serine rich. Examples of the peptide linker include, but are not limited to, G, GG, and GGGGSGG.
In one embodiment of the invention, the variant may be variant, allelic variant or combination of variants and/or allelic variants. In a further embodiment, the variant, allelic variant or combination of variants and/or allelic variants comprise one or more amino acid substitution relative to reference ACE2 protein sequence (
In one embodiment of the invention, the antibody is an immunoglobulin. The immunoglobulin may comprise an immunoglobulin heavy chain. The immunoglobulin may comprise an immunoglobulin light chain. The immunoglobulin may comprise an immunoglobulin heavy chain and an immunoglobulin light chain. Examples of the immunoglobulin include, but are not limited to, IgM, IgG, IgA, IgD and IgE. In a preferred embodiment of the invention, the immunoglobulin is IgG. Examples of the IgG include, but are not limited to, IgG1, IgG2, IgG3 and IgG4.
In one embodiment, the immunoglobulin binds an antigen on SARS-CoV-2 virus or SARS-CoV-2 spike glycoprotein (S-protein). In another embodiment, the immunoglobulin is derived from a hybridoma. In yet another embodiment, the immunoglobulin is produced by recombinant DNA method or molecular biology method. In another embodiment, the immunoglobulin is derived from a Fab library. In another embodiment, the immunoglobulin is derived from a single chain variable antibody fragment (scFv) phage display library.
In one embodiment, the Fab library or scFv phage display library comprises a binding protein for SARS-CoV-2 virus or SARS-CoV-2 protein, wherein the binding protein does not compete with ACE2 binding of SARS-CoV-2 virus or SARS-CoV-2 protein. In a further embodiment, the binding protein is CR3022 scFv which binds SARS-CoV-2 virus and SARS-CoV-2 S-protein (ter Meulen J, van den Brink E N, Poon L L M, Marissen W E, Leung C S W, et al. (2006) Human monoclonal antibody combination against SARS coronavirus: Synergy and coverage of escape mutants. PLoS Med 3(7): e237. DOI: 10.1371/journal.pmed.0030237).
In one embodiment, the immunoglobulin is obtained after converting CR3022 scFv to an immunoglobulin format. In another embodiment, the immunoglobulin is a recombinant protein. In another embodiment, the immunoglobulin is from a mammal or classified as being from a mammal. Examples of the mammal include, but are not limited to, mouse, rat, dog, cat, civet, pangolin, bat, pig, guinea pig, goat, sheep, donkey, horse, camel, chimpanzee, monkey, gorilla, cattle, and human. In a preferred embodiment, the mammal is human. In another embodiment, the immunoglobulin is from a chicken or classified as being from a chicken. In another embodiment, the immunoglobulin is a full-length immunoglobulin. In another embodiment, the full-length immunoglobulin is derived from converting a Fab or scFv to a full-length immunoglobulin. In a further embodiment, the Fab or scFv binds SARS-CoV-2 virus or SARS-CoV-2 S-protein but does not compete with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein. In another embodiment, the scFv that binds SARS-CoV-2 virus or SARS-CoV-2 S-protein is CR3022 scFv. In yet another embodiment, the scFv that binds SARS-CoV-2 virus or SARS-CoV-2 S-protein is a variant of CR3022 scFv, wherein one or more amino acid change in complement-determining regions (CDRs) increases binding affinity of the variant to SARS-CoV-2 virus or SARS-CoV-2 S-protein without competing with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein.
3) In one embodiment of the invention, the antibody fragment is a fragment or portion of an immunoglobulin. Examples of the fragment or portion of an immunoglobulin include, but are not limited to, Fab, Fab′, F(ab′)2, Fc, single chain variable fragment (scFv), diabody and recombinantly produced immunoglobulin fragment and a combination thereof. In another embodiment, the antibody fragment is a scFv, which does not compete with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein. Further, in another embodiment, the scFv is CR3022 scFv.
In one embodiment, the scFv is a variant of CR3022 scFv, wherein one or more amino acid change in CDRs increases binding affinity of the variant to SARS-CoV-2 virus or SARS-CoV-2 S-protein without competing with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein. In another embodiment, the antibody fragment is not a scFv but is derived from a scFv and wherein the antibody fragment does not compete with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein. In a further embodiment, the scFv is CR3022 scFv. In another embodiment wherein the scFv is a variant of CR3022 scFv, one or more amino acid change in CDRs increases binding affinity of the variant to SARS-CoV-2 virus or SARS-CoV-2 S-protein without competing with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein.
In one embodiment of the invention, the antibody fragment is a Fab. In another embodiment, the antibody fragment is a Fab′. In yet another embodiment, the antibody fragment is a F(ab′)2. In another embodiment, the antibody fragment is a diabody or a scFv.
In one embodiment of the invention, the antibody fragment binds SARS-CoV-2 virus or SARS-CoV-2 S-protein without competing with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein. In another embodiment, the antibody fragment is derived from CR3022 scFv. In another embodiment, the antibody fragment is derived from a variant of CR3022 scFv, wherein one or more amino acid change in CDRs increases binding affinity of the variant to SARS-CoV-2 virus or SARS-CoV-2 S-protein without competing with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 protein. In another embodiment, the antibody fragment is a Fe. In another embodiment, the antibody fragment is recombinantly produced immunoglobulin fragment obtained by recombinant DNA method or molecular biology method.
In one embodiment of the invention, the antibody or antibody fragment comprises a Fc with functional Fc effector functions. In another embodiment, the antibody or antibody fragment comprises a Fc mutated so as to reduce or abolish Fc effector function. In another embodiment, the Fc effector function is to support binding of Fc receptor and/or complement protein 1q (C1q). In another embodiment, the Fc effector function is antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or a combination thereof. In another embodiment, the mutated Fc has one or more amino acid change. In a further embodiment, the amino acid change decreases or abolishes binding of the Fc receptor or complement protein 1q (C1q) to the antibody or antibody fragment. In another embodiment, the amino acid change decreases or abolishes binding of the Fcγ receptor or complement protein 1q (C1q) to IgG or IgG fragment. In another embodiment, the Fcγ receptor is any of Fcγ receptor 1, Fcγ receptor II and Fcγ receptor III and a combination thereof.
In one embodiment, the amino acid change decreases or abolishes antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or a combination thereof. In another embodiment, the amino acid change is at aspartic acid 265, asparagine 297 or both for IgG or equivalent, wherein equivalent is one or more amino acid change at other amino acid position of IgG reducing or abolishing Fe effector function or at a corresponding position or other position for IgM, IgD, IgA or IgE. In another embodiment, the amino acid change is D265A or N297G or both. In yet another embodiment, the amino acid change is D265A and N297G.
In one embodiment, the combination comprises two or more antibody fragments. In another embodiment, the combination comprises a Fc and a diabody or scFv. In a further embodiment, the Fc and the diabody or scFv are covalently linked. In another embodiment, the Fc and the diabody or scFv are covalently linked through a linker. In yet another embodiment, the linker is a peptide linker. In another embodiment, the Fc is linked to the amino terminus of the diabody or scFv.
In one embodiment of the invention, the isolated SARS-CoV-2 binding protein complex is a bi-specific protein. In another embodiment, the bispecific protein binds two different determinants on SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, one specificity is conferred by an antigen-binding determinant of an immunoglobulin component and other specificity is conferred by an ACE2 component, wherein antigen binding site and ACE2 binding site of SARS-CoV-2 virus or SARS-CoV-2 S-protein do not overlap and both sites can be occupied at the same time by the antigen-binding determinant of an immunoglobulin and ACE2.
In one embodiment of the invention, the antigen-binding determinant of an immunoglobulin component consists of or comprises a light chain and a heavy chain of an immunoglobulin. In another embodiment, the light chain consists of or comprises a variable domain, VL, and a constant domain, CL. In another embodiment, the heavy chain consists of or comprises a variable domain, VH, and three constant domains, CH1, CH2 and CH3. In another embodiment, the heavy chain further comprises a hinge region. In another embodiment, the heavy chain further comprises am additional constant domain, CH4.
In one embodiment of the invention, the antigen-binding determinant of an immunoglobulin does not compete with ACE2 binding at SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the antigen-binding determinant is that of CR3022 scFv or comprises CDRs of CR3022 scFv. In yet another embodiment, the CDRs of CR3022 scFv are defined by Kabat method or IMGT method.
In one embodiment, the antibody, antibody fragment, immunoglobulin, diabody, scFv or Fc is human or humanized. In another embodiment, the ACE2 component consists of or comprises ACE2 extracellular domain, its variant or fragment thereof and an immunoglobulin heavy chain of a Fc fragment. In another embodiment, the ACE2 extracellular domain, its variant or fragment thereof is linked at its C-terminus to the immunoglobulin heavy chain of a Fe fragment. In another embodiment, the ACE2 extracellular domain or fragment thereof has a sequence as described in any of the figures or SEQ ID NO: 2-11. In a preferred embodiment, the ACE2 extracellular domain fragment is SEQ ID NO: 3. In another embodiment, the variant may be a variant, allelic variant or combination of variants and/or allelic variants. In another embodiment, the variant, allelic variant or combination of variants and/or allelic variants comprise one or more amino acid substitutions relative to reference ACE2 protein sequence (
In one embodiment, these amino acid substitutions may comprise an alteration at an HEXXH zinc-binding motif corresponding to amino acids 374 to 378 of
The amino acid substitutions at reference ACE2 protein may be at any of S19, 121, E22, E23, Q24, A25, K26, T27, L29, D30, K31, N33, H34, E35, L39, F40, Y41, Q42, Q60, N64, A65, W69, F72, E75, Q76, L79, A80, M82, Q89, N90, L91, T92, V93, Q102, T324, Q325, N330, L351, H378, M383, A386, P389, R393, T445, Y510, S511, R518, S19P, S19V, S19W, S19Y, S19F, I21V, E22T, E23F, E23K, E23M, E23T, E23Q, E23C, Q24T, A25L, A25T, A25F, A25V, K26V, K26A, K26D, K26E, K26R, K26I, K26R, K31 W, T27K, T27M, T27L, 127A. T27D, T27H, 127W, T27Y, T27F, T27C, L29F, D30E, D301, D30V, K31Y, N33C, N33D, N33I, H34S, 1134V, H34A, 134P, E35C, E35D, E35M, E35V, L391, L39V, L39K, L39R, F40L, Y41R, Q42V, Q42K, Q42H, Q42M, Q42L, Q42C, Q421, Q60R, N64K, A65W, W69L, W69C, W691, W69V, W69T, W69K, F72W, F72Y, E75A, E75K, E75R, E75S, E75T, E75Q, E75H, E75W, E75G, Q76M, Q76R, Q76Y, Q76I, Q76V, Q76T, L791, L79P, L79V, L79T, L79W, L79Y, L79F, A80G, M82C, Q89L, Q89D, Q89P, N90M, N90L, N90I, N90V, N90A, N90S, N90T, N90Q, N90D, N90E, N90K, N90R, N90H, N90W, N90Y, N90F, N90P, N90G, N90C, L91P, T92M, T92L, T92I, T92V, T92A, T92N, T92Q, T92D, T92E, T92K, T92R, T92H, T92W, T92Y, T92F, T92P, T92G, T92C, V93P, Q102P, T324A, T324E, T324P, Q325P. N330L, N330H, N330W, N330Y, N330F, L351F, H378R, M383T, A386I, A386L, P389D, R393K, T445M, Y510H, S511D and R518G, or a combination thereof.
In a specific embodiment, the allelic variant of a reference ACE2 protein comprises an amino acid change at any of S19, T27, N33, A80 and T92 or a combination thereof. For example, the amino acid change may include, but are not limited to any of S19P, T27A, A33, A80G and T92I and a combination thereof. In another embodiment, the allelic variant comprises an amino acid change at any of I21, K26, N64, Q102 and H378 or a combination thereof. For example, the amino acid change may include, but are not limited to any of I21V, K26R, N64K, Q102P and H378R or a combination thereof. In yet another embodiment, the variant comprises an amino acid change at any of E23, K26, F40, Q60, M383, T445 and Y510 or a combination thereof. For example, the amino acid change may include, but are not limited to any of E23K, K26E, F40L, Q60R, M383T, T445M and Y510H or a combination thereof.
In a specific embodiment, the variant of a reference ACE2 protein comprises an amino acid change at any of S19, 121, E23, K26, T27, N33, F40, N64, A80, N90, T92, Q102, H378, M383 and T445 or a combination thereof. For example, the amino acid change may include, but are not limited to, any of S19P, I21V, E23K, K26E, K26R, T27A, N33I, F40L, N64K, A80G, N90I, N90T, T92I, Q102P, H378R, M383T and T445M or a combination thereof. In another embodiment, the variant comprises amino acid changes at amino acid S19, 121, E23, K26, 127, N33, F40, N64, A80, N90, T92, Q102, 1378, M383 and T445. For example, the variant may comprise amino acid changes S19P, I21V, E23K, K26E, T27A, N33I, F40L, N64K, A80G, N90I, N90T, T92I, Q102P, H378R, M383T and T445M or, the variant may comprise amino acid changes S19P, I21V, E23K, K26R, T27A, F40L, N64K, N90I, N90T, T92I, Q102P, H378R, M383T and T445M.
In another embodiment, the ACE2 variant lacks peptidase or carboxypeptidase activity. In another embodiment, the variant comprises H374N, H378N or both. In another embodiment, the variant comprises H374N and H378N.
In one embodiment, the ACE2 extracellular domain, its variant or fragment thereof is directly linked to the immunoglobulin heavy chain of a Fe fragment without a linker to produce a single polypeptide chain. In another embodiment, a linker is used link to the immunoglobulin heavy chain. In a further embodiment, the linker is a peptide linker. In another embodiment, the peptide linker is between one to twenty amino acids. In another embodiment, the peptide linker is glycine and/or serine rich. Examples of the peptide linker include, but are not limited to G, GG, and GGGGSGG.
In one embodiment, the immunoglobulin heavy chain of a Fc fragment comprises CH2 and CH3 constant domains. In another embodiment, the immunoglobulin heavy chain of a Fc fragment further comprises a hinge region. In another embodiment, the immunoglobulin heavy chain of a Fe fragment further comprises CH4 constant domain. In another embodiment, the immunoglobulin heavy chain of a Fc fragment comprises CH2 and CH3 constant domains and a hinge region.
In one embodiment, the bispecific protein consists or comprises an immunoglobulin heavy chain comprising a variable domain, VH, three constant domains, CH1, CH2 and CH3, and a hinge region and an immunoglobulin light chain comprising a variable domain, VL, and a constant domain, CL, to form an antigen-binding determinant which binds to SARS-CoV-2 virus or SARS-CoV-2 S-protein but does not compete with ACE2 binding; and a third polypeptide comprising an ACE2 extracellular domain fragment comprising one or more amino acid change reducing or abolishing peptidase or carboxypeptidase activity linked to an immunoglobulin heavy chain, constant region fragment, an Fc fragment, comprising a hinge region and CH2 and CH3 constant domains.
Examples of the ACE2 extracellular domain fragment include, but are not limited to, a polypeptide from amino acid residue 1-740 of SEQ ID NO: 1, a polypeptide from amino acid residue 1-615 of SEQ ID NO: 1, a polypeptide from amino acid residue 1-393 of SEQ ID NO: 1, a polypeptide with SEQ ID NO: 2, a polypeptide with SEQ ID NO: 3, and a polypeptide with SEQ ID NO: 4 and variant thereof and wherein the polypeptide comprises one or more amino acid change that reduces or abolishes peptidase or carboxypeptidase activity.
In one embodiment, the ACE2 extracellular domain fragment is a polypeptide from amino acid residue 1-615 of SEQ ID NO: 1, a polypeptide with SEQ ID NO: 2 or variant thereof and wherein the polypeptide comprises one or more amino acid change that reduces or abolishes peptidase or carboxypeptidase activity.
In another embodiment, the ACE2 extracellular domain fragment comprises a polypeptide with SEQ ID NO: 2 or variant thereof and wherein the polypeptide comprises one or more amino acid change that reduces or abolishes peptidase or carboxypeptidase activity. In another embodiment, the ACE2 extracellular domain fragment is a polypeptide from amino acid residue 1-615 of SEQ ID NO: 1 and wherein the polypeptide comprises one or more amino acid change that reduces or abolishes peptidase or carboxypeptidase activity. Examples of the one or more amino acid change that reduces or abolishes peptidase or carboxypeptidase activity may include, but are not limited to, H374N, H378N, H378R, both H1374N and H378N, and both H374N and H378R.
In one embodiment, the variant comprises one or more amino acid substitution which increases binding or binding affinity of the ACE2 fragment for SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the immunoglobulin heavy chain constant domains additionally comprise one or more amino acid changes based on a “knob-in-hole” protein design principle, wherein the changes favor heterodimer formation between the immunoglobulin heavy chain comprising a heavy chain variable domain and the fragment of an immunoglobulin heavy chain linked to ACE2. In a further embodiment, the amino acid changes are in CH3 constant domain. In a further embodiment, the CH3 constant domain of a first heavy chain comprises at least one amino acid change to introduce a “knob” or “hole” and the CH3 constant domain of a second heavy chain comprises a complementary “hole” or “knob,” respectively, so as to permit fitting of a “knob” into a “hole,” thereby, favoring heterodimerization over homodimerization of a mixture of two different immunoglobulin heavy chains. In another embodiment, the complex additionally comprises at least one amino acid change in the CH3 constant domain of the second heavy chain so as to form the complementary “hole” or “knob.”
In one embodiment, the immunoglobulin component and ACE2 component, the immunoglobulin or the immunoglobulin heavy chain of the Fe fragment comprises a Fc heterodimer with functional Fc effector functions. In another embodiment, the bispecific protein complex, the immunoglobulin component and ACE2 component, the immunoglobulin or the immunoglobulin heavy chain of the Fc fragment comprises a Fc heterodimer mutated so as to reduce or abolish Fc effector function.
In one embodiment, the Fc effector function is to support binding of Fc receptor and/or complement protein 1q (C1q). In another embodiment, the Fc effector function is antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or a combination thereof. In another embodiment, the mutated Fc has one or more amino acid change. In a further embodiment, the amino acid change decreases or abolishes binding of the Fc receptor or complement protein 1q (C1q) to an immunoglobulin or immunoglobulin fragment. In another embodiment, the amino acid change decreases or abolishes binding of the Fcγ receptor or complement protein 1q (C1q) to IgG or IgG fragment. In another embodiment, the Fcγ receptor is any of Fcγ receptor 1, Fcγ receptor II and Fcγ receptor III and a combination thereof. In another embodiment, the amino acid change decreases or abolishes antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) or a combination thereof. In another embodiment, the amino acid change is at aspartic acid 265, asparagine 297 or both for IgG or equivalent, wherein equivalent is one or more amino acid change at other amino acid position of IgG reducing or abolishing Fc effector function or at a corresponding position or other position for IgM, IgD, IgA or IgE. In another embodiment, the amino acid change is any of D265A, N297G and both. In another embodiment, the amino acid change is D265A and N297G. In another embodiment, the bispecific protein further lacks or has reduced Fc effector function. In another embodiment, the bispecific protein further comprises D265A and N297G amino acid substitutions in heavy chain constant region. In another embodiment, the bi-specific protein comprises a homodimer of a polypeptide comprising an ACE2 extracellular domain fragment or its variants, a Fc immunoglobulin fragment, and a diabody or scFv. In another embodiment, the polypeptide comprises from the amino-to-carboxyl terminus: the ACE2 extracellular domain fragment or its variants, the Fc immunoglobulin fragment, and a diabody or scFv.
In one embodiment, the ACE2 extracellular domain fragment consists of or comprises amino acid residues 1-614 of SEQ ID NO: 1 or a polypeptide of SEQ ID NO: 3. In another embodiment, the ACE2 extracellular domain fragment additionally has reduced or lacks peptidase or carboxypeptidase activity. In another embodiment, the ACE2 extracellular domain fragment additionally comprises H374N and H378N amino acid substitutions, or alternatively, H374N and H378R amino acid substitutions. In another embodiment, the ACE2 variant increases binding affinity or binding to SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the immunoglobulin fragment, Fc, comprises a hinge region and CH2 and CH3 constant domains of a heavy chain immunoglobulin. In another embodiment, the Fc additionally has reduced or lacks Fc effector function. In another embodiment, the Fe additionally comprises D265A and N297G amino acid substitution.
In another embodiment, the diabody or scFv binds SARS-CoV-2 virus or SARS-CoV-2 S-protein at an antigenic site other than a site bound by ACE2 extracellular domain fragment and does not compete with ACE2 binding. In another embodiment, the diabody or scFv is derived from CR3022 scFv or comprises the CDRs of CR3022 scFv. In another embodiment, one or more peptide linkers may be used to link the ACE2 extracellular domain fragment or its variants, the Fc immunoglobulin fragment, and the diabody or scFv. In another embodiment, the protein is an antibody comprising two identical immunoglobulin heavy chains stabilized by intermolecular disulfide bonds at the hinge region, two identical immunoglobulin light chains with each light chain associated with a heavy chain so as to form a functional antigen-binding determinant and an ACE2 extracellular domain or its fragment, wherein the ACE2 extracellular domain or its fragment, optionally with a signal sequence, is linked to the amino terminus of each heavy chain.
In one embodiment, the protein is an antibody comprising two identical immunoglobulin heavy chains stabilized by intermolecular disulfide bonds at the hinge region, two identical immunoglobulin light chains with each light chain associated with a heavy chain so as to form a functional antigen-binding determinant and an ACE2 extracellular domain or its fragment, wherein the ACE2 extracellular domain or its fragment, optionally with a signal sequence, is linked to the carboxy terminus of each heavy chain. In another embodiment, the protein is an antibody comprising two identical immunoglobulin heavy chains stabilized by intermolecular disulfide bonds at the hinge region, two identical immunoglobulin light chains with each light chain associated with a heavy chain so as to form a functional antigen-binding determinant and an ACE2 extracellular domain or its fragment, wherein the ACE2 extracellular domain or its fragment, optionally with a signal sequence, is linked to the amino terminus of each light chain. In another embodiment, the protein is or comprises a homodimer of an immunoglobulin heavy chain fragment from a Fc immunoglobulin fragment (Fc heavy chain fragment) comprising a hinge region and two constant domains, CH2 and CH3, and an ACE2 extracellular domain or its fragment linked to amino terminus of the Fc heavy chain fragment, and further comprising an immunoglobulin heavy chain fragment from a Fab fragment (Fab heavy chain fragment), a scFv, a diabody or a target protein binding domain linked to carboxyl terminus of the Fc heavy chain fragment, wherein the homodimer comprises two Fc heavy chain fragments held together by disulfide bonds at the hinge region. In another embodiment, the protein is or comprises a homodimer of a polypeptide comprising a first component comprising an immunoglobulin heavy chain fragment from a Fc immunoglobulin fragment (Fc heavy chain fragment) comprising a hinge region and two constant domains, CH2 and CH3, a second component comprising an immunoglobulin heavy chain fragment from a Fab fragment (Fab heavy chain fragment), a scFv, a diabody or a target protein binding domain and a third component an ACE2 extracellular domain or its fragment, wherein the polypeptide comprises from amino-to-carboxyl terminus direction the second component, the first component and the third component, and wherein the homodimer is stabilized by disulfide bonds at the hinge region contained in the Fc heavy chain fragment of the first component.
In one embodiment, the protein is a bispecific protein with two binding specificities formed by a heterodimer comprising or consisting of a first polypeptide comprising an first immunoglobulin heavy chain fragment from a Fc immunoglobulin fragment (first Fe heavy chain fragment) comprising a hinge region and two constant domains, CH2 and CH3, and an immunoglobulin heavy chain fragment from a Fab fragment (Fab heavy chain fragment), a scFv, a diabody or a target protein binding domain linked to amino terminus of the first Fc heavy chain fragment, and a second polypeptide comprising a second immunoglobulin heavy chain fragment from a Fc immunoglobulin fragment (second Fc heavy chain fragment) comprising a hinge region and two constant domains, CH2 and CH3, and an ACE2 extracellular domain or its fragment linked to amino terminus of the second Fc heavy chain fragment, and further wherein heterodimer formation is favored between the first Fc heavy chain fragment and the second Fc heavy chain fragment by the introduction of complementary “knobs” and “holes” in the CH3 constant domain of the two different heavy chain fragments and wherein the heterodimer is stabilized by presence of disulfide bonds between the two hinge regions.
In a further embodiment, the bispecific protein comprises the Fab heavy chain fragment additionally comprises an immunoglobulin light chain, wherein the light chain associates with the first polypeptide so as to form a functional antigenic binding determinant. In another embodiment, the antigenic binding determinant is directed to SARS-CoV-2 virus or SARS-CoV-2 S-protein.
In one embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 740 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 740 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fe immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains and wherein the Fc further comprises D265A and N297G to reduce or abolish antibody effector function. In another embodiment, the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In one embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fe immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains and wherein the Fc further comprises D265A and N297G to reduce or abolish antibody effector function. In another embodiment, the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
45) In one embodiment, the ACE2 extracellular domain fragment additional comprises one or more amino acid changes which increases binding or binding affinity of the ACE2 fragment for SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the amino acid changes are at any of S19, 121. E23, K26, K26, T27, N33, F40, N64, A80, N90, T92, Q102, H378, M383 and T445 and a combination thereof. In another embodiment, the amino acid change is any of S19P, I21V, E23K, K26E, K26R, T27A, N33I, F40L, N64K, A800, N90I, N90T, T92I, Q102P, H378R, M383T and T445M and a combination thereof. In another embodiment, the ACE2 extracellular domain fragment additional comprises amino acid changes at S19, K26, T27, N90 and H378. In another embodiment, the amino acid changes are S19P, K26R, T27A, N90I and H378R. In another embodiment, the amino acid changes are S19P, K26R, T27A, N90T and H378R. In another embodiment, the ACE2 extracellular domain fragment additional comprises amino acid changes at S19, K26, T27, T92 and H378. In another embodiment, the amino acid changes are S19P, K26R, T27A, N92I and H378R. In another embodiment, the ACE2 extracellular domain fragment additional comprises amino acid changes at S19, T27 and N90. In another embodiment, the amino acid changes are S19P, T27A and N90I. In another embodiment, the amino acid changes are S19P, T27A and N90T. In another embodiment, the amino acid changes increase binding or binding affinity of the ACE2 fragment for SARS-CoV-2 virus or SARS-CoV-2 S-protein.
In one embodiment, the ACE2 extracellular domain fragment additional comprises amino acid changes to reduce or abolish peptidase or carboxypeptidase activity. In another embodiment, the ACE2 extracellular domain fragment additional comprises amino acid change at H374, H378 or both. Examples of the amino acid change include, but are not limited to, H374N, H378N, H378R, both H374N and H378N, and both H374N and H378R.
In another embodiment, the ACE2 extracellular domain fragment additional comprises either both H374N and H378N or both H374N and H378R amino acid substitution. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N and H378N amino acid substitutions.
In one embodiment, the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In one embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains and wherein the ACE2 fragment additionally comprises one or more amino acid changes selected from the group consisting of S19P, I21V, E23K, K26E, K26R, T27A, N33I, F40L, N64K, A80G, N90I, N90T, T92I, Q102P, H378R, M383T and T445M and a combination thereof. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fe fragment additionally comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment additionally comprises one or more amino acid changes selected from the group consisting of S19P, I21V, E23K, K26E, K26R, T27A, N33I, F40L, N64K, A80G, N90I, N90T, T92I, Q102P, H378R, M383T and T445M and a combination thereof. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fe immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the ACE2 fragment additionally comprises one or more amino acid changes selected from the group consisting of S19P, I21V, E23K, K26E, K26R, T27A, N33I, F40L, N64K, A80G, N90I, N90T, T92L, Q102P, H378R, M383T and T445M and a combination thereof, and wherein the ACE2 fragment additionally comprises H374N and either H378N or N378R amino acid substitutions.
In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, wherein the ACE2 fragment additionally comprises one or more amino acid changes selected from the group consisting of S19P, I21V, E23K, K26E, K26R, T27A, N33I, F40L, N64K, A80G, N90I, N90T, T92L, Q102P, H378R, M383T and T445M and a combination thereof, and wherein the ACE2 fragment additionally comprises H374N and either H378N or H378R amino acid substitutions.
In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fe immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, and wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and 1-1378R amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fe immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N amino acid substitutions.
In another embodiment, the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, and wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fe immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33L, A80G, N90I, T92I and H378R amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A800, N90I, T92I and H378R amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fe immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N amino acid substitutions.
In one embodiment, the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In one embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, and wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N amino acid substitutions.
In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fe immunoglobulin heavy chain fragment comprises a hinge region, and CH12 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, wherein the ACE2 fragment additionally comprises S19P, K26R, T27A, N33I, A80G, N90I, T92I and H378R amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N amino acid substitutions.
In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, wherein the ACE2 fragment additionally optionally comprises S19P, K26R, T27A, N33I, or N33I, A80G, and T92I and I378R amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N amino acid substitutions.
In another embodiment, the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, and wherein the ACE2 fragment additionally comprises S19P, T27A and N90I amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fe immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment additionally comprises S19P, T27A and N90I amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fe immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the ACE2 fragment additionally comprises S19P, T27A and N90I amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N and H378N amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, wherein the ACE2 fragment additionally comprises S19P, T27A and N90I amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N and H378N amino acid substitutions.
In one embodiment the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In one embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, and wherein the ACE2 fragment additionally comprises S19P, T27A and N90T amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fe immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment additionally comprises S19P, T27A and N90T amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fe immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the ACE2 fragment additionally comprises S19P, T27A and N90T amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N and H378N amino acid substitutions. In another embodiment, the protein consists of or comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 linked to amino terminus of a Fc immunoglobulin heavy chain fragment, wherein the Fc immunoglobulin heavy chain fragment comprises a hinge region, and CH2 and CH3 constant domains, wherein the Fc fragment additionally comprises D265A and N297G amino acid substitutions, wherein the ACE2 fragment additionally comprises S19P, T27A and N90T amino acid substitutions, and wherein the ACE2 fragment additionally comprises H374N and H378N amino acid substitutions.
In another embodiment, the protein has the following amino acid sequence:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the immunoglobulin is human or humanized. In another embodiment, the ACE2 fragment is a fragment of human ACE2 protein. In another embodiment, the protein is a homodimer comprising intermolecular disulfide bonds at the hinge region of two polypeptide chains derived from the Fc immunoglobulin heavy chain fragment. In another embodiment, the homodimer is mono-specific. In another embodiment, the homodimer is bivalent. In another embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs and a Fc immunoglobulin fragment, wherein the segmented ACE2 protein secondary structural motifs are ACE2 helix 2 peptide as provided in SEQ ID NO: 6, ACE2 helix 1 peptide as provided in SEQ ID NO: 7 and ACE2 beta turn peptide as provided in SEQ ID NO: 8, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, ACE2 helix 2 peptide-ACE2 helix 1 peptide-ACE2 beta turn peptide and linked by glycine containing linkers to form helix 2-helix 1-beta turn structure (HHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, and wherein the HHB synthetic binding domain is linked to amino terminus of the Fc fragment to form HHB-Fc hybrid protein. In a further embodiment, the HHB synthetic binding domain binds SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the HHB-Fc hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In another embodiment, the homodimer is mono-specific but bivalent. In another embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs and a Fc immunoglobulin fragment, wherein the segmented ACE2 protein secondary structural motifs are ACE2 helix 2 peptide as provided in SEQ ID NO: 6, ACE2 helix 1 peptide as provided in SEQ ID NO: 7 and ACE2 beta turn peptide as provided in SEQ ID NO: 8, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, ACE2 helix 2 peptide-ACE2 helix 1 peptide-ACE2 beta turn peptide and linked by glycine containing linkers to form helix 2-helix 1-beta turn structure (HHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, wherein the Fc fragment further comprises D265A and N297G amino acid substitutions reducing or abolishing Fc effector function, and wherein the H4B synthetic binding domain is linked to amino terminus of the Fc fragment to form HHB-Fc DANG hybrid protein.
In another embodiment, the HHB-Fc DANG hybrid protein consists of or comprises an amino acid sequence as shown:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the HHB-Fc DANG hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In a further embodiment, the homodimer is mono-specific but bivalent.
In another embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs, a Fc immunoglobulin fragment and a signal sequence (SS), wherein the segmented ACE2 protein secondary structural motifs are ACE2 helix 2 peptide as provided in SEQ ID NO: 6, ACE2 helix 1 peptide as provided in SEQ ID NO: 7 and ACE2 beta turn peptide as provided in SEQ ID NO: 8, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, ACE2 helix 2 peptide-ACE2 helix 1 peptide-ACE2 beta turn peptide and linked by glycine containing linkers to form helix 2-helix 1-beta turn structure (HHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, wherein the Fc fragment further comprises D265A and N297G amino acid substitutions reducing or abolishing Fc effector function, and wherein the signal sequence is found at the amino terminus of HHB synthetic binding domain which is linked at its carboxyl terminus to amino terminus of the Fc fragment to form SS-HHB-Fc DANG hybrid protein.
In another embodiment, the SS-HHB-Fc DANG hybrid protein consists of or comprises an amino acid sequence as shown:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the SS-HHB-Fc DANG hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In another embodiment, the homodimer is mono-specific but bivalent.
In one embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs and a Fe immunoglobulin fragment, wherein the segmented ACE2 protein secondary structural motifs are minimal ACE2 helix 2 peptide as provided in SEQ ID NO: 9, minimal ACE2 helix 1 peptide as provided in SEQ ID NO: 10 and minimal ACE2 beta turn peptide as provided in SEQ ID NO: 11, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, minimal ACE2 helix 2 peptide-minimal ACE2 helix 1 peptide-minimal ACE2 beta turn peptide and linked by glycine containing linkers to form minimal helix 2-helix 1-beta turn structure (minHHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, and wherein the minHHB synthetic binding domain is linked to amino terminus of the Fc fragment to form minHHB-Fc hybrid protein. In another embodiment, the minHHB synthetic binding domain binds SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the minHHB-Fc hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In yet another embodiment, the homodimer is mono-specific but bivalent.
In one embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs and a Fc immunoglobulin fragment, wherein the segmented ACE2 protein secondary structural motifs are minimal ACE2 helix 2 peptide as provided in SEQ ID NO: 9, minimal ACE2 helix 1 peptide as provided in SEQ ID NO: 10 and minimal ACE2 beta turn peptide as provided in SEQ ID NO: 11, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, minimal ACE2 helix 2 peptide-minimal ACE2 helix 1 peptide-minimal ACE2 beta turn peptide and linked by glycine containing linkers to form minimal helix 2-helix 1-beta turn structure (minHHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, wherein the Fc fragment further comprises D265A and N297G amino acid substitutions reducing or abolishing Fc effector function, and wherein the minHHB synthetic binding domain is linked to amino terminus of the Fc fragment to form minHHB-Fc DANG hybrid protein. In another embodiment, the minHHB-Fc DANG hybrid protein consists of or comprises an amino acid sequence as shown:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In one embodiment, the minHHB-Fc DANG hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In another embodiment, the homodimer is mono-specific but bivalent.
72) In another embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs, a Fe immunoglobulin fragment and a signal sequence (SS), wherein the segmented ACE2 protein secondary structural motifs are minimal ACE2 helix 2 peptide as provided in SEQ ID NO: 9, minimal ACE2 helix 1 peptide as provided in SEQ ID NO: 10 and minimal ACE2 beta turn peptide as provided in SEQ ID NO: 11, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, minimal ACE2 helix 2 peptide-minimal ACE2 helix 1 peptide-minimal ACE2 beta turn peptide and linked by glycine containing linkers to form minimal helix 2-helix 1-beta turn structure (minHHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, wherein the Fc fragment further comprises D265A and N297G amino acid substitutions reducing or abolishing Fc effector function, and wherein the signal sequence is found at the amino terminus of minHHB synthetic binding domain which is linked at its carboxyl terminus to amino terminus of the Fe fragment to form SS-minHHB-Fc DANG hybrid protein.
In another embodiment, the SS-minHHB-Fc DANG hybrid protein consists of or comprises an amino acid sequence as shown:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs and a Fc immunoglobulin fragment, wherein the segmented ACE2 protein secondary structural motifs are minimal ACE2 helix 1 peptide as provided in SEQ ID NO: 10 and ACE2 beta turn peptide as provided in SEQ ID NO: 8, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, minimal ACE2 helix 1 peptide-ACE2 beta turn peptide and linked by glycine containing linkers to form minimal helix 1-beta turn structure (minHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, and wherein the minHB synthetic binding domain is linked to amino terminus of the Fc fragment to form minHB-Fc hybrid protein. In another embodiment, the minHB synthetic binding domain binds SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the minHB-Fc hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In another embodiment, the homodimer is mono-specific but bivalent.
In another embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs and a Fc immunoglobulin fragment, wherein the segmented ACE2 protein secondary structural motifs are minimal ACE2 helix 1 peptide as provided in SEQ ID NO: 10 and ACE2 beta turn peptide as provided in SEQ ID NO: 8, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, minimal ACE2 helix 1 peptide-ACE2 beta turn peptide and linked by glycine containing linkers to form minimal helix 1-beta turn structure (minHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH3 constant domains, wherein the Fc fragment further comprises D265A and N297G amino acid substitutions reducing or abolishing Fc effector function, and wherein the minHB synthetic binding domain is linked to amino terminus of the Fc fragment to form minHB-Fc DANG hybrid protein.
In another embodiment, the minHB-Fc DANG hybrid protein consists of or comprises an amino acid sequence as shown:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the minHB-Fc hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In another embodiment, the homodimer is mono-specific but bivalent.
In one embodiment, the protein comprises a synthetic binding domain comprising a combination of segmented ACE2 protein secondary structural motifs, a Fc immunoglobulin fragment and a signal sequence (SS), wherein the segmented ACE2 protein secondary structural motifs are minimal ACE2 helix 1 peptide as provided in SEQ ID NO: 10 and ACE2 beta turn peptide as provided in SEQ ID NO: 8, wherein the structural motifs are linked in the order from amino-to-carboxyl direction, minimal ACE2 helix 1 peptide-ACE2 beta turn peptide and linked by glycine containing linkers to form minimal helix 1-beta turn structure (minHB), wherein the Fc fragment comprises an immunoglobulin heavy chain constant region fragment comprising a hinge region and CH2 and CH13 constant domains, wherein the Fc fragment further comprises D265A and N297G amino acid substitutions reducing or abolishing Fc effector function, and wherein the signal sequence is found at the amino terminus of minHB synthetic binding domain which is linked at its carboxyl terminus to amino terminus of the Fc fragment to form SS-minHB-Fc DANG hybrid protein. In another embodiment, the minHB-Fc DANG hybrid protein consists of or comprises an amino acid sequence as shown:
In another embodiment, the protein may include the amino acid sequence as shown in any of
In another embodiment, the minHB-Fc hybrid protein forms a homodimer stabilized by intermolecular disulfide bonds at the hinge region of two polypeptide chains. In another embodiment, the homodimer is mono-specific but bivalent.
80) In one embodiment, the protein comprises a comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 or its variant, a heavy chain constant region fragment corresponding to a Fc portion and a non-ACE2-competing anti-SARS-CoV-2 virus or S-protein diabody or scFv, wherein the ACE2 fragment is linked to amino terminus of the heavy chain constant region fragment, which is in turn linked at its carboxyl terminus to the amino terminus of the diabody or scFv, wherein the ACE2 fragment further comprises H374N and H378N amino acid substitutions, wherein the Fc portion further comprises D265A and N297G amino acid substitutions, and wherein the ACE2 fragment is linked to amino terminus of the heavy chain constant region fragment, which is in turn linked at its carboxyl terminus to the amino terminus of the diabody or scFv to produce a ACE2 extracellular domain fragment-Fc-diabody or scFv fusion protein.
In another embodiment, the protein comprises a homodimer of two identical polypeptides, wherein the polypeptide comprises an ACE2 signal sequence and extracellular domain fragment from amino acid residue 1 to 615 of SEQ ID NO: 1 or its variant, a heavy chain constant region fragment corresponding to a Fc portion and a non-ACE2-competing anti-SARS-CoV-2 virus or S-protein diabody or scFv, wherein the ACE2 fragment is linked to amino terminus of the heavy chain constant region fragment, which is in turn linked at its carboxyl terminus to the amino terminus of the diabody or scFv, wherein the ACE2 fragment further comprises H374N and H378N amino acid substitutions, wherein the Fc portion further comprises D265A and N297G amino acid substitutions, wherein the Fc portion additionally comprises intermolecular disulfide bonds stabilizing the homodimer, and wherein the ACE2 fragment is linked to amino terminus of the heavy chain constant region fragment, which is in turn linked at its carboxyl terminus to the amino terminus of the diabody or scFv to produce a homodimer of ACE2 extracellular domain fragment-Fe-diabody or scFv fusion protein. In another embodiment, the ACE2 extracellular domain fragment-Fe-diabody or scFv fusion protein has reduced or lacks ACE2 peptidase or carboxypeptidase activity. In another embodiment, the ACE2 extracellular domain fragment-Fc-diabody or scFv fusion protein has reduced or lacks Fc effector function. In another embodiment, the variant of ACE2 extracellular domain fragment increases or enhances binding of ACE2 to SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the ACE2 extracellular domain fragment-Fc-diabody or scFv fusion protein is bispecific. In another embodiment, the ACE2 extracellular domain fragment-Fc-diabody or scFv fusion protein is bivalent. In another embodiment, the diabody or scFv is derived from CR3022 scFv or comprises CDRs of CR3022 scFv. In another embodiment, the diabody or scFv and Fc portion is human or humanized.
The invention further provides a bispecific knob-hole format ACE2 extracellular domain anti-SARS-Cov-2 S-protein antibody. In one embodiment, the antibody comprises a complex of three polypeptide chains, wherein the first polypeptide comprises a fusion of ACE2 extracellular domain fragment or its variant to amino terminus of an immunoglobulin heavy chain fragment corresponding to Fc portion comprising a hinge region and CH2 and CH3 constant domains, a second polypeptide comprising an immunoglobulin heavy chain comprising a heavy chain variable domain, a hinge region and CH1, CH2 and CH3 constant domains, and a third polypeptide comprising an immunoglobulin light chain comprising a light chain variable domain and a light chain constant region, wherein the CH3 domain of the 1st and 2nd polypeptides are mutated so as to create complementary “knobs” and “holes” based on “knob-in-hole” protein design in order to favor formation of heterodimer between the 1st and 2nd polypeptides, wherein the heterodimer additionally comprises intermolecular disulfide bonds in the hinge region, and wherein the 3rd polypeptide associates with the 2nd polypeptide in order to form an antigen-binding determinant.
In one embodiment, the antigen-binding determinant binds to SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the antigen-binding determinant does not compete with ACE2 binding to SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the antigen-binding determinant is derived from CR3022 scFv or comprises CDRs of CR3022 scFv. In another embodiment, the variable domain of the light chain or heavy chain is derived from CR3022 scFv or comprises one or more CDRs of CR3022 scFv. Examples of the ACE2 extracellular domain fragment include, but are not limited to, a polypeptide from amino acid residue 1-740 of SEQ ID NO: 1, a polypeptide from amino acid residue 1-615 of SEQ ID NO: 1, a polypeptide from amino acid residue 1-393 of SEQ ID NO: 1, a polypeptide with SEQ ID NO: 2, a polypeptide with SEQ ID NO: 3 and a polypeptide with SEQ ID NO: 4.
In one embodiment, the variant of the ACE2 extracellular domain fragment comprises one or more amino acid change in ACE2 fragment which increases binding or binding affinity of the fragment for SARS-CoV-2 virus or SARS-CoV-2 S-protein. In another embodiment, the 1st and 2nd polypeptides additionally comprise D265A and N297G amino acid substitutions in the Fc portion. In another embodiment, the immunoglobulin and ACE2 are human or humanized.
Another embodiments of the invention is an ACE2ecd(1-615)-(T92I)-H374N-H378N-Fc-(DANG)-3B11scFv and DPP4ecd(39-766)-S630A-Fc-(DANG)-CR3022scFv as shown in
The invention provides a pharmaceutical composition comprising any of the compositions of the invention described herein including isolated SARS-CoV-2 binding protein complexes and bispecific antibodies of the invention above, and one or more pharmaceutically acceptable excipients or carriers.
The invention further provides a pharmaceutical composition comprising the bispecific knob-hole format ACE2 extracellular domain anti-SARS-Cov-2 S-protein antibody of the invention above, and one or more pharmaceutically acceptable excipients or carriers.
In one embodiment, the one or more pharmaceutically acceptable excipients are formulated for delivery as a nasal or oral spray. In another embodiment, the one or more pharmaceutically acceptable excipients or carriers are formulated or carriers are formulated for delivery as a throat lozenge or a cough drop. In another embodiment, the one or more pharmaceutically acceptable excipients or carriers are formulated as a mouth wash. In another embodiment, the one or more pharmaceutically acceptable excipients or carriers are formulated as an injectable drug.
In one embodiment, the one or more pharmaceutically acceptable excipients or carriers are formulated for parenteral administration. Examples of parenteral administration include, but are not limited to, intradermal, subcutaneous, intramuscular, intravenous, intra-arterial, intrathecal, intraperitoneal and intra-articular administration.
In another embodiment, the one or more pharmaceutically acceptable excipients are formulated for oral administration. Examples of forms of oral administration include, but are not limited to, tablet, capsule, soft-gelled capsule, hard-shelled capsule, orally disintegrating tablet, buccal tablet, sublingual table, mini-tablet, effervescent tablet, immediate release tablet, controlled release tablet, immediate-and-controlled release tablet, think film, medicated gum, granule, troche, lozenge, solution, suspension, syrup, emulsion, elixir, and buccal spray.
In another embodiment, the one or more pharmaceutically acceptable excipients are formulated for nasal administration. Examples of forms of nasal administration include, but are not limited to, nasal drop or nasal spray.
In another embodiment, the one or more pharmaceutically acceptable excipients are formulated for inhalation. Examples of forms of inhalation include, but are not limited to, dry powder, lyophilized powder and liquid spray.
In another embodiment, the one or more pharmaceutically acceptable excipients are formulated for ocular administration. Examples of forms of ocular administration include, but are not limited to, solution, emulsion, suspension, ointment, contact lens, implant, insert and intravitreal.
In another embodiment, the one or more pharmaceutically acceptable excipients are formulated for otic administration. Examples of forms of otic administration include, but are not limited to, topical, intratympanic and intracochlear.
In another embodiment, the one or more pharmaceutically acceptable excipients are formulated for topical or transdermal administration. Examples of forms of topical or transdermal administration include, but are not limited to, ointment, cream, lotion, gel, spray and patch.
In another embodiment, the one or more pharmaceutically acceptable excipients are formulated for rectal or vaginal administration. Examples of forms of rectal or vaginal administration include, but are not limited to, suppository, enema, tablet, pessary, gel, cream, foam and sponge
The invention further provides a nucleic acid sequence encoding an isolated SARS-CoV-2 binding protein complex of the invention as described herein.
Examples of nucleic acid sequences encoding full length, wild-type human ACE2 protein (SEQ ID NO: 1; UniProtKB ID: Q9BYF1-1) may be accessed under GenBank Accession number: AF291820.1 or AF241254.1. Such coding sequences can be modified to introduce desired mutations as shown in the variants described herein that increases binding or binding affinity for SARS-CoV-2 virus or SARS-CoV-2 S-protein. In addition, the coding sequences provided for full length human ACE2 protein can be truncated using recombinant DNA methods to produce desired ACE2 fragments, so as to practice the full breath of the instant invention. Such fragments may be linked in frame with other coding sequences to produce desired fusion proteins as described herein following introduction to DNA vector, typically providing regulatory signals such as transcriptional promoter/enhancer and terminator, for expression in host systems or in vitro by in vitro transcription-translation system. Further, the nucleic acid sequences which encode amino acid sequences corresponding to polypeptides disclosed in the instant invention can be identified using the GenBank Accession numbers described herein and the gene transcript identifiers. Additionally, based on publicly available codon usage tables, nucleic acid sequence encoding polypeptides of interest can be designed for optimal gene expression for a variety of organisms, including humans (Athey, J. et al. (2017) A new and updated resource for codon usage tables. BMC Bioinformatics. 18 (391): 391; Alexaki, A. et al. (2019) Codon and Codon-Pair Usage Tables (CoCoPUTs): Facilitating Genetic Variation Analyses and Recombinant Gene Design. J. Mol. Biol. 431 (13): 2434-2441).
The invention further provides a nucleic acid encoding a bispecific knob-hole format ACE2 extracellular domain anti-SARS-Cov-2 S-protein antibody of the invention as described herein.
Additionally, the invention provides a vector comprising a nucleic acid of the invention above. The invention also provides a cell comprising a nucleic acid of the invention above. The invention further provides a cell comprising a vector of the invention.
Further, the invention also provides a host vector system, comprising a nucleic acid molecule of the invention above and a host cell. In one embodiment, the host cell is a prokaryote or eukaryote.
The invention also provides methods for making a SARS-CoV-2 binding protein. In one embodiment, the method comprises growing the cells of the invention above under suitable conditions so as to produce the isolated SARS-CoV-2 binding protein.
The invention also provides methods for making a bispecific knob-hole format ACE2 extracellular domain anti-SARS-Cov-2 S-protein antibody. In one embodiment, the method comprises growing the cells of the invention above under suitable conditions so as to produce the isolated SARS-CoV-2 binding protein.
The invention also provides methods for producing a protein comprising growing the host vector systems of the invention in cells above under suitable conditions so as to produce the protein in the host and recovering the protein so produced.
Any of the compositions of the invention described herein including the isolated SARS-CoV-2 complexes, bispecific antibodies and conjugates/fusion proteins containing the ACE2 variants of the invention may be provided in a pharmaceutically acceptable excipient or carrier, and may be in various formulations. As is well known in the art, a pharmaceutically acceptable excipient or carrier is a relatively inert substance that facilitates administration of a pharmacologically effective substance. For example, an excipient can give form or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995).
Pharmaceutically acceptable excipients/carriers are generally non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. Examples of pharmaceutically acceptable carriers include water, saline, Ringer's solution, dextrose solution, ethanol, polyols, vegetable oils, fats, ethyl oleate, liposomes, waxes polymers, including gel forming and non-gel forming polymers, and suitable mixtures thereof. The carrier may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient.
Generally, these compositions are formulated for administration by injection or inhalation, e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc. Accordingly, these compositions are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history.
The invention provides a specific formulation comprising an isolated SARS-CoV-2 binding protein complex of the invention mentioned above. In one embodiment, the formulation is a hand or body lotion, cream, emulsion, ointment, gel, spray or patch.
The invention also provides a formulation comprising the bispecific knob-hole format ACE2 extracellular domain anti-SARS-Cov-2 S-protein antibody of the invention mentioned above.
In one embodiment, the formulation may be an eye drop comprising an isolated SARS-CoV-2 binding protein and a stabilizing solution, optionally with a preservative and/or a carrier. In another embodiment, the formulation is a nasal spray or mouth spray. In another embodiment, the formulation is a nasal wash or mouth wash.
The invention provides methods for treating a subject infected with SARS-CoV-2 virus with any of the compositions of the invention.
In one embodiment of the invention, the method comprises administering an effective amount of a soluble fragment of angiotensin-converting enzyme 2 (ACE2) so as to inhibit or reduce SARS-CoV-2 virus interaction with ACE2 receptor of the subject so as to limit, inhibit or reduce infection in the subject, thereby treating the subject infected with SARS-CoV-2 virus.
In another embodiment, the method comprises administering an effective amount of an ACE2-Fc fusion protein containing the protease domain 19-617 or deletion of the domain, so as to inhibit or reduce a SARS-CoV-2 virus interaction with ACE2 receptor of the subject so as to limit, inhibit or reduce infection in the subject, thereby treating the subject infected with SARS-CoV-2 virus.
In another embodiment, the method comprises administering an effective amount of an ACE2-Fc with c-terminal anybody fusion (Fab or ScFV) that bind to viral proteins (S-protein, M-protein or N-protein), so as to inhibit or reduce SARS-CoV-2 virus interaction with ACE2 receptor of the subject so as to limit, inhibit or reduce infection in the subject, thereby treating the subject infected with SARS-CoV-2 virus.
The invention also provides methods for inhibiting or reducing SARS-CoV-2 virus infection of a susceptible subject. In one embodiment, the method comprises administering an effective amount of a soluble fragment of angiotensin-converting enzyme 2 (ACE2) so as to inhibit or reduce SARS-CoV-2 virus interaction with ACE2 receptor of the subject, thereby inhibiting or reducing SARS-CoV-2 virus infection of a susceptible subject.
In one embodiment of any of the method above, the amino acid sequence of ACE2 is provided in SEQ ID NO:1 (UniProtKB ID: Q9BYF1-1):
In one embodiment of any of the method above, the soluble fragment consists of amino acid residues 18-708. In another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 805 amino acid residues of ACE2. In yet another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 741 amino acid residues of ACE2. In another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 617 amino acid residues of ACE2. In another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 400 amino acid residues of ACE2. In another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 250 amino acid residues of ACE2. In another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 150 amino acid residues of ACE2. In another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 75 amino acid residues of ACE2. In another embodiment of any of the method above, the soluble fragment consists or comprises a protein fragment of at least 35 amino acid residues but less than 50 amino acid residues of ACE2. In another embodiment of any of the method above, the soluble fragment consists or comprises an ACE2 protein fragment of at least 35 amino acid residues but less than 50 amino acid residues of ACE2.
In accordance with the practice of the invention, in one embodiment of any of the method above, the soluble fragment consists or comprises N-terminal domain of ACE2 peptidase domain. In a further embodiment, the peptidase domain consists of amino acid residues 18-606. In another embodiment, the N-terminal domain of ACE2 peptidase domain consists of the SARS-CoV-2 receptor binding site as shown in the SARS-CoV-2 virus RBD footprint of
In accordance with the practice of the invention, in one embodiment of any of the method above, the soluble fragment has a higher affinity than the same fragment derived from UniProtKB ID: Q9BYF1-1 (SEQ ID NO: 1). In a further embodiment, the soluble fragment having a higher affinity comprises one or more amino acid changes. Examples of the one or more amino acid changes include, but are not limited to, S19P, 121T/V, E23K, A25T, K26E or K26R, T27A, F40L, Q60R, N64K, W69C, T92I, Q102P, Q325R, M366T, D367V, H374R, H378R, M383T, E398D, E398K, T445M, 1446M, and Y510H.
In accordance with the practice of the invention, in one embodiment of any of the method above, the soluble fragment is monomeric. In another embodiment of any of the method above, the soluble fragment is coupled to one or more soluble fragment, so as to produce two or more soluble ACE2 fragments which are linked to each other. In another embodiment of any of the method above, the soluble fragment is coupled to a biologically compatible macromolecule. In another embodiment of any of the method above, the soluble fragment is a chimeric protein. In another embodiment of any of the method above, the soluble fragment is a recombinant protein.
In accordance with the practice of the invention, in one embodiment of any of the method above, the subject is a mammal. In a further embodiment, the mammal is a human. Examples of mammals include, but are not limited to, a human or an animal such as a non-human primate, pig, mouse, rat, dog, cat, horse, monkey, ape, rabbit or cow.
The invention also provides methods for monitoring the course of a SARS-CoV-2 infection in a subject using any of the compositions of the invention. In one embodiment, the method comprises obtaining a sample from the subject, determining amino acid sequence of ACE2 of the subject, comparing identity of amino acid so determined to reference amino acids known to affect SARS-CoV-2 interaction with ACE2, wherein finding an amino acid change favoring interaction with surface spike glycoprotein, S protein, of SARS-CoV-2 are any of S19P, I21T/V, E23K, A25T, K26E or K26R, T27A, F40L, Q60R, N64K, W69C, T92I, Q102P, Q325R, M366T, D367V, H374R, H378R, M383T, E398D, E398K, T445M, I446M, and Y510H, and wherein an amino acid change resulting in less favorable interaction with S protein of SARS-CoV-2 are any of K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51D or N51S, M62I or M62V, A65S, K68E, F72H, M82I, Y83H, P84T, V93G, N290H, G326E, E329G, P346S, G352V, D355N, T371K, Q388L, P389H, F504I or F504L, H505R, D509Y, S511P, R514G, Y515C and R518T and predicting a subject to have a more severe course of infection for the subject with an amino acid change favoring interaction with S protein of SARS-CoV-2 or a milder course of infection for the subject with an amino acid change resulting in less favorable interaction with S protein of SARS-CoV-2.
The invention additionally provides methods for assessing risk of being infected by SARS-CoV-2 virus in a subject using any of the compositions of the invention. In one embodiment, the method comprises obtaining a sample from the subject, determining amino acid sequence of ACE2 of the subject, comparing identity of amino acid so determined to reference amino acids known to affect SARS-CoV-2 interaction with ACE2, wherein finding an amino acid change resulting in increased risk of being infected are any of S19P, I21T/V, E23K, A25T, K26E or K26R, T27A, F40L, N64K, Q60R, N64K, W69C, T92I, Q102P, Q325R, M366T, D367V, H374R, H378R, M383T, E398D, E398K, T445M, I446M, and Y510H, and wherein an amino acid change resulting in decreased risk of being infect are any of K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51D or N51S, M62I or M62V, A65S, K68E, F72H, M82I, Y83H, P84T, V93G, N290H, G326E, E329G, P346S, G352V, D355N, T371K, Q388L, P389H, F504I or F504L, H505R, D509Y, S5M1P, R514G, Y515C and R518T and predicting a subject to have an increased or decreased risk based on finding a match falling into the two groups.
The invention further provides methods for determining presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a sample using any of the compositions of the invention. In one embodiment, the method comprises applying a fixed volume of a sample to the lateral flow diagnostic cassette of the invention mentioned above. In another embodiment, the method further comprises adding a fixed volume of the buffer. In another embodiment, the method further comprises waiting for a prescribed amount of time. In another embodiment, the method further comprises examining the cassette for emergence of visible lines. In another embodiment, the method further comprises determining the number and location of one or more lines; wherein presence of one line further away from the sample well indicates absence of or below detection limit for SARS-CoV-2 virus or SARS-CoV-2 S-protein, presence of two lines each line closest to edge of window of the cassette indicate presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein, and presence of three lines or no line indicates a lack of confidence in the test result, thereby determining presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a sample.
In one embodiment, the sample is a liquid or liquid-air mixture. Examples of the liquid or liquid-air mixture include, but are not limited to, blood, serum, bodily fluid, saliva, nasal drip, respiratory droplet, aerosol, sputum, phlegm, mucus, secretion, urine, fecal material, tissue culture media, spent media, biological extract, known SARS-CoV-2-containing fluid, and suspect SARS-CoV-2 containing fluid. In a preferred embodiment, the sample is human blood, serum, or a bodily fluid.
In another embodiment of the method for determining presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a subject, the method comprises attaching a nose cone of the lateral flow diagnostic kit of the invention for directing nasal spray to the sample well or a mask of the invention.
In another embodiment, the method for determining presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a subject further comprises placing the sample well of the lateral flow diagnostic cassette of the lateral flow diagnostic kit of the invention directly under the second opening. In another embodiment, the method further comprises forcefully expelling air through a nostril attached to the nose cone or coughing through the mouth covered with the mask. In another embodiment, the method further comprises repeating the expelling step mentioned above if required or desired. In another embodiment, the method further comprises adding a fixed volume of the buffer of the invention mentioned above. In another embodiment, the method further comprises waiting for a prescribed amount of time. In another embodiment, the method further comprises examining the cassette for emergence of visible lines. In another embodiment, the method further comprises determining the number and location of one or more lines; wherein presence of one line further away from the sample well indicates absence of or below detection limit for SARS-CoV-2 virus or SARS-CoV-2 S-protein, presence of two lines each line closest to edge of window of the cassette indicate presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein, and presence of three lines or no line indicates a lack of confidence in the test result, thereby determining presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a sample.
In another embodiment of the method, the method comprises immobilizing the isolated SARS-CoV-2 binding protein complex of the invention mentioned above or a fragment thereof lacking a signal sequence on a surface of a solid support. In another embodiment, the method further comprises contacting the isolated SARS-CoV-2 binding protein of the immobilization step above with the sample. In another embodiment, the method further comprises washing unbound sample off the immobilizing surface. In another embodiment, the method further comprises contacting the immobilizing surface with a biotinylated CR3022 antibody in a full-length immunoglobulin format wherein biotin is conjugated to Fc portion of the immunoglobulin. In another embodiment, the method further comprises washing unbound biotinylated CR3022 antibody off the immobilizing surface. In another embodiment, the method further comprises contacting the immobilizing surface with streptavidin conjugate horse radish peroxidase. In another embodiment, the method further comprises washing unbound streptavidin conjugate horse radish peroxidase off the immobilizing surface. In another embodiment, the method further comprises contacting the immobilizing surface with a chromogenic or fluorogenic substrate for horse radish peroxidase for a fixed length of time. In another embodiment, the method further comprises determining presence of a colored or fluorescent product; wherein presence of a colored or fluorescent product above negative control background indicates presence of SARS-CoV-2 virus or SARS-CoV-2 S-protein in the sample.
The invention further provides methods for quantifying amount of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a sample. In one embodiment, the method comprises immobilizing the isolated SARS-CoV-2 binding protein complex of the invention mentioned above or a fragment thereof lacking a signal sequence on a surface of a solid support. In another embodiment, the method further comprises contacting the isolated SARS-CoV-2 binding protein of the immobilization step above with the sample or a reference SARS-CoV-2 virus or SARS-CoV-2 S-protein serially diluted. In another embodiment, the method further comprises washing unbound sample off the immobilizing surface. In another embodiment, the method further comprises contacting the immobilizing surface with a biotinylated CR3022 antibody in a full-length immunoglobulin format wherein biotin is conjugated to Fc portion of the immunoglobulin. In another embodiment, the method further comprises washing unbound biotinylated CR3022 antibody off the immobilizing surface. In another embodiment, the method further comprises contacting the immobilizing surface with streptavidin conjugate horse radish peroxidase. In another embodiment, the method further comprises washing unbound streptavidin conjugate horse radish peroxidase off the immobilizing surface. In another embodiment, the method further comprises contacting the immobilizing surface with a chromogenic or fluorogenic substrate for horse radish peroxidase for a fixed length of time. In another embodiment, the method further comprises detecting and quantifying amount of colored or fluorescent product produced by the sample and the serially diluted reference. In another embodiment, the method further comprises estimating the amount of SARS-CoV-2 virus or SARS-CoV-2 S-protein in the sample by comparing amount of colored or fluorescent product for the sample with that quantified for the serially diluted reference, thereby, quantifying the amount of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a sample.
In one embodiment of the method, the sample is human blood, serum, or a bodily fluid. In another embodiment, the sample is a liquid or liquid-air mixture. Examples of the liquid or liquid-air mixture include, but are not limited to, blood, serum, bodily fluid, saliva, nasal drip, respiratory droplet, aerosol, sputum, phlegm, mucus, secretion, urine, fecal material, tissue culture media, spent media, biological extract, known SARS-CoV-2-containing fluid, and suspect SARS-CoV-2 containing fluid.
The present invention provides kits (i.e., a packaged combination of reagents with instructions) containing the active agents of the invention (i.e., any of the compositions of the invention described herein) useful for detecting, diagnosing, monitoring or treating COVID-19 diseases and/or conditions.
The kit can contain a pharmaceutical composition that includes one or more agents of the invention effective for detecting, diagnosing, monitoring or treating COVID-19 and an acceptable carrier or adjuvant, e.g., pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
The agents may be provided as dry powders, usually lyophilized, including excipients that upon dissolving will provide a reagent solution having the appropriate concentration.
The kit comprises one or more containers with a label and/or instructions. The label can provide directions for carrying out the preparation of the agents for example, dissolving of the dry powders, and/or detecting, diagnosing, monitoring or treating COVID-19.
The label and/or the instructions can indicate directions for in vivo use of the pharmaceutical composition. The label and/or the instructions can indicate that the pharmaceutical composition is used alone, or in combination with another agent to detecting, diagnosing, monitoring or treating COVID-19.
The label can indicate appropriate dosages for the agents of the invention as described supra.
Suitable containers include, for example, bottles, vials, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The container can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a needle such as a hypodermic injection needle).
The invention further provides a lateral flow diagnostic kit for detection of SARS-CoV-2 virus or SARS-CoV-2 S-protein in a sample. In one embodiment, the kit comprises: a cassette comprising a sample well and one or more windows encasing a solid support for one or more capillary beds arranged in the order of: i) a first sample pad for absorption of sample, initiating capillary action and directly forming floor of the sample well; ii) a second conjugation pad comprising a mixture of gold-labelled SARS-CoV-2 binding protein comprising a human ACE2 extracellular domain fragment and a human Fc fragment and a gold-labelled rabbit IgG positive control antibody for interrogating the sample; iii) a third membrane pad visible through one or more windows for inspecting test lines, wherein the membrane pad comprises three separate lines of immobilized antibodies in the order from closest to furthest from the sample well: immobilized CR3022 antibody for binding SARS-CoV-2 virus or SARS-CoV-2 S-protein, IgG1 antibody for negative control, and anti-rabbit IgG for positive control; iv) a fourth absorption pad to wick excess fluid. The kit further comprises a buffer for maintaining capillary action to be applied after the sample to the sample well, and instruction for use.
In another embodiment of the kit, the isolated SARS-CoV-2 binding protein is that of the protein of the invention mentioned above. In another embodiment, the CR3022 antibody is an scFv, an immunoglobulin or an immunoglobulin fragment comprising CDRs of CR3022. In another embodiment, the sample is a liquid or liquid-air mixture. Examples of the liquid or liquid-air mixture include, but are not limited to, blood, serum, saliva, nasal drip, respiratory droplet, aerosol, sputum, secretion, urine, fecal material, bodily fluid, tissue culture media, spent media, biological extract, known SARS-CoV-2-containing fluid, and suspect SARS-CoV-2 containing fluid.
In another embodiment, the kit further comprises a nose cone for directing nasal spray to the sample well. In another embodiment, the nose cone comprises one opening that fits into one nostril, or over at least one nostril, and a second opening to place over the sample well, and a channel between the two openings so as to direct air forcedly expelled through a nostril of the subject to the sample well. In another embodiment, the nose cone comprises a porous or non-porous material. In another embodiment, the nose cone comprises a contiguous channel wall or a channel wall designed to release air. In another embodiment, the nose cone fit tightly or snuggly at both openings the channel comprises a semi-porous material or a vent to release air. In another embodiment, the kit further comprises a mask for directing a cough to the sample well. In another embodiment, the mask comprises one opening that fits tightly or snuggly on a face covering the mouth, and a second opening to place over the sample well, and a channel between the two openings so as to direct air forcedly expelled through the mouth of the subject to the sample well. In a further embodiment, the mask comprises a porous or non-porous material. In a further embodiment, the mask comprises a contiguous channel wall or a channel wall designed to release air. In another embodiment, the mask fits tightly at both openings the channel comprises a hole sufficient to release air or a vent to release air. In a further embodiment, the sample is a liquid or liquid-air mixture. Examples of the liquid or liquid-air mixture include, but are not limited to, blood, serum, bodily fluid, saliva, nasal drip, respiratory droplet, aerosol, sputum, phlegm, mucus, secretion, urine, fecal material, tissue culture media, spent media, biological extract, known SARS-CoV-2-containing fluid, and suspect SARS-CoV-2 containing fluid. In a preferred embodiment, the sample is human blood, serum, or a bodily fluid.
The invention also provides kits comprising the isolated SARS-CoV-2 binding protein complex of the invention above and a label or instructions for use.
Additionally, the invention provides kits comprising the bispecific knob-hole format ACE2 extracellular domain anti-SARS-Cov-2 S-protein antibody of the invention and a label or instruction for use.
Additionally, the invention provides the nucleic acid of the invention above and a label or instruction for use.
Additionally, the invention provides kits comprising the vector of the invention above and a label or instruction for use.
Additionally, the invention provides kits comprising the cell of the invention above and a label or instruction for use.
In a further embodiment, the present invention provides kits (i.e., a packaged combination of reagents with instructions) containing the active agents of the invention useful for assessing risk or course of a SARS-CoV-2 infection such as oligonucleotide or nucleic acid fragment for assessing polymorphism of ACE2 gene.
The kit can contain a pharmaceutical composition that includes one or more agents of the invention (such as oligonucleotide or nucleic acid fragment for assessing polymorphism of ACE2 gene) effective for treating or assessing risk or course of a SARS-CoV-2 infection and an acceptable carrier or adjuvant, e.g., pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
The agents may be provided as dry powders, usually lyophilized, including excipients that upon dissolving will provide a reagent solution having the appropriate concentration.
The kit may comprise one or more containers with a label and/or instructions. The label can provide directions for carrying out the preparation of the agents for example, dissolving of the dry powders, and/or treatment or assessing risk or course of a SARS-CoV-2 infection.
The label and/or the instructions can indicate directions for in vivo use of the pharmaceutical composition. The label and/or the instructions can indicate that the pharmaceutical composition is used alone, or in combination with another agent to treat or assess risk or course of a SARS-CoV-2 infection.
The label can indicate appropriate dosages for the agents of the invention as described supra.
Suitable containers include, for example, bottles, vials, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The container can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a needle such as a hypodermic injection needle).
According to another aspect of the invention, kits for assessing risk or course of a SARS-CoV-2 are provided. In one embodiment, the kit comprises oligonucleotide or nucleic acid fragment for assessing polymorphism of ACE2 gene and instruction for use. In a further embodiment, the polymorphism is directed to the coding region of the ACE2 gene. In another embodiment, the polymorphism is directed to the SARS-CoV-2 S protein interaction site on ACE2 protein as provided in
According to another aspect of the invention, kits for detecting SARS-CoV-2 comprising an ACE2 variant from any of the Tables herein and an informational insert are also provided.
The invention also provides a filter, membrane, fabric, polyester, cloth, cotton, mask, screen, fiber, carbon fiber, granule, nanoparticle, gold particle, nanotube, computer chip, surface plasmon resonance (SPR) chip, biosensor chip, glass, plastic, non-porous material or porous material coated, modified or impregnated with The isolated SARS-CoV-2 binding protein complex of the invention mentioned above, so as to trap or capture SARS-CoV-2 virus or SARS-CoV-2 S-protein.
Additionally, the invention provides a filter, membrane, fabric, polyester, cloth, cotton, mask, screen, fiber, carbon fiber, granule, nanoparticle, gold particle, nanotube, computer chip, surface plasmon resonance (SPR) chip, biosensor chip, glass, plastic, non-porous material or porous material coated, modified or impregnated with the bispecific knob-hole format ACE2 extracellular domain anti-SARS-Cov-2 S-protein antibody of the invention mentioned above, so as to trap or capture SARS-CoV-2 virus or SARS-CoV-2 S-protein.
It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may, of course, 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 present invention, which will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to 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 invention, the preferred methods, devices and materials are now described.
All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the reagents, cells, constructs, and methodologies that are described in the publications, and which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.
We queried multiple genomic databases including gnomaAD (Karczewski et al., 2019) (https://gnomad.broadinstitute.org/), DicoverEHR (Dewey et al., 2016), RotterdamStudy (Ikram et al., 2017), ALSPAC (Fraser et al., 2013) and Asian specific databases which included GenomeAsia100k (GenomeAsia, 2019), HGDP (Bergstrom et al., 2020), TOMMO-3.5kjpnv2 (Tadaka et al., 2019) IndiGen (https://indigen.igib.in/) and other aggregated data for ACE2 protein altering variations in populations groups across the world. The ACE2 genotypes in this study were from over 290,000 samples representing over 400 population groups across the world.
To assess genetic variation in the coding region of ACE2, we calculated the fixation index (Fst) from 2,381 unrelated individuals across 26 populations in the 1000 Genomes Project Phase 3 and 57,783 female individuals across eight populations in gnomAD. For 1000 Genome data, we used the Weir and Cockerham (1984) method as implemented in vcftools (Version 0.1.17); the weighted Fst were calculated from 88 variants. For gnomAD (v2.1.1), because we only have access to the allele counts, we used the original formulation by Wright (1969) and reported the weighted mean Fst as described in Bhatia et al. (2013); 277 variants were used. Because Fst values vary based on variants used (Bhatia et al. 2013), we calculated the Fst in a set of randomly selected genes on the same chromosomes matched by the length decile to use for comparison. To assess if variants in the peptidase domain has lower genetic variation, we used the one-sided Wilcoxon rank-sum test to compare 15 variants in the peptidase domain against 50 variants outside. Variants with Fst<1e-4 were removed as they were uninformative.
We used data from the 1000 Genomes Project (Genomes Project et al., 2015) to estimate the time of mutation of all variants located within a 1 Mb region around the ACE2 gene on Chromosome X, from the female-only subset of 1,271 individuals (
A total of 295 Human ACE2 orthologs were obtained from NCBI (Table 2 for accession numbers). A snake ACE2 ortholog protein was obtained from the published Indian cobra genome (Suryamohan et al., 2020). Multiple sequence alignment of residues surrounding the ACE2 NxT/S motif was performed using MCoffee (www.tcoffee.org). Phylogenetic trees were constructed using the PhyML webserver (www.phylogeny.fr).
Each identified variant was mapped, modeled, and analyzed in Pymol using the recently deposited crystal structures 6VW1 and 6LZG of human ACE2 bound to either chimeric SARS CoV-2 RBD (6VW1) or complete SARS CoV-2 RBD (6LZG).
Extracellular domain (amino acids 1-615; NP_001358344) of human ACE2 (hACE2) WT or variants with a c-terminal 8×-His or human-Fc tag was synthesized (IDT, USA) and cloned into a CMV promoter driven mammalian expression vector. Human codon optimized CoV-2-S-RBD (amino acids 319-541; YP_009724390) sequence with a c terminal 8×His-tag were synthesized and cloned into a CMV promoter driven mammalian expression vector. The prefusion SARS-CoV-2 S-protein trimer stabilized ectodomain (amino acids 1-1208; YP_009724390), as previously described (Wrapp et al., 2020), containing K986P, V987P, RRAR to GSAS (residues 682-685) at the furin cleavage site, a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrep-tag and a 8×Hi-tag was synthesized and expressed using a CMV promoter. Sequence verified plasmids prepared using NucleoBond® Xtra Midi kit (Takara Bio USA, Inc) were transfected into 293 cells using FectoPro (Polyplus, USA). Proteins were purified from media 3-5 days post transfection using Protein A GraviTrap column or His GraviTrap column (GE Healthcare).
The affinity of S-RBD or S1 for hACE2-Fc WT or variants was measured using a standard ELISA assay. Briefly, purified CoV2-S-RBD (2 μg/mL) or S1 (2 μg/mL) or the prefusion S-protein trimer (2 μg/mL) was coated onto 96-well ELISA plates (Fisher Scientific, #07-000-102) and incubated at 4° C. for 18 h. The coated plates were washed three times with 200 μl of PBST and then blocked with 200 ul of 3% BSA (Sigma-Aldrich #A8327) in PBST (Sigma-Millipore #524653) and incubated for 1h at room temperature. After washing the plates three times with 200 μl of PBST an increasing concentration of hACE2-Fc proteins were added and incubated for 1 h at room temperature. The unbound hACE2-Fc was removed by washing the plate three times with 200 μl of PBST. The bound hACE2 was detected using Goat-anti-human-IgG-Fc HRP (Jackson Immuno Research #109-035-008; 1:5000 dilution) using 50 μl TMB substrate (Pierce/Thermo Fisher Scientific #34028). After 3 minutes, the reaction was stopped using 50 μL of 2N H2SO4. The optical density of the reaction was measured at 450 nm using a plate reader (Molecular Devices Gemini XPS). The data was analyzed and EC50 was calculate using Prism (GraphPad).
The SARS-CoV-2 S-protein interacts with the ACE2 PD to enter the human host cells. Analysis of the RBD domain of SARS-CoV-2, SARS-CoV and bat CoV RaTG13 S-proteins identified changes that have increased the affinity of CoV-2 S1 RBD to human ACE2, which likely contributes to its increased infectivity (Shang et al., 2020; Wrapp et al., 2020). It is very likely that there exists ACE2 variants in human populations, though not under selection, that may increase or decrease its affinity to SARS-CoV-2 S-protein and thereby render individuals more resistant or susceptible to the virus. To investigate this, we assessed ACE2 protein-altering variations from a number of databases including gnomAD (Karczewski et al., 2019), RotterdamStudy (Ikram et al., 2017), ALSPAC (Fraser et al., 2013) and Asian-specific databases which included GenomeAsia100k (GenomeAsia, 2019), TOMMO-3.5kjpnv2 (Tadaka et al., 2019), and IndiGen (https://indigen.igib.in/), and HGDP (Bergstrom et al., 2020) (Table 1). We found a total of 298 unique protein altering variants across 256 codons distributed throughout the 805 amino acid long human ACE2 (
Structural studies involving SARS-CoV and SARS-CoV-2 S-protein and complex with human ACE2 have identified three regions in an ˜120 amino acid claw-like exposed outer surface of the human ACE2 (ACE2-claw) that contributes to its binding to the S-protein (Shang et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020). The key residues at the ACE-2 S-protein-RBD interface include S19, Q24, T27, F28, D30, K31, H34, E35, E37, D38, Y41, Q42, L45, L79, M82, Y83, T324, Q325, G326, E329, N330, K353, G354, D355, R357, P389, and R393 (
To investigate the effect of the ACE2 polymorphisms on receptor recognition by the SARS-CoV-2 RBD, we modeled the identified ACE2 variants using published cryo-EM and crystal structures of ACE2/SARS-CoV-2 RBD complexes (Shang et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020). Based on the evaluation of the structures and a functional analysis of a synthetic human ACE2 mutant library for RBD binding affinity (Chan et al., 2020b), we broadly classified ACE2 polymorphic variants into two categories with respect to their predicted effect on ACE2-RBD binding as enhancing or disrupting (
To validate our structural predictions, we measured the effect of select ACE2 polymorphisms on its binding affinity to CoV-2 S-protein. We expressed and purified the S1 subunit of the S-protein, CoV-2 S-RBD, and a trimer stabilized form of S-protein (S-trimer;
We tested the affinity of these ACE2 variants to a panel of S-protein constructs using an enzyme-linked immunosorbent assay (ELISA). We used dimeric ACE2-Fc to assess its binding to the S-protein variants. We found the ACE2-Fc WT dimer bound to the isolated S-RBD (EC50 1.01 nM) and S-trimer (EC50 0.95 nM) more strongly compared to the S1 subunit (EC50 10.4 nM) (Table 3). This is consistent with previous studies that showed a decreased ACE2 affinity for SARS-CoV S1 subunit compared to S-RBD, indicating a conformational difference between these variants (Hoffmann et al., 2020; Li et al., 2005a; Wong et al., 2004). In the trimeric state, in contrast to the monomeric full length S1-protein, the RBD within the S1 subunit in one or more of the constituent S-proteins is known to adopt a receptor-accessible ‘RBD-out’confirmation, supporting its high affinity for ACE2 that is comparable to that observed for isolated RBD (Walls et al., 2019; Wrapp et al., 2020; Yan et al., 2020).
The affinity of the S-RBD or S-trimer for ACE2-Fc variants based on ELISA is shown in Table 1 and
A recent mutagenesis screen using a synthetic human ACE2 mutant library identified variants that either increased or decreased its binding to SARS-CoV-2 S-protein (Procko, 2020). Using a sequencing-based enrichment assay, the fold enrichment or depletion of the mutant sequences was measured in this study (Procko, 2020). Mapping the enrichment z-scores from this study (Procko, 2020) to the spectrum of natural ACE2 polymorphisms, we identified several rare ACE2 variants (
Included among the ACE2 polymorphic variants that increase ACE2/S-protein interaction are S19P, I21T/V, E23K, A25T, K26E or K26R, T27A, N33, F40L, N64K, Q60R, N64K, W69C, A80G, T92I, Q102P, Q325R, M366T, D367V, 1-1374R, H378R, M383T, E398D, E398K, T445M, I446M, and Y510H. Among these, the T92I polymorphism stands out in particular because it is part of a NxT/S (where x is any amino acid except proline) consensus N-glycosylation motif (Gavel and von Heijne, 1990) where N90 is the site of N-glycan addition. The ACE2 NxT/S motif, while conserved in 96 out of 296 jawed vertebrate with ACE2 sequence available is absent or altered in several species, including the civet cat (Paguma larvata) and several bat species where residue N90 is mutated, a proline is present at position 91 or the T92 is altered to any amino acid except serine (
Microcaecilia
unicolor
Pseudonaja
textilis
Odocoileus
virginianus
texanus
Opisthocomus
hoazin
Nothoprocta
perdicaria
Thamnophis
sirtalis
Oreochromis
aureus
Poecilia
mexicana
Austrofundulus
limnaeus
Cottoperca
gobio
Camarhynchus
parvulus
Protobothrops
mucrosquamatus
Ursus
arctos
horribilis
Nanorana
parkeri
Gekko
japonicus
Buceros
rhinoceros
silvestris
Apteryx
mantelli
Aquila
chrysaetos
canadensis
Aquila
chrysaetos
Myotis
davidii
Miniopterus
natalensis
Balaenoptera
acutorostrata
scammoni
Anser
cygnoides
domesticus
Struthio
camelus
australis
Grammomys
surdaster
Corvus
cornix
Orycteropus
afer
Sinocyclocheilus
anshuiensis
Paramormyrops
kingsleyae
Seriola
lalandi
dorsalis
Enhydra
lutris
kenyoni
Variants that are predicted to reduce the virus S-protein interactions and thereby decrease S/ACE2 binding affinity include K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, K68E, F72V, Y83H, G326E, G352V, D355N and Q388L. Below we discuss the structural basis for the inhibitory effect on ACE2/S-protein binding for this selected set of mutations, as well as for the enhancing effect of the selected polymorphisms that were shown to increase ACE2/S-protein binding in vitro (Procko, 2020).
The host-virus evolutionary arms race over time leads to natural selection that alters both the host and the viral proteins allowing both to increase their fitness (Daugherty and Malik, 2012). In this context, multiple studies have analyzed and identified the origin, evolution and successful adaption of the SARS coronaviruses as human pathogens (Andersen et al., 2020; Guo et al., 2020). Viral genome sequencing and analysis have identified bats as the most likely natural host of origin for both SARS-CoV and the recent SARS-CoV-2 (Guo et al., 2020). In particular, several studies have focused on the viral S-protein RBD that interacts with its host ACE2 receptor and identified key changes between the bat CoVs and other suspected intermediary host CoVs found in the civet and pangolin (Andersen et al., 2020; Chen et al., 2020; Shang et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020). These studies have identified S-protein changes that have rendered the human cells permissive to the SARS-CoV and SARS-CoV-2 infection (Chen et al., 2020; Shang et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020).
Thus far, the role of variations in human ACE2 receptor in susceptibility to both SARS CoVs had not been comprehensively examined. While a recent in silico study analyzed limited ACE2 population variation data set and concluded that these polymorphisms did not confer resistance to the virus (Cao et al., 2020a), other studies have implicated ACE2 variants in altering binding to S-protein (Benetti et al., 2020; 338 Cirulli et al., 2020; Devaux et al., 2020; Hou et al., 2020; Hussain et al., 2020). In this study, we comprehensively examined human ACE2 variation data compiled from multiple data sets and identified polymorphisms that will either likely render individuals more susceptible to the SARS-CoV-2 or protect them from the virus. Using published protein structures and data from a high-throughput functional mutagenesis screen that used deep sequencing to assess enrichment or depletion of S-protein binding to ACE2 variants (
We identified several ACE2 polymorphic variants that increase ACE2/S-protein interaction including S19P, I21V, E23K, K26R, K26E, T27A, N64K, T92I, Q102P, M383T and H378R. Among these, the T92I polymorphism is part of a NxT/S consensus N-glycosylation motif (Gavel and von Heijne, 1990). The ACE2 NxT/S motif, while conserved in 96 out of 296 jawed vertebrates, it is absent or altered in several species, including the civet cat (Paguma larvata). The NxT/S motif is altered in several bat species and this includes substitution at N90, presence of a proline at position 91 or any amino acid except serine at T92, any of which will abolish the glycosylation at N90 (
We also show that another ACE2 residue, K26, plays an important role in controlling the susceptibility to viral infections. Our biochemical binding assays showed increased affinity of K26R ACE2 for S-protein (Table 3 and
Currently, there are no approved targeted therapeutics for curing SARS-CoV-2 infection. Therefore, development of therapeutics to treat patients and mitigate the COVID-19 pandemic is urgently needed (Cascella et al., 2020; Jiang, 2020). Several small molecules and neutralizing antibodies for treatment are in development (Li and De Clercq, 2020; Zhou et al., 2020b). Soluble ACE2 and ACE2-Fc fusion protein have been proposed as decoy SARS-CoV-2 receptor therapeutic (Hofmann et al., 2004; Kruse, 2020; Lei et al., 2020). Soluble ACE2, as a therapy for pulmonary arterial hypertension, has been shown to be safe in early human clinical studies (Guignabert et al., 2018; Haschke et al., 2013). A rationally designed, catalytically inactive, human ACE2 that carries one or more of the natural variants predicted to show improved binding to SARS viral S-protein RBD that could be safely developed as a soluble protein with or without an Fc domain for treatment of COVID-19 is proposed herein.
Even though a human recombinant soluble ACE2 is in clinical trials to treat SARS-CoV-2 infection (Zoufaly et al., 2020), a catalytically-inactive soluble ACE2 might be preferred from a safety perspective, as S-protein binding enhances ACE2's carboxypeptidase activity (Lu and Sun, 2020). Additionally, as ACE2 enzymatic activity modulates multiple biological pathways (Arendse et al., 2019), a catalytically inactive form should be considered for treating SARS-CoV-2 infection, as is disclosed herein. Such a recombinant ACE2 protein can be engineered to create a pan-CoV neutralizing drug (see for example,
Full length human ACE2 protein encoded by human ACE2 gene is illustrated in
Exemplary IgG-ACE2 fusion proteins comprising a human ACE2 full-length extracellular domain (ecd) or a truncated ACE2 ecd and an IgG are shown in
This subject patent application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/005,163, filed Apr. 3, 2020, and U.S. Provisional Application No. 63/019,952, filed May 4, 2020, the contents of which are herein incorporated by reference in their entireties into the present patent application for all purposes. Throughout this application various publications are referenced. All publications, gene transcript identifiers, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, gene transcript identifiers, patent, or patent application was specifically and individually indicated to be incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/25824 | 4/5/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63005163 | Apr 2020 | US | |
| 63019952 | May 2020 | US |
