Patent Application
20240182549
The present invention relates to peptides and peptide-antibody fusions that bind to the SARS-CoV-2 spike (S protein) and the receptor binding domain (RBD), and methods for using the compositions containing these antibodies in the treatment of COVID-19.
COVID-19, the disease caused by the SARS-CoV-2 beta-coronavirus has caused a global pandemic, infecting more than 487 million people worldwide and causing the deaths of over 6 million people and the hospitalization of scores more as of the time of this application. SARS-CoV-2 is also mutating, creating new variants of the original virus that are resistant against treatments and vaccines. Despite the granting of Emergency Use Authorization by the U.S. Food and Drug Administration to three antibody treatments (casirivimab/imdevimab, bamlanivimab/etesevimab, sotrovimab) in addition to convalescent plasma therapy, as well as the emergence of a few more promising antivirals (molnupiravir and paxlovid) than the approved remdesivir there is still no pharmaceutical standard of care treatment for the infection creating a massive unmet medical need for safe, specific and effective anti-viral therapeutics against the original virus and variants of the virus.
SARS-CoV-2 emerged in the city of Wuhan, Hubei province of China sometime in late 2019 and rapidly spread throughout the world, becoming pandemic in March of 2020. Sequence analysis revealed it is most closely related to two coronaviruses from bat (bat-SL-CoVZC45 and bat-SL-CoVZXC21) as opposed to the recent human coronaviruses SARS-CoV and MERS-CoV (Lu, 2020, PMID: 32007145), suggesting it is a zoonotic coronavirus strain.
As with other coronaviruses, SARS-CoV-2 uses its surface spike glycoprotein (S protein) to interact with and enter host cells. The SARS-CoV-2 S protein gene has the lowest sequence identity compared to related coronaviruses. However, like other SARS-CoV strains, it makes use of angiotensin converting enzyme 2 (ACE2) found on the surface of alveolar, endothelial, and smooth muscle cells in the lower respiratory tract to enter the host cell. (Hoffman, Cell, 2020, PMID: 32142651). The S protein is a hetero-trimeric class I viral fusion glycoprotein (Bosch, 2003, PMID: 12885899), which contains a large ectodomain, a single-pass transmembrane segment, and a small intracapsid tail. The ectodomain is subdivided into N-terminal (S1) and C-terminal (S2) regions. In the absence of host cell target, the S protein exists in a metastable prefusion conformation, where it adopts a stubby, clover-like structure. The S1 domain is responsible for ACE2 recognition through its “receptor binding domain” (RBD). Binding of the RBD to ACE2 induces proteolytic cleavage at the S1/S2 interface. The S2 domain then undergoes large conformational changes, forming an extended “postfusion” structure, which is believe to promote fusion of the viral capsid to the host cell membrane (Walls, 2017, PMID: 32155444), allowing the virus access to the intracellular space. One significant change in the SARS-CoV-2 S protein compared to SARS-CoV is an insertion of a furin cleavage site at the S1/S2 interface, which allows for more efficient proteolytic processing facilitating more efficient infection of the host cell, (Coutard, 2020, PMID:32057769). Along with the RBD, S1 also contains an N-terminal domain (NTD).
Since the zoonotic spillover of the original Wuhan strain of SARS-CoV-2, a variant of the virus (D614G) has become globally dominant (Korber, Cell, 2020, PMID: 32697968). Additional variants have emerged with mutations in the S protein in various regions around the world including a B.1.1.7 variant from UK (https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563), the B.1.351 variant from South Africa (Tegally, Nat Med, 2021, PMID: 33531709) the P.1 variant from Manaus, Brazil (https://virological.org/t/genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-manaus-preliminary-findings/586), a MB61 variant from Italy (Fiorentini, Lancet, Inf Dis, 2020) and others (Arora:2021jy). These variants have been noted to confer resistance to neutralization by therapeutic antibodies, convalescent plasma and vaccine sera (Wang, Nature, 2021, PMID: 33684923). Resistance can arise from mutations in the S protein and RBD that impair binding of neutralizing antibodies and strategies for overcoming resistance are required. One strategy that has been used to overcome mutational resistance in the context of other viruses is the development of multi-specific, broadly neutralizing antibodies (Khan, J Virol, 2018, PMID: 29976677, Steinhardt, Nat Comm, 2018, PMID: 29491415).
Development of therapeutics and vaccines to treat COVID-19 is essential toward restoring normal economic and social function. Indeed, several therapies have already been developed in many jurisdictions around the world, including recent approval of novel mRNA vaccines, (Jackson, 2020, PMID: 32663912) monoclonal antibody (mAb) therapeutics, and small molecular antivirals (remdesivir, molnupiravir, and paxlovid). While these developments have curbed the pandemic significantly, the recurring nature of coronaviruses necessitates interventions applicable to novel coronavirus variants and strains, which have already emerged and spread.
The term “amino acid” includes all of the naturally occurring amino acids as well as modified amino acids.
An “affinity matured” antibody or “maturation of an antibody” refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent or source antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen or to other desired properties of the molecule.
The term “antibody” as used herein is intended to include human antibodies, humanized antibodies, monoclonal antibodies, polyclonal antibodies, antibody binding fragments, single chain and other chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. A “whole antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL or CL1. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Herein, the assignment of amino acids to each domain is as defined in accordance with INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (IMGT) numbering (Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Development and Comparative Immunology. 2003; 27:55-77). In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. The exemplary antibody clones described herein are generally referred to with a number starting with 150 followed by a two-digit number (e.g., 15033), and sometimes may have a number following a dash (e.g., 15033-7). The nomenclature used herein could also be shortened to just the two-digit number, with the number following the dash, if applicable (e.g., 33 or 33-7, respectively).
The term “antibody binding fragment”, “binding fragment” or “antigen binding fragment” as used herein is intended to include without limitations Fab, Fab′, F(ab′)2, scFv, scFab, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and single-domain antibodies, e.g., VHH. Antibodies can be fragmented using conventional techniques. For example, F(ab′)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, scFab, VHH, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques, see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 and Holliger et al. (1993) PNAS. USA 90:6444-6448, and “Single domain antibodies,” Methods in Molecular Biology, Eds. Saerens and Muyldermans, 2012, Vol 911.
An antigen binding site or “binding site” is formed by amino acid residues of the N-terminal variable regions of the heavy chain (VH) and N-terminal variable regions of the light chain (VL). The three CDRs of a light chain and the CDRs of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen. In single domain antibodies, also known as nanobodies, the binding site is formed by only one heavy variable domain (VH). “VHH” as used herein refers to a human VH that has been engineered to be independent of the light chain (Nilvebrant et al. Curr Pharm Des. (2016) 22(43):6527-6537; Barthelemy et al. Journal of Biological Chemistry (2007) 283:3639-3654).
The term “capture antibody” as used herein refers to an antibody or binding fragment thereof bound to a solid support and used to capture the target antigen in a sample, for example a COVID-19 polypeptide, e.g., an S polypeptide, and its S1 and S2 subunits, by forming a complex with the target antigen.
By “consisting essentially of”, it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention. For example, in some cases antibodies or binding fragments or multivalent binding molecules “consisting essentially of” a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based upon the sequence from which it was derived, e.g. about 5 residues, 4 residues, 3 residues, 2 residues or about 1 residue less than the recited bounding amino acid residue, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited bounding amino acid residue.
By “consisting of”, it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, antibodies or binding fragments thereof or multivalent binding molecules “consisting of” a disclosed sequence consists only of the disclosed amino acid sequence.
A “conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size; alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, and valine are all typically considered to be small.
The “COVID-19 S polypeptide” as used herein is an extensively glycosylated S protein of SARS-CoV-2 that protrudes from the viral membrane, to mediate host-cell entry. The S polypeptide contains 1,282 amino acids organized into 2 subunits (S1 and S2) and the receptor binding domain (RBD), is located in the S1 subunit.
The term “COVID variant” or “variants of SARS-CoV-2” as used herein refer to variants of the original Wuhan strain of SARS-CoV-2. For example, variants include coronaviruses with mutations in the S protein and/or RBD that may impair binding of neutralizing antibodies. These variants include the ones already identified, such as B.1, D614G (Plante, J. A., et al., 2021, Spike mutation D614G alters SARS-CoV-2 fitness. Nature, 592(7852), 116-121.), B.1.1.7 (Davies, N. G., et al., 2021, Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England. Science (New York, N.Y.), 372(6538), eabg3055.), B.1.351 (Yadav, P. D., et al., 2021, Imported SARS-CoV-2 V501Y.V2 variant (B.1.351) detected in travelers from South Africa and Tanzania to India. Travel medicine and infectious disease, 41, 102023.), B.1.128 (P.1) (Faria, N. R., et al., 2021, Genomics and epidemiology of the P.1 SARS-CoV-2 lineage in Manaus, Brazil. Science (New York, N.Y.), 372(6544), 815-821.), MB61 (Fiorentini, S., et al., 2021, First detection of SARS-CoV-2 spike protein N501 mutation in Italy in August, 2020. The Lancet. Infectious diseases, 21(6), e147.), B.1.617.1 (Melloul, M., et al., 2021, Genome Sequences of the Delta Variant (B.1.617.2) and the Kappa Variant (B.1.617.1) Detected in Morocco. Microbiology resource announcements, 10(39), e0072721,), B.1.617.2 (Melloul, M., et al., 2021, Genome Sequences of the Delta Variant (B.1.617.2) and the Kappa Variant (B.1.617.1) Detected in Morocco. Microbiology resource announcements, 10(39), e0072721.), B.1.617.2+(Kannan, S. R., et al., 2021, Evolutionary analysis of the Delta and Delta Plus variants of the SARS-CoV-2 viruses. Journal of autoimmunity, 124, 102715.), B.1.427/429 (Carroll, T., et al., 2022, The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters. PLoS pathogens, 18(2), el 009914.), and B.1.1.529 (Rahmani, S., & Rezaei, N. (2022). Omicron (B.1.1.529) variant: Development, dissemination, and dominance. Journal of medical virology, 94(5), 1787-1788.), and any yet to be identified variants with mutations to the S protein and/or RBD. The term “denatured” as used herein refers to a polypeptide that has lost tertiary and/or secondary structure (e.g. fully unfolded protein), for example when exposed to denaturing conditions in SDS sample loading buffer.
The term “detectable tag” or “detectable label” as used herein refers to moieties such as peptide sequences, a radio-opaque or a radioisotope label, such as 3H, 14C; 32P; 35S; 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion that can be appended or introduced into recombinant protein.
The term “detection antibody” as used herein refers to an antibody or binding fragment thereof that binds a capture antibody or a target antigen, or optionally binds a target antigen already in a complex with a capture antibody. For example, the detection antibody binds the capture antibody or a coronavirus polypeptide, or binds a coronavirus:capture antibody complex at an epitope on the coronavirus that is different than the one recognized by the capture antibody. The coronavirus may be, e.g. SARS-CoV-2 and the coronavirus polypeptide may be, e.g., a SARS-CoV-2 S protein
“Diabody” or “Diabodies” as used herein are dimeric antibody fragments. In each polypeptide of the diabody, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fv fragments, the linker between the VL and VH is too short for intramolecular pairing and as such each antigen-binding site is formed by pairing of the VH and VL of one polypeptide with the VH and VL of the other polypeptide. Diabodies thus have two antigen-binding sites, and can be monospecific or bispecific. (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123; Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5) incorporated herein by reference.
As used herein an “effective amount” of an agent, e.g., the antibodies, binding fragments thereof, and multivalent binding molecules described herein or a pharmaceutical composition comprising the antibodies or molecules, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result e.g., to ameliorate at least one physical adverse effect of COVID-19 infection or proliferation. This might be an amount sufficient to interfere with the binding of COVID-19 or a COVID-19 protein to its target, and/or delay onset of, one or more symptoms of the disease. The amount required to be administered will depend on the binding affinity of the antibody or multivalent binding molecule for its specific antigen, and will also depend on the rate at which an administered antibody or molecule is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight and from about 2 mg/kg to about 30 mg/kg. Common dosing frequencies may range, for example, from twice daily to once a week.
The term “epitope” as used herein refers to the site on the antigen that is recognized by the antibodies, antibody binding fragments, or multivalent binding molecule disclosed herein. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ≤10 μM; e.g., ≤100 nM, preferably ≤10 nM and more preferably ≤1 nM.
The term the “Fe region” or “Fe domain” refers to the constant region of immunoglobulin molecules is also called the fragment crystallizable region. The Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains and the Fc domains of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity.
The term “heavy chain complementarity determining region” as used herein refers to regions of hypervariability within the heavy chain variable region of an antibody molecule. The heavy chain variable region has three complementarity determining regions termed heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2) and heavy chain complementarity determining region 3 (CDR-H3) from the amino terminus to carboxy terminus. All CDRs and framework regions (FRs) disclosed herein, amino acid sequences of CDRs and FRs disclosed herein, and CDR-encoding or FR-encoding nucleic acid sequences disclosed herein, are defined herein in accordance with IMGT numbering (Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Development and Comparative Immunology. 2003; 27:55-77).
The term “heavy chain variable region” or “heavy chain variable domain” as used herein refers to the variable domain of the heavy chain comprising the heavy chain complementarity determining region 1 (CDR1), heavy chain complementarity determining region 2 (CDR2) and heavy chain complementarity determining region 3 (CDR3).
The term “host cell” refers to a cell into which a recombinant DNA expression cassette or vector can be introduced to produce a recombinant cell. Host cell includes but is not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as S2 drosophila cells and Sf9, or animal cells, including human cells, e.g., fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.
The term “isolated antibody” or “isolated binding fragment thereof” or “isolated and purified antibody” or “isolated and purified binding fragment thereof” refers to an antibody or binding fragment thereof that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized, and/or other antibodies, for example antibodies directed to a different epitope.
The term “KD” refers to the dissociation constant of a complex for example of a particular antibody-antigen interaction.
The term “light chain complementarity determining region” as used herein refers to regions of hypervariability within the light chain variable region of an antibody molecule. Light chain variable regions have three complementarity determining regions termed light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR2) and light chain complementarity determining region 3 (CDR-L3) from the amino terminus to the carboxy terminus.
The term “light chain variable region” or “light chain variable domain” as used herein refers to the variable domain of the light chain comprising the light chain CDR1, light chain CDR2 and light chain CDR3.
The term “monoclonal antibody” (MAb), as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
The term “native” or “natively folded” as used herein refers to a protein in its native conformation (e.g. 3D conformation) or in a conformation sufficient to confer functionality, including for example partially unfolded protein capable of binding a receptor or ligand.
The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term “nucleic acid” includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The term “isolated nucleic acid sequences” as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) from which the nucleic acid is derived.
“Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate regulatory sequences is dependent on the host cell chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
As used herein the term “paratope” includes the antigen binding site in the variable region of an antibody or a multivalent binding molecule of this invention that binds to an epitope.
The term “peptide” as used herein refers to a chain of amino acid residues linked by peptide bonds. The peptides can be as short as 8 amino acids in length, and as long as 24 amino acids. The peptides described herein can be identified by a lowercase “p” followed by the number identified in
The term “polypeptide” as used herein refers to a polymer consisting of a large number of amino acid residues bonded together in a chain. The polypeptide can form a part or the whole of a protein. The polypeptide may be arranged in a long, continuous and unbranched peptide chain. The polypeptide may also be arranged in a biologically functional way. The polypeptide may be folded into a specific three-dimensional structure that confers it a defined activity. The term “polypeptide” as used herein is used interchangeably with the term “protein”.
The term “polypeptide fragment” as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence.
The term “isolated polypeptide” as used herein refers to substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
The term “reference agent” as used herein refers to an agent that can be used in an assay and that can be, e.g., a standard amount of COVID-19 or a COVID-19 polypeptide, e.g., a S polypeptide, used as a reference, e.g., for detecting COVID-19 or a COVID 19 polypeptide in a sample or for screening or for diagnosing a subject having or suspected of having a COVID-19 infection.
The term “sample from a subject” or “biological sample” as used herein refers to any biological fluid, cell or tissue sample from a subject, which can be assayed for a COVID-19 polypeptide. For example, the sample can comprise lung or nasal secretions, lung lavage, urine, serum, plasma or cerebrospinal fluid. The sample can for example be a “post-treatment” sample wherein the sample is obtained after one or more treatments, or a “base-line sample” which is for example used as a base line for assessing disease progression.
The term “sandwich ELISA” as used herein refers to an ELISA comprising a solid support and a capture antibody or binding fragment thereof, or a multivalent binding molecule described herein, immobilized onto the solid support. In such an ELISA an amount of target antigen (e.g. a COVID-19 S polypeptide or fragment thereof) in a sample is bound by the capture antibody. The bound antigen is detected by a second antibody or binding fragment thereof or a multivalent binding molecule described herein, which recognizes an epitope that is different from the one recognized by the capture antibody. The capture antibody:target complex (e.g., multivalent binding molecule:COVID-19 polypeptide complex) is detected by the detection antibody which can be covalently linked to an enzyme or can itself be detected by addition of a secondary antibody which is linked to an enzyme. For example, the capture antibody and/or the detection antibody can comprise CDR regions disclosed herein.
“SARS-CoV-2” or “COVID-19” as used herein is the novel coronavirus belonging to Coronaviridae family, beta-coronavirus genus and Sarbecovirus subgenus and the cause of the ongoing pandemic of Coronavirus Disease 2019 (COVID-19).
“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide (Fv fragment has the antigen-binding site made of the VH and VL regions, but they lack the constant regions of Fab (CH1 and CL) regions) further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.).
The “COVID-19 S polypeptide” as used herein is an extensively glycosylated S protein of SARS-CoV-2 that protrudes from the viral membrane, to mediate host-cell entry. The S polypeptide contains 1,282 amino acids organized into 2 subunits (S1 and S2) and the receptor binding domain (RBD), is located in the S1 subunit.
“Single-domain antibody” (sdAb), or “nanobody”, is an antibody fragment consisting of a single monomeric variable antibody domain. “VHH” or “VHH fragment” as used herein refers to a human VH that has been engineered to be independent of the light chain (Nilvebrant et al. Curr Pharm Des. (2016) 22(43):6527-6537; Barthelemy et al., Journal of Biological Chemistry (2007) 283:3639-3654).
The terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject, e.g., a human being, a monkey, an ape, or a dog or cat, for whom diagnosis, treatment, or therapy is desired.
The terms “treatment”, “treating” and the like are used herein to generally means obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., slowing or arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered before the symptomatic stage of the disease, during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
The term “variant” as used herein includes one or more amino acid and/or nucleotide modifications in a sequence (polypeptide or nucleic acid respectively) for example, one or more modifications of a light chain or a heavy chain complementarity determining region (CDR) disclosed herein that perform substantially the same function as the light chain and heavy chain CDRs disclosed herein in substantially the same way. For instance, variants of the CDRs disclosed herein have the same function of being able to specifically bind to an epitope on a coronavirus, e.g., a COVID-19 polypeptide, e.g., an epitope on the RBD of the S protein, or in the case of nucleotide modifications, encode CDRs that have same function of being able to specifically bind to an epitope on the coronavirus polypeptide. For example, codon optimized and degenerate sequences are included. Variants of CDRs disclosed herein include, without limitation, amino acid substitutions, including conservative amino acid substitutions, as well as additions and deletions to the CDR sequences disclosed herein. For example, the substitution, addition or deletion can be of 1, 2, 3, 4, 5, 6, 7, or 8 amino acids and/or the corresponding number of nucleotides. The substitutions may be conservative amino acid substitutions.
As used in this invention, the term “vector” refers to a nucleic acid delivery vehicle or plasmid that can be engineered to contain a nucleic acid molecule, e.g., a nucleic acid sequence encoding the antibody binding molecules or the multivalent binding molecules described herein. The vector that can express protein when inserted with a polynucleotide is called an expression vector. Vectors can be inserted into the host cell by transformation, transduction, or transfection, so that the carried genetic substances can be expressed in the host cell. Vectors are well known to the technical personnel in the field, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1 derived artificial chromosome (PAC); phage such as λphage or M13 phage and animal viruses etc. Animal viruses may include but not limited to, reverse transcriptase virus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e. g. herpes simplex virus), chicken pox virus, baculovirus, papilloma virus, and papova virus (such as SV40). A vector can contain multiple components that control expression of the antibody or multivalent binding molecules described herein, including but not limited to, promoters, e.g., viral or eukaryotic promoters, e.g., a CMV promoter, signal peptides, e.g., TRYP2 signal peptide, transcription initiation factor, enhancer, selection element, and reporter gene. In addition, the vector may also contain replication initiation site(s).
The term “level” as used herein refers to an amount (e.g. relative amount or concentration) of COVID-19 or a COVID-19 S polypeptide that is detectable or measurable in a sample. For example, the COVID-19 or a COVID-19 S polypeptide level can be a concentration such as pM or a relative amount such as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and/or 10 times a control level, where for example, the control level is the level of COVID-19 or a COVID-19 S polypeptide in a healthy or asymptomatic subject.
In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. For example, a composition comprising antibodies or binding fragments is a composition that may comprise other elements in addition to the antibodies or binding fragments, e.g. functional moieties such as polypeptides, small molecules, or nucleic acids bound, e.g. covalently bound, to antibodies or binding fragments; agents that promote the stability of the antibodies or binding fragments composition, agents that promote the solubility of the antibodies or binding fragments composition, adjuvants, etc. as will be readily understood in the art, with the exception of elements that are encompassed by any negative provisos. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.
In understanding the scope of the present disclosure, the term “consisting” and its derivatives, as used herein, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.” Further, it is to be understood that “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “about” means plus or minus 0.1 to 10%, 1-10%, or preferably 1-5%, of the number to which reference is being made.
Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Considerable effort has gone towards isolation and characterization of neutralizing mAbs (nAbs) (Hansen, 2020, PMID: 32540901) (Liu, 2020, PMID: 32698192) (Kreye, 2020, PMID: 33058755) (Zhou, 2020, PMID: 33657424) (Du, 2020, PMID: 32243608) (Ju, 2020) (Wu, 2020, PMID: 33275640) (Hurlburt, 2020, PMID: 33110068). nAbs as therapeutics are an intriguing strategy, because they can be rapidly isolated and characterized from the peripheral blood of previously infected humans. Moreover, since these mAbs are produced by human immune systems and their nature is such that they are highly specific for their target, chances of side effects due to broad targeting and patient immune response are limited. This significantly reduces time from lead identification to clinical approval. Within months of the emergence of COVID-19, several nAbs leads were isolated and characterized, and three have recently received approval for clinical use. These nAbs represent the first non-repurposed therapeutics approved for treatment of COVID-19, demonstrating the reduced timeline from lead development to approval compared to other drug classes such as small molecules. Isolating nAbs from human subjects, however, necessitates infection, and is thus inherently limited in its developmental pathway.
One way around the issue of requiring human infection is through the use of recombinant phage-displayed mAb libraries. All that is necessary for this process is the genetic sequence of the viral antigen to be targeted. This allows for the production of critical antigens through recombinant protein production technology, against which the phage-displayed libraries are bio-panned to isolate specific high-affinity nAbs. Our lab was able to implement this technique to isolate nAbs of comparable affinity and quality to those isolated from peripheral blood of COVID-19 infection patients over the same timeline.
Intriguingly, despite divergence in methodology, nearly all of the nAbs against CoV-2 bind highly overlapping epitopes found on the S protein RBD. The mode of action of these nAbs is through steric blocking of the ACE2-RBD interaction to prevent infection. One exception are a set of nAbs that bind S protein in the NTD, and likely neutralize the virus through allosteric effects which stabilize CoV-2 S protein confirmations unable to bind ACE2, or unable to be cleaved and transition to the post-fusion state (Yuan, 2020, PMID: 32661058). Binding of nAbs to such a limited set of epitopes raises of the possibility of mutations which allow the virus to escape blocking by a large set of nAbs through a reduction or elimination of binding to these Abs. Already, a covid variant of CoV-2 which is more transmissible has emerged where in a residue in the RBD, N501, is mutated to a tyrosine (https://cmmid.github.io/topics/covid19/reports/uk-novel-variant/2020_12_23_Transmissibility_and_severity_of_VOC_202012_01_in_England.pd f). This position is significant for ACE2 binding (Starr, 2020, PMID: 32841599), and thus one explanation for increased transmissibility is that the N501Y mutation increases affinity for ACE2. It is unclear how N501Y will affect neutralization efficiency of nAbs, however location of the mutation and mechanism of the vast majority of nAb action suggests it will hinder neutralization efficiency of at least of subset of nAbs, and that other RBD mutations that impact nAb binding to SARS-CoV-2 S protein are sure to emerge in the future.
One strategy for overcoming resistance conferred by escape mutations is to devise recombinant nAbs into formats that increase avidity, potency and breadth of neutralization against viruses, such as variants of SARS-CoV-2. Our lab and others have shown this strategy to have promise (Miersch et al, https://doi.org/10.1101/2020.10.15.341636.). Taking it a step further, we can expand the number of epitopes of high valency formats via the fusion of peptides to neutralizing antibodies. Recent work has shown that peptide fragments are able to bind both RBD and non-RBD epitopes on the S protein (Cao, 2020, PMID: 32425270) also (Pomplun ACS, 2021, PMID: 33527085).
Previous studies have fused a single inhibitory peptide (HR2P) derived from the heptad repeat 2 (HR2) domain of MERS-CoV to the C-terminus of recombinant antibody fragments that targeted the receptor-binding site (Wang, 2019, PMID: 31690009 and 30621343). The peptide was identified by a structure-based approach for its ability to form a stable six-helical bundle by binding the MERS-CoV S protein independently to block viral fusion and replication (Lu, Nat Commun, 2014). Authors concluded from their study that bispecific inhibitors, formed by fusion of this peptide to the C-terminus of a recombinant antibody targeting the receptor-binding site increased the efficacy against MERS-CoV.
We developed novel CoV-2 S protein binding peptides making use of a naïve phage-displayed 16mer peptide library. We show that these peptides bind epitopes in both the RBD and S protein outside of the RBD. Fusion of these peptides to the N-terminus of nAbs improves their apparent affinity, potency and breadth of neutralization, without compromising their developability profile. Thus, these peptides represent a novel class of SARS-CoV-2 S protein binders, which can be formatted in various ways to improve functional properties of nAbs.
In a first embodiment of the invention, at least one peptide that binds the CoV-2-S protein is fused to an anti-CoV-2 antibody. In another embodiment, the fusion includes at least one peptide fused to the N-terminus of at least one of the light chains of the anti-CoV-2 antibody. In another embodiment, the fusion includes at least one peptide fused to the N-terminus of at least one of the heavy chains of the anti-CoV-2 antibody. In another embodiment, the fusion includes at least one peptide fused to the N-terminus of each of the heavy chains of the anti-CoV-2 antibody. In another embodiment, the fusion includes at least one peptide fused to the N-terminus of each of the light chains of the anti-CoV-2 antibody. In another embodiment, the fusion molecule comprises an anti-CoV-2 antibody and two different peptides. In another embodiment, the fusion molecule comprises an anti-CoV-2 antibody and at least one peptide fused on each of the heavy chain and the light chains, where the peptide fused to the heavy chain can be the same or different than the peptide fused to the light chain.
In another embodiment of the invention, at least one peptide that binds to the protein of a virus of interest is fused to an antibody that binds to the same virus of interest. In another embodiment of the invention, the antibody is a neutralizing antibody. An embodiment of this invention is that the epitope to which the antibody binds is within proximity to the binding site of the peptide so as to enable avidity effects by tethering the peptide to the antibody N-terminus with a linker peptide of reasonable length. Suitable linkers are described herein. Identification of peptides that enhance antibody avidity for the viral target can be screened by any method capable of resolving functional differences, for example, by fusing the peptide to the N-terminus of the antibody light or heavy chain and screening their binding kinetics to reveal enhancements relative to the parental antibody without the linker and with the linker alone, or by neutralization assays of authentic or pseudovirus, or the methods described herein for CoV-2.
In another embodiment of the invention, at least one peptide that binds the CoV-2-S protein is fused to an anti-viral synthetic antibody. In another embodiment, the fusion comprises at least one peptide fused to a multivalent binding molecule comprising two or more antibody binding fragments that bind a coronavirus, wherein the binding fragments of the multivalent binding molecule are joined by a peptide linker. In an embodiment at least one peptide described herein is fused to the multivalent binding molecule, where the multivalent binding molecule is at least bivalent or at least trivalent. In an embodiment of the invention, the peptide can be fused to any free N-terminus. The peptides fused to each N-terminus can be the same or different. The peptides can be fused to all of the free N-terminus locations, or to any combination of the free N-terminus locations. In one embodiment, there is a peptide attached to each free N-terminus of the multivalent binding molecule. Exemplary embodiments using anti-SARS-CoV-2 S protein IgG-Fab multivalent molecules are shown in
The peptide can comprise any peptide that is found to bind to the CoV-2 S protein in either the RBD or non-RBD regions of the S protein. The peptide can be fused to the N-terminus of the anti-CoV-2 antibody. The peptide embodied by this invention can range in size from 8 to 24 amino acids. The peptide is a suitable size to allow for binding to the S protein. For example, the peptide can be 16 amino acids, like those described in
In another embodiment of the invention, the peptide comprises a sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% identical to the peptides identified in
The peptide and the antibody can be joined via a linker (i.e., a flexible molecular connection, such as a flexible polypeptide chain). The linker can be any suitable linker, such that the fusion protein can bind to the RBD or non-RBD regions of the S protein. The linker can be any suitable length, but is preferably at least about 10 (e.g., at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, or ranges thereof) amino acids in length. In another embodiment, the linker is in excess of 70 Å in order for a peptide tethered to the N-terminus of either the heavy or the light chain to reach adjacent S protein epitopes. In one embodiment, the linker is a flexible non-cleavable linker. An example of a suitable linker includes, but is not limited to, (G4S)4. The average extended length of a non-structural peptide is ˜3.5 Å per residue. Given this (G4S)4 linker is 20 residues in length and that is glycine serine rich content would render it unstructured, it should have a length of approximately 75 Å, making it a suitable linker to tether a peptide and antibody, such as Antibody 15033 described herein.
The anti-CoV-2 antibody can be any antibody with anti-CoV-2 properties, for example, the antibodies described herein. In one embodiment, the anti-CoV-2 antibody is one that binds the RBD. In one embodiment, the anti-CoV-2 antibody is an antibody or antibody binding fragment that binds SARs-CoV-2 and/or SARs-CoV-2 S polypeptide, e.g., S protein subunits S1 or S2. In another embodiment, the anti-CoV-2 antibody is a synthetic multivalent antibody that binds SARS-CoV-2.
In another embodiment of the invention, the fusion molecule comprises at least one peptide described in
Structural analysis of 15033 Fabs bound to CoV-2 S protein reveals a strategy for neutralization efficiency improvement through peptide tethering.
We developed a panel of nAbs against CoV-2 through binding of various epitopes on the S protein (Miersch, S., et al., 2021, Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of molecular biology, 433(19), 167177.). The most efficient neutralizer with ideal drug-like properties was Ab 15033, which was subsequently improved to yield 15033-7. The structure of 15033-7 Fabs bound to CoV-2 S protein ectodomain was solved by cryo-EM. These structures revealed that 15033 Fabs are able to bind S protein in a variety of ways. Structures with 2 or 3 Fabs bound were observed, and with 3 Fabs bound, it was observed that on RBD could adopt the ‘up’ or ‘down’ conformation. Negative stain EM revealed that a 15033 IgG could bind one S protein, and the orientation of the class average of this structure suggested that each Fab of the IgG bound to an ‘up’ RBD. Thus, we selected the two Fab bound to two ‘up’ cryo EM structure as a model for IgG binding to the S protein for analysis.
The N-terminus of the Fabs fall more closely to the NTD region of the S protein monomer adjacent to the one to which they are bound. The N-terminus of the heavy chain is slightly closer to the adjacent NTD than the light chain (67 Å versus 71 Å, respectively) (
Based on this we determine that we would need a linker in excess of 70 Å in order for a peptide tethered to the N-terminus of either the heavy or the light chain to reach adjacent RBD or non-RBD epitopes. The average extended length of a non-structured peptide is ˜3.5 Å per residue. In previous work we have made use of a four repeat, four glycine plus one serine linker (G4S)4. Given this linker is 20 residues in length and that is glycine serine rich content would render it unstructured, it should have a length of approximately 75 Å, making it suitable to tether a peptide to 15033 such that both the 15033 paratope and the peptide epitopes were within the range of distances conferred by the linker;
Isolation of CoV-2 S protein-binding peptides from a phage displayed peptide library.
In order to isolate novel SARS-CoV-2 S protein binding peptides, a randomized 16mer phage-displayed library was panned against purified CoV-2 S protein RBD or CoV-2-S protein-ECD antigens. The library was an equal mixture of 16mers fully randomized at each position, and libraries where two positions in the 16mer were fixed as cysteines, with the remaining position were fully randomized, resulting in a closed loop (constrained) peptide through intramolecular disulphide bridge formation. The distance between cysteines ranged from 4 to 12, thus the library was a mixture of 10 libraries total, in equal amounts. After four rounds of selection, 160 unique clones against CoV-2 S protein ECD and 128 unique clones against CoV-2 S protein RBD were sequenced and characterized. Table 1 shows the number of unique clones identified for SARS-CoV-2-S protein ectodomain and SARS-CoV-2 RBD and the particular families in which they fall.
Each unique SARS-CoV-2 S protein antigen favored binding from specific library families. Table 1 shows that of the 160 unique clones from SARS-CoV-2 S protein ectodomain, about 63% belonged to the unconstrained family (U) and about 22% belonged to the X5-C-X4-C-X5 family (C4). The term “unconstrained” means that the peptide does not contain cysteine residues and is therefore not constrained by a disulfide bond. With respect to SARS-CoV-2 RBD selections about 27% belonged to the X—C—X12-C-X family (C12) and about 42% belonged to the X2-C—X10-C—X2 family (C10).
The unconstrained family of peptides can be subdivided into 3 distinct subfamilies. The first, herein referred to as the WΦ family, is characterized by a WΦxxLxxMM motif. The second, herein referred to as the WE family, contained a WExxLVxML motif. And the final, herein referred to as the W family, contains a WxxDxxxLxxML motif. In terms of cysteine constrained peptides there were 4 families. The S-C4 family contains a DWCXΦWXC consensus from position 4, the S-C7 family contained predominantly one peptide with a FPEW motif in between the flanking cysteines, S-C10 contains a CWExxxxxWxC motif, and finally the S-C12 family contains a CWEWxxxxxxWxxC motif from position 2. As can be seen in
We sought to characterize the epitopes targeted by these peptides. The RBD fused to an Fe tag (RBD-Fc) as well as the full-length S protein ectodomain fused to a His tag (S protein ectodomain) were cloned and purified from Expi293 cells, and the EC50 of phage-displayed peptides to these antigens was determined through ELISA (
Given these results, we selected a panel of peptides from each family, 10 peptides in total, to build peptide antibody fusions to either the heavy or light chain. Peptides sequences are as per
We next determined whether these peptides improved affinity of 15033 for purified recombinant SARS-CoV-2 S protein. Results from
From this panel of antibodies we selected those that improved affinity over 100-fold when fused to either the light or heavy chain, or both, for further characterization. Since the end goal of these molecules is to serve as lead therapeutics for treatment of COVID infection, we performed a series of experiments to determine their drug-like properties. SDS-PAGE analysis revealed that the IgG's were purified to homogeneity and that proper disulphide bridge formation within the IgG's molecules had occurred (
Another variable is the IgG chain to which the peptide was fused. Peptide fusion to the light chain appears to have a less disruptive effect on IgG structure compared to fusion to the heavy chain (
Improvement in CoV-2 neutralization efficiency through fusion of S protein binding peptides to nAbs. Fusion of S protein-binding peptides enhanced IgG potency in a SARS-CoV-2 pseudoviral infection assay. Following confirmation of enhanced avidity, which is known to often correlate with enhanced anti-viral potency, the functional activity was assessed in a pseudovirus infection assay that uses an HIV-gag virus-like particle pseudotyped with SARS-CoV-2 S protein that also encodes a luciferase gene (Miersch BioRxiv, 2020). Uptake of the particle results in the translation of an active luciferase enzyme that can be detected upon cell lysis in the presence substrate and whose activity is proportional to the amount of internalized VLP. Pre-incubation of VLP's with either p16 or p102 peptide-fused IgG 15033 on either the heavy or light chain revealed a marked improvement in neutralization potency in comparison to parent IgG without peptide (
We next sought to determine whether fusion of these peptides could improve SARS-CoV-2 potency against authentic virus (Washington strain). Authentic SARS-CoV-2 virus was incubated with increasing concentrations of our peptide-fused anti-CoV-2 anti-bodies and then added to ACE2 expressing Vero E6 cell cultures. Vero E6 infection was measured and plotted as a function of incubating peptide-Ab concentration. These plots were used to determine IC50 values for comparison of neutralization efficiency. We used 15033 IgG with no peptide fusion as a baseline comparison, and for upper bound comparison, we included 15033 in a Fab-IgG tetravalent format, previously shown to dramatically improve neutralization efficiency. We found significant improvements in the IC50 values of all our peptide fusions compared to the 15033 parental alone, except for p96, which showed no apparent improvement in potency when fused to the light chain, and decreased potency when fused to the heavy chain (
Encouraged by these results we opted to fuse p16 and p102 to the 15033-7 tetravalent formats and express the fusions with our diabody-Fc-Fab (db-Fc-Fab), and Fab-diabody-Fc (Fab-db-Fc) modalities to determine if it would improve neutralization performance of either of these molecules. SEC and non-specificity profiles revealed that fusion of p16 or p102 to either 15033-7 light or heavy chain, or 15033 Fab-IgG light chain did not disrupt these molecules structure, stability, or non-specific reactivity profile. (
Conceived of as a tool to rapidly enhance the affinity of virtually any anti-S protein antibody, we identified a small subset of 10 candidates (indicated by an asterisk* in FIG. 2) based on apparent favourable physicochemical properties and consensus sequences, and fused them (with a 20 (G4S)4 amino acid linker) to the N-terminus of the light and heavy chains of a highly potent CoV-2 neutralizing antibody (IgG 15033) (
Virtually identical results were obtained and comparison to a previously assayed tetravalent antibody known to possess higher potency demonstrated that even a small peptide can enhance avidity and anti-viral potency similar to larger modular formats (
Results demonstrated that even a single peptide fused to the N-terminus of the antibody was capable of enhancing both potency and dramatically overcoming resistance to antibody-mediated neutralization conferred by mutation in the B.1.351 variant (
Peptides selected from peptide-phage libraries are fused to the N-terminus of the heavy (H) and/or light (L) chain of IgG-Fab via molecular cloning of genes encoding the peptides in to constructs for expression of the antibody (
Peptides selected from peptide-phage libraries are fused to the N-terminus of the heavy (H) and/or light (L) chain of Fab-IgG via molecular cloning of genes encoding the peptides in to constructs for expression of the antibody (
Peptides selected from peptide-phage libraries are fused to the N-terminus of the heavy (H) and/or light (L) chain of diabody-Fc-Fab via molecular cloning of genes encoding the peptides in to constructs for expression of the antibody (
Peptides selected from peptide-phage libraries are fused to the N-terminus of the heavy (H) and/or light (L) chain of Fab-diabody-Fc via molecular cloning of genes encoding the peptides in to constructs for expression of the antibody (
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
The present disclosure relates to an antibody and/or binding fragment thereof as well as methods of making and use for example for diagnosing and/or prognosing COVID-19 infection and/or proliferation.
Described herein are antibodies and antibody binding fragments that specifically bind COVID-19 or a COVID-19 S polypeptide, e.g., S protein subunits S1 and S2. The antibodies described herein exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, and also bound to the receptor binding domain (RBD).
Accordingly, an embodiment of this invention is an antibody and/or binding fragment thereof that specifically binds COVID-19 or a COVID-19 S polypeptide, e.g., S protein subunits S1 or S2.
In some embodiments, the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention may be at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy chain (hIg1) CDRs of an antibody of Table 2. In some embodiments, the antibodies and antigen-binding fragments thereof contain a heavy chain variable region having a CDR amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% 98%, or at least 99% identical to the amino acid sequence of the heavy chain (hIg1) CDRs of an antibody of Table 2. In some embodiments, the amino acid sequence of the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention consist essentially of the amino acid sequence of the heavy chain (hIg1) CDRs of an antibody of Table 2.
In some embodiments, the light chain CDRs of the antibodies and antigen-binding fragments of this invention may be at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light chain (hk) CDRs of an antibody of Table 2. In some embodiments, the antibodies and antigen-binding fragments thereof contain a light chain variable region having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2. In some embodiments, the amino acid sequence of the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention consist essentially of the amino acid sequence of light chain (hk) CDRs of an antibody of Table 2.
In some embodiments, the antibodies and antigen-binding fragments thereof comprise (a) a VH domain comprising three heavy chain variable region CDRs, the CDRs having an amino acid sequence at least 90%/o, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of three heavy chain (hIg1) CDRs of an antibody of Table 2, and (b) a VL domain comprising three light chain CDRs having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2.
In some embodiments, the antibodies and antigen-binding fragments thereof comprise (a) a VH domain comprising three heavy chain variable region CDRs, the CDRs having an amino acid sequence of three heavy chain (hIg1) CDRs of an antibody of Table 2, and/or (b) a VL domain comprising three light chain CDRs having an amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2.
In an embodiment of the invention the antibody comprises an amino acid sequence at least 90%/o, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of a molecule of Table 4.
In an embodiment of the invention the antibody comprises an amino acid sequence of the molecules of Table 4. In an embodiment of the invention the antibody consists essentially of an amino acid sequence of the molecules of Table 4. Although the amino acid and nucleotide sequences of the molecules in Table 4 comprise the heavy chain and light chain CDRs of antibody 15031, embodiments of this invention include the amino acid and nucleotide of the molecules in Table 4 wherein the CDRs of 15031 are replaced with the CDRs of the antibodies set forth in Tables 2 and 3, for example, 15032, 15033, 15033-7, 15034, 15046, and 15049.
A further aspect is an antibody and/or binding fragment of this invention, wherein the antibody and/or binding fragment thereof specifically binds to COVID-19 or a COVID-19 S polypeptide in an unfixed or mildly fixed sample.
In an embodiment, the antibody and/or binding fragment thereof comprises a VL domain comprising a CDR3 and a VH domain comprising a CDR1, a CDR2 and a CDR3, wherein the amino acid sequences of the CDRs comprise one or more of the CDR sequences set forth in Table 2.
In an embodiment, the antibody and/or binding fragment thereof comprises a light chain variable region comprising an hk CDR3 of Table 2 and the heavy chain variable region comprises a hIg1 CDR1, a CDR2 and a CDR3 of an antibody set forth in Table 2.
In an embodiment, the antibody, binding fragment thereof, or CDRs disclosed herein may have one or more amino acid substitutions, including but not limited to conservative amino acid substitutions.
The antibody comprising the light chain and heavy chain CDRs set forth in Table 2 can be any class of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; and any isotype, including: IgG1, IgG2, IgG3 and IgG4.
Chimeric antibodies can be prepared using routine recombinant techniques. For example, a Fab of an IgG of Table 4 may be reformatted into another full length IgG by subcloning the variable domains of the immunoglobulin's light and heavy chains into mammalian expression vectors and producing the IgG protein using human embryonic kidney cells (HEK293T). As described elsewhere any cell type suitable for expressing an antibody can be used.
In one embodiment, the antibody may be a full-length immunoglobulin molecule, e.g., a chimeric antibody, a human antibody, a humanized antibody, a polyclonal antibody, or a minibody, and the antibody binding fragment may be a Fab, a Fab′, a F(ab)2, a F(ab′)2, a Fv, a disulfide linked Fv, a scFv, a disulfide linked scFv, a single chain domain antibody scFab or VHH, or a diabody, that does not comprise an Fc domain or fragment thereof.
The antigen binding fragments, e.g., Fab, Fab′ and F(ab′)2, scFv, scFab, dsFv, ds-scFv, VHH, or diabodies, and other fragments can be synthesized or expressed by recombinant techniques. Antibodies can also be fragmented using conventional techniques. For example, F(ab′)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. Papain digestion can lead to the formation of Fab fragments.
In an embodiment, the antibody is a synthetic monoclonal antibody or a chimeric antibody comprising one or more CDRs selected from the CDRs of Table 2. In an embodiment, the antibody is a chimeric antibody comprising a VL domain comprising the hk CDR1, CDR2 and CDR3 from an antibody of Table 2 and/or a VH domain comprising the hIg1 CDR1, CDR2 and CDR3 from an antibody of Table 2.
In an embodiment, the COVID-19 binding molecule is a VHH fragment, a scFv, an Fab or a diabody that comprises the hIg1 CDR1, CDR2 and CDR3 and/or the hk CDR1, CDR2 or CDR3 from an antibody of Table 2.
The antibodies and antibody fragments described herein may be isolated antibodies and antibody fragments.
In an embodiment of the full-length antibodies of this invention the Fc domain of the antibody is engineered such that it does not target the cell that binds the antibody for ADCC or CDC-dependent death. In an embodiment of the invention the Fc domain is dimerized via a knob-in-hole configuration. Methods are well known in the art for mitigating antibody effector function, including for example amino acid substitutions in the Fc regions, e.g., the N297G (NG) and D265A, N297G (DANG) variants or L234A, L235A, P329G (LALA-PG) substitutions, see e.g., Lo et al. “Effector Attenuating Substitutions that Maintain Antibody Stability and Reduce Toxicity in Mice. The Journal of Biological Chemistry Vol. 292, No. 9, pp. 3900-3908, Mar. 3, 2017, incorporated herein by reference.
In an embodiment of this invention the antibody and/or binding fragment thereof is linked, e.g., conjugated or recombinantly fused, to a therapeutic moiety, e.g., a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioactive metal ion.
In a further embodiment, the antibody and/or binding fragment is labelled and/or conjugated or fused to a tag, for example to produce a detection tool or a diagnostic agent. For example, the detectable tag can be a purification tag such as a His-tag, a HA-tag, a GST-tag, biotin or a FLAG-tag.
The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as 3H, 14C; 32P; 35S; 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion.
Another aspect of the disclosure relates to an antibody complex comprising the antibody and/or binding fragment described herein and COVID-19 or a COVID-19 S polypeptide.
Yet another aspect of this invention is a nucleic acid encoding an antibody and/or binding fragment described herein. In an embodiment, the nucleic acid encodes an antibody and/or binding fragment comprising a light chain variable region comprising a CDR1, CDR2 and CDR3 and a heavy chain variable region comprising a CDR1 CDR2 and CDR3, wherein one or more of the CDRs are the light chain and heavy chain CDRs set forth in Table 2.
The degeneracy of the genetic code allows for different nucleic acids to encode the same amino acid sequence. Accordingly, also included are nucleotide sequences that encode an antibody disclosed herein that specifically binds COVID-19 or a COVID-19 S polypeptide.
Also included in another embodiment are codon degenerate or optimized sequences. In another embodiment, the nucleic acid sequences are at least 70%, at least 75%, at least 80%, at least 90% and at least 95% identical to nucleic acid sequences of Table 3 or Table 4 encoding the molecules set forth in Table 2 or Table 4.
In an embodiment of this invention, the nucleic acid is an isolated nucleic acid.
Another aspect of this invention is a vector comprising the nucleic acid herein disclosed. In an embodiment, the vector is an isolated vector.
The vector can be any vector suitable for producing an antibody and/or binding fragment thereof, including for example vectors described herein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses).
A further aspect of this invention is a recombinant cell producing the antibody and/or binding fragment thereof herein disclosed or the vector herein disclosed.
The recombinant cell can be generated using any cell suitable for producing a polypeptide, for example suitable for producing an antibody and/or binding fragment thereof.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.
More particularly, bacterial host cells suitable for producing recombinant antibody producing cells include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genus Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art. Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the R-lactamase (penicillinase) and lactose promoter system, the trp promoter and the tac promoter. Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322, the pUC plasmids pUC18, pUC19, pUC118, pUC119, and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).
Suitable yeast and fungi host cells include, but are not limited to Saccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for the transformation of yeast and fungi are well known to those of ordinary skill in the art.
Suitable mammalian cells include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), NS-1 cells and any derivatives of these lines.
In an embodiment, the mammalian cells used to produce a recombinant antibody are selected from CHO, HEK293 cells or FREESTYLE™ 293-F cells (Life technologies). FREESTYLE 293-F cell line is derived from the 293 cell line and can be used with the FREESTYLE™ MAX 293 Expression System, FREESTYLE™ 293 Expression System or other expression systems.
Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences.
In an embodiment, the vector is designed for production of a light chain or a heavy chain, e.g., an IgG1 heavy chain.
Suitable insect cells include cells and cell lines from Bombyx or Spodotera species. Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series and the pVL series.
The recombinant expression vectors may also contain genes which encode a fusion moiety (i.e. a “fusion protein”) which provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including for example tags and labels described herein. Further, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
“Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate regulatory sequences is dependent on the host cell chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
In an embodiment, expression of the antibody or binding fragment thereof is under the control of an inducible promoter. Examples of inducible non-fusion expression vectors include pTrc (28) and pET 11d.
The recombinant expression vectors may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein suchas—galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest. Other selectable markers include fluorescent proteins such as GFP which may be cotransduced with the nucleic acid of interest.
Yet another aspect is a composition, e.g., a pharmaceutical comprising the antibody and/or binding fragment thereof, the nucleic acid herein disclosed or the recombinant cell herein disclosed, optionally in combination with a suitable diluent or carrier or excipient. The pharmaceutical composition may consist or consist essentially of the antibodies or binding fragment or the nucleic acid molecules expression cassettes and vectors encoding the antibodies or binding fragment described herein, and a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers, diluents and excipients, and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the agonist, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. Delivery systems for administering therapeutic agents, e.g., antibodies and fragments thereof, are known in the art, see e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), Langer, 1990, Science 249:1527-1533; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574; Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides, poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of the antibodies or binding fragment being administered.
The composition can be a lyophilized powder or aqueous or non-aqueous solution or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes. Other components that may be present in such compositions include, e.g., water, surfactants (such as TWEEN™), alcohols, polyols, glycerin and vegetable oils.
Suitable diluents for nucleic acids include but are not limited to water, saline solutions and ethanol. Suitable diluents for polypeptides, including antibodies or fragments thereof and/or cells include but are not limited to saline solutions, pH buffered solutions and glycerol solutions or other solutions suitable for freezing polypeptides and/or cells.
The composition can further comprise stabilizing agents, for example reducing agents, hydrophobic additives, and protease inhibitors which are added to physiological buffers.
Additional aspects of the invention are methods for using, producing or isolating an antibody and/or binding fragment thereof described herein with specific binding affinity to COVID-19 or a COVID-19 S polypeptide.
It has been reported the symptoms of COVID-19 infections include, e.g., fever, cough, shortness of breath, chills, repeated shaking with chills, muscle pain, headache, sore throat, loss of taste or smell, and can lead to physical complications, e.g., pneumonia and trouble breathing, organ failure in several organs, heart problems, severe lung condition, e.g., acute respiratory distress syndrome, blood clots, acute kidney injury and additional viral and bacterial infections. An embodiment of the invention relates to a method for treating, preventing, mitigating, or delaying the progression of a COVID-19 infection, or ameliorating a symptom or complication of a COVID-19 infection, comprising administering an effective amount of an antibody or binding fragment of this invention to a subject having or suspected of having a COVID-19 infection.
A method for treating, preventing, mitigating, or delaying the progression of a COVID-19 infection includes reducing viral load in a subject in need thereof. The antibody or binding fragment of the present invention may be administered systemically or locally, e.g., by injection (e.g. subcutaneous, intravenous, intraperitoneal, intrathecal, intraocular, etc.), inhalation, implantation, topically, or orally. Depending on the route of administration, the antibody or binding fragment may be coated in a material to protect the molecules from the action of acids and other natural conditions which may inactivate the molecules. The antibody or binding fragment described herein may be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition comprising the binding molecule can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The antibody or binding fragment of this invention can also be administered in conjunction with other therapeutic regimens or agents for treatment of COVID-19 infections and the associated symptoms or complications, e.g., convalescent serum therapy, small molecules, oleandrin, neutralizing monoclonal antibodies, recombinant antibodies, cytokines, or antivirals, e.g., LY-CoV555 (Eli Lilly), e.g., recombinant anti-CD47/PD-L1 bispecific antibody (IBI322)(Innovent Biologics, Inc.), remdesivir, Actemra, Oluminant (baricitinib), merimepodib, dexamethaone (glucocorticoid), Aviptadil (RLF-100), hydroxycloropuine, Avigan (favipiravir, Avifavir), Bucillamine, niclosamide, aviptadil, PlKfyve kinase inhibitor, tyrosine kinase inhibitor efineptakin alfa, interferon beta, or SLV213 (Selva Therapeutics).
Effective dosages and schedules for administering the antibodies and fragments thereof of this invention and nucleic acids that encode them described herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of such antibodies and fragments thereof that must be administered will vary depending on, for example, the subject that will receive the binding molecule, the route of administration, the particular type of antibody or fragment thereof used and other drugs being administered. Guidance in selecting appropriate doses for antibodies and fragments thereof of this invention is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith, Antibodies in Human Diagnosis and Therapy, Haber, eds., Raven Press, New York (1977) pp. 365-389. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the inflammation in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the antibody, fragments thereof, nucleic acid and vector is within the skill of the art.
An embodiment of the invention relates to a method for preventing or delaying the cleavage of the S2 subunit, or the fusion of COVID-19 with the cell and internalization of COVID-19 by a cell, e.g., a cell having an ACE2 receptor, by contacting the cell with an effective amount of an antibody or antibody binding fragment of this invention.
Yet another aspect relates to a method for screening or for diagnosing a subject having or suspected of having a COVID-19 infection, the method comprising measuring the level of COVID-19 or a COVID-19 S polypeptide in a sample from a subject by contacting the sample with an antibody or binding fragment of this invention, wherein detecting a level of COVID-19 or a COVID-19 S polypeptide bound by the antibody or binding fragment is indicative that the subject is infected with COVID-19. The method may further comprise the step of comparing the level of COVID-19 or a COVID-19 S polypeptide in the sample bound by the antibody or binding fragment with a negative or positive control, wherein an increased level of COVID-19 or a COVID-19 S polypeptide in the sample bound by the antibody or binding fragment as compared to a negative control, e.g., a value derived from a group of subjects without COVID-19 infection, or convalescing from a COVID-19 infection, is indicative that the subject is infected with COVID-19 or wherein a level of COVID-19 similar to or exceeding the level of COVID-19 of the positive control, e.g., a value derived from a group of subjects known to have a COVID-19 infection, is indicative that the subject is infected with COVID-19.
An additional aspect of the invention is a method for prognosing COVID-19 progression or lack thereof (e.g. recovery or worsening of disease). Accordingly an aspect is a method of prognosing a likelihood of recovery after COVID-19 infection, the method comprising: a) measuring a level of COVID-19 or a COVID-19 S polypeptide in a sample from a subject by contacting the sample with the antibodies or antibody fragments described herein; and b) comparing the level of COVID-19 or a COVID-19 S polypeptide bound by the antibodies or fragments thereof in the sample with a control, for example a control value derived from a group of subjects who recovered from or in whom the COVID-19 disease symptoms regressed, wherein a decreased level of COVID-19 in the sample bound by the antibodies or binding fragments compared to the control is indicative that the subject has an increased likelihood of recovery after COVID-19 infection. The level of COVID 19 may be measured by, e.g., measuring the amount of COVID-19 or a COVID-19 S polypeptide bound to the antibodies or antibody fragments of this invention by e.g., immunoprecipitation, an immunoassay, e.g. ELISA, immunoblot detection. The antibody-based detection can also be combined with a mass spectrophometric assay, for example as in the case of a particle-based flow cytometric assay.
In an embodiment, the control is a control value derived from a group of subjects recovered from COVID-19 infection and an increased level of COVID-19 or a COVID-19 S polypeptide in the sample compared to the control is indicative that the subject has a decreased likelihood of recovery after COVID-19 infection and/or an increased likelihood of COVID-19 infection and associated adverse effects progressing.
A further embodiment of the invention is a method for prognosing the likelihood of progression of a COVID-19 infection and associated adverse effects, the method comprising: a) measuring a level of COVID-19 or a COVID-19 S polypeptide in a sample from a subject by contacting the sample with the antibodies or antibody fragments described herein; and b) comparing the level of COVID-19 or a COVID-19 S polypeptide in the sample bound to the antibodies or antibody fragments described herein with a control, wherein an increased level of COVID-19 or a COVID-19 S polypeptide in the sample compared to the control, for example wherein the control is a control value derived from a group of subjects who did not recover or progressed is indicative that the subject has an decreased likelihood of recovering from a COVID-19 infection.
In an embodiment, the control is a control value derived from a group of subjects who recovered and an increased level of COVID-19 or a COVID-19 S polypeptide in the sample compared to the control is indicative that the subject has a decreased likelihood of recovery after COVID-19 and/or an increased likelihood of COVID-19 infection and associated adverse effects progressing.
The sample can for example be taken after the subject has received a treatment and compared for example to a pre-treatment sample. Alternatively, the patient can be monitored after a repeating interval to assess for example if treatment or other intervention is necessary. In an embodiment, the test is repeated and plotted to assess the subject's progression.
The sample may be a tissue sample, e.g. lung tissue, or a biological fluid such as blood, or a fraction thereof such as plasma or serum, or a lung secretion or a pulmonary lavage. In an embodiment the biological fluid is saliva or a nasal secretion.
In another embodiment, the sample is selected from a fresh sample such as a fresh (e.g. not frozen) biological fluid sample or tissue sample, a onetime frozen biological fluid sample or tissue sample (e.g. frozen a single time at the time of obtaining the sample)) or a repeat frozen sample (e.g. frozen and thawed and frozen biological fluid sample or repeat frozen tissue sample). In an embodiment, the sample comprises native or natively folded COVID-19 polypeptide. In an embodiment, the sample is a fixed sample such as a mildly fixed sample wherein the fixation induces limited denaturation and/or unfolding.
In another embodiment, the isolated and purified antibody and/or binding fragment thereof is affinity matured. Affinity maturation can be performed as described for the initial selection, with antigen adsorbed to plastic plates, using a for example a phage library comprising variants of the CDR sequences.
A person skilled in the art will appreciate that several methods can be used to isolate and produce antibodies and/or binding fragments thereof with specific binding affinity to COVID-19 or a COVID-19 polypeptide, e.g. COVID-19 S polypeptide. A method that can be used is a phage display method. For example, COVID-19 or a COVID-19 S polypeptide is produced in order to isolate and characterize the antibody and/or binding fragment thereof. Phage from a human Fab phage-displayed library are selected following several rounds of panning. Phage with specific binding affinity to the COVID-19 or a COVID-19 S polypeptide, as determined by ELISA, are sequenced and cloned into vectors designed for production of light chain or heavy chain. The heavy chain can be for example an IgG, or an IgG isotype such as an IgG1 or an IgG4. Antigen binding fragments and IgG polypeptides are then affinity purified by using, for example, Protein A affinity columns.
In another embodiment, a nucleic acid encoding an antibody or antibody binding fragment described herein is expressed in a host cell to make the antibody and/or binding fragment. In an embodiment, the method comprises: a) expressing in a host cell a nucleic acid encoding an antibody and/or binding fragment; b) culturing the host cell to produce the antibody and/or binding fragment; and c) isolating and/or purifying the antibody and/or binding fragment from the host cell.
In some embodiments, a nucleic acid encoding a single chain antibody is expressed. In other embodiments, multiple nucleic acids are expressed, for example a nucleic acid encoding an antibody light chain and a nucleic acid encoding an antibody heavy chain.
Suitable host cells and vectors are described above. Vectors and nucleic acids encoding an antibody or antibody biding fragment described herein may be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection and other liposome-based transfection agents, electroporation or microinjection.
Nucleic acid encoding an antibody or antibody biding fragment described herein may be directly introduced into mammalian cells using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors.
The COVID-19 or COVID-19 S polypeptide specific antibodies disclosed herein can be used in a variety of assays for binding, detecting and measuring COVID-19 or a COVID-19 S polypeptide in a sample. For example, the antibodies can be used in an ELISA, as well as immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA) (Muhammad S. Alam, November 2018) Current Protocols in Immunology Vol 123, Issue 1., mass spectroscopy-based techniques, particle-based flow cytometric assay, fluorescence-activated cell sorting (FACS) and ELISA.
Immunodetection methods as described herein generally involve the detection or measuring of antibody:COVID-19 or a COVID-19 S polypeptide, e.g. an S1 of S2 subunit, complexes using antibodies and/or binding fragments thereof disclosed herein. The detection of such complexes is well known in the art and may be achieved through different methods, for example by using a detectable label or marker, such as a radioactive, fluorescent or enzymatic tag. Detection of these complexes may also involve the use of a ligand such as a secondary antibody and/or binding fragment thereof specific for COVID-19 or a COVID-19 S polypeptide or for the antibody:COVID-19 or a COVID-19 S polypeptide complex.
They can also be used to make detection assays, for example a sandwich ELISA, as one antibody can be used as a capture reagent to isolate the COVID-19 or a COVID-19 S polypeptide while another antibody binding a distinct epitope can be used as a detection reagent.
Accordingly, another aspect is an immunoassay comprising one or more antibodies and/or binding fragments thereof described herein.
In an embodiment, the immunoassay is an enzyme linked immunosorbent assay (ELISA). Antibodies and/or binding fragments thereof may be used in the context of detection assays such as ELISAs, for example sandwich ELISAs. The antibodies and binding fragments of this invention may be used as capture and detection antibodies for the detection of COVID-19 or a COVID-19 S polypeptide in solution. For example, the full-length antibodies or binding fragments of this invention that recognize the S protein, e.g., the S1 subunit, are immobilized and incubated with COVID-19 or a COVID-19 S polypeptide or subunit. The samples are then incubated with biotinylated IgGs recognizing epitopes on the virus, polypeptide, or subunit.
In an embodiment, the ELISA is a sandwich ELISA comprising a capture antibody and a detection antibody that bind to different epitopes. For example, the capture antibody is an antibody or binding fragment which specifically binds a first epitope of COVID-19 or a COVID-19 S polypeptide, e.g., a S1 subunit or the RBD, and/or the detection antibody is an antibody or binding fragment which specifically binds a second epitope of the COVID-19 or a COVID-19 S polypeptide, e.g., the S1 subunit or the RBD, wherein the first and second epitopes are different. In an embodiment, one or both of the capture or detection antibodies is an antibody and/or binding fragment thereof herein.
In one embodiment, the immunoassay is for the detection and/or measuring of COVID-19 or a COVID-19 S polypeptide, e.g., the S1 or S2 subunit, in a sample, wherein the method of making the immunoassay comprises: a) coating a solid support with the capture antibody; b) contacting the capture antibody with the sample under conditions to form a capture antibody:COVID-19 or a COVID-19 S polypeptide complex; c) removing unbound sample; d) contacting the capture antibody:COVID-19 or a COVID-19 S polypeptide complex with the detection antibody; e) removing unbound detection antibody; and f) detecting and/or measuring the capture antibody:COVID-19 or a COVID-19 S polypeptide complex, wherein one or both of the capture or detection antibodies is an antibody and/or binding fragment thereof herein.
In an embodiment, the ELISA is a competitive ELISA. In an embodiment, the ELISA is a direct ELISA. In an embodiment, the ELISA is an indirect ELISA.
As used herein, “solid supports” include any material to which COVID-19 or a COVID-19 S polypeptide and antibodies and/or binding fragments thereof herein disclosed are capable of binding to. For example, the solid support can include plastic, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses and polyacrylamides. For example, the solid support is a microtiter plate, magnetic beads, latex beads or array surfaces.
In embodiments of the immunoassays described herein, the sample may be contacted with an antibody and/or binding fragment thereof under appropriate conditions, for example, at a given temperature and for a sufficient period of time, to allow effective binding of COVID-19 or a COVID-19 S polypeptide to the antibody or binding fragment, thus forming an antibody:COVID-19 or a COVID-19 S polypeptide complex, such as a capture antibody:COVID-19 or a COVID-19 S polypeptide complex. For example, the contacting step is carried out at room temperature for about 30 minutes, about 60 minutes, about 2 hours or about 4 hours. For example, the contacting step is carried out at about 4° C. overnight.
The antibody and/or binding fragment as described herein may be, e.g., complexed with COVID-19 or a COVID-19 S polypeptide in a suitable buffer. For example, the buffer has a pH of about 5.0 to about 10.0. For example, the buffer has a pH of 4.5, 6.5 or 7.4. For example, the buffer is a HBS-EP buffer, a KRH buffer or Tris-buffered saline. For example, the buffer comprises BSA and/or TWEEN™ 20.
Any unbound material in a sample may be removed by washing so that only the formed antibody:COVID-19 or a COVID-19 S polypeptide complex remains on the solid support. For example, the unbound sample is washed with phosphate-buffered saline, optionally comprising bovine serum albumin (BSA).
In an embodiment, the detection antibody is labelled and/or conjugated to a tag.
For example, the detection antibody directly labelled and/or conjugated. For example, the detection antibody is indirectly labelled and/or conjugated. Indirect labels include for example fluorescent or chemiluminescent tags, metals, dyes or radionuclides attached to the antibody. Indirect labels include for example horseradish peroxidase, alkaline phosphatase (AP), beta-galactosidase and urease. For example, HRP can be used with a chromogenic substrate, for example tetramethybenzidine, which produces a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
An embodiment of the invention is an assay for detecting and/or measuring level of COVID-19 or a COVID-19 S polypeptide in a sample, the assay comprising: a) contacting a sample with the antibody and/or binding fragment described herein under conditions to form an antibody:COVID-19 or a COVID-19 S polypeptide complex; and b) detecting and/or measuring the antibody:COVID-19 or a COVID-19 S polypeptide complex. A further embodiment is an assay for detecting and/or measuring COVID-19 or a COVID-19 S polypeptide the method comprising: a) contacting a sample, the sample being a biological fluid, with the antibody and/or binding fragment of the antibody as described herein under conditions to form an antibody:COVID-19 or a COVID-19 S polypeptide complex; and b) detecting and/or measuring the antibody:COVID-19 or a COVID-19 S polypeptide complex. The assay for detecting and/or measuring the level of COVID-19 or a COVID-19 S polypeptide is performed under non-denaturing or mildly denaturing conditions.
In an embodiment, the antibody:COVID-19 or a COVID-19 S polypeptide complex is detected directly for example by using an antibody labeled with a detectable tag or fusion moiety. In an embodiment, the complex is detected indirectly using a secondary antibody specific for the antibody:COVID-19 or a COVID-19 S polypeptide complex.
In an embodiment, the assay for detecting or measuring the level of COVID-19 or a COVID-19 S polypeptide is an immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA), mass spectroscopy-based techniques and fluorescence-activated cell sorting (FACS), and ELISA.
Detecting can be performed using methods that are qualitative or measured using quantitative methods, for example by comparing to a standard or standard curve.
The sample used in the methods described herein may be a biological sample from a subject, e.g., a human or other primate, or the sample may be from another source. The biological sample from a subject may be a tissue or a fluid, e.g., blood and its component parts, serum or plasma, saliva, a lung secretion or a lung lavage, or urine etc. Other samples may be, e.g., wastewater or a body of water where wastewater drains, e.g., a river, canal, pond, bay etc.
A further aspect of this invention relates to a kit comprising i) an antibody and/or binding fragment described herein, ii) a nucleic acid encoding the an antibody and/or binding fragment described herein, iii) a composition comprising the an antibody and/or binding fragment described herein or the nucleic acid encoding the antibody or fragment, or iv) a recombinant cell described herein, comprised in a vial such as a sterile vial or other housing and optionally a reference agent, and/or instructions for use thereof.
In an embodiment, the kit comprises components and/or is for use in performing an assay described herein.
For example, the kit is an ELISA kit and can comprise a first antibody, e.g. a capture antibody, for example attached to a solid support, and a second antibody, e.g. a detection antibody, that binds to COVID-19 or a COVID-19 S polypeptide and/or binds the capture antibody: COVID-19 or a COVID-19 S polypeptide complex, and that is conjugated to a detectable label. The first and/or second antibody may be an antibody or antibody fragment of this invention. Any combination of antibodies or antibody fragments described herein can be used.
In an embodiment, the kit is a diagnostic kit and the instructions are directed to a method described herein.
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
The following non-limiting examples are illustrative of the present disclosure:
To identify antibodies that neutralize SARS-CoV-2 as a basis for therapeutic development, we conducted phage-display selections against the RBD of the S protein, isolated numerous clones that bound to the SARS-CoV-2 RBD or the full-length S protein ectodomain, and screened to identify those that blocked ACE2 binding. In these studies, a library containing 1010 Fab-phage clones was used for selections (Persson et al. (2013) CDR-H3 diversity is not required for antigen recognition by synthetic antibodies. J. Mol. Biol. 425, 803-811.). Among the high affinity Fab-phage clones, those that blocked ACE2 were selected for further characterization and expressed as full-length human IgG proteins with a framework engineered to possess high thermostability and low immunogenicity for therapeutic applications (Persson et al. (2013)). Several IgGs were found to exhibit sub-nanomolar affinities for the SARS-CoV-2 S protein and from this group antibody number 15033 emerged as a best neutralizer with an ability to neutralize SARS-CoV-2 with high potency.
The previously reported piggyBac transposase-based expression plasmid PB-T-PAF (Li et al., (2013) Simple piggyBac transposon-based mammalian cell expression system for inducible protein production. Proc. Natl. Acad. Sci. U.S.A. 110, 5004-5009) was modified to generate two new vectors, one containing a CMV promotor (PB-CMV) and the other a TRE promotor (PB-TRE). To facilitate nuclear export of mRNA, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was also added to each of these plasmids. Each of the protein ORFs was cloned into both expression vectors. The PB-CMV version was used for large-scale transient expression. The PB-TRE version was used for establishing inducible stable cell lines using the piggyBac transposase-based method (Li, Z., et al. (2013) Simple piggyBac transposon-based mammalian cell expression system for inducible protein production. Proc. Natl. Acad. Sci. U.S. A. 110, 5004-5009.).
cDNAs encoding the SARS-CoV and SARS-CoV-2 S protein proteins were human codon optimized and synthesized by Genscript. cDNA encoding human ACE2 was obtained from MGC clone 47598. The soluble SARS-CoV-2 S protein ectodomain trimer included residues 1-1211, followed by a foldon trimerization motif (Tao et al. (1997) Structure of bacteriophage T4 fibritin: A segmented coiled coil and the role of the C-terminal domain. Structure 5, 789-798), a 6×His tag and an AviTag biotinylation motif (Fairhead, M., and Howarth, M. (2015) Site-specific biotinylation of purified proteins using BirA. Methods Mol. Biol. 1266, 171-184.). Residues 682-685 (RRAR) were mutated to SSAS to remove the furin cleavage site on the SARS-CoV-2 S protein protein. Residues 986-987 (KV) were mutated to two proline residues to stabilize the pre-fusion form of the S protein (Pallesen et al. (2017) Immunogenicity and structures of a rationally designed prefusion MERS-CoV S protein antigen. Proc. Natl. Acad. Sci. U. S.A. 114, E7348-E7357.). The SARS-CoV-2 receptor binding domain (RBD, residues 328-528), the soluble human ACE2 construct (residues 19-615), and the SARS-CoV RBD (residues 315-514) were each followed by a 6×His tag and an AviTag.
Freestyle 293-F cells were grown in Freestyle 293 expression medium (Thermo Fisher) in suspension culture. Upon transfection, 300 mL cells were seeded to 1-L shaker flasks at a cell density of 106 cells/mL. 15 mL Opti-MEM medium (Thermo Fisher) containing 300 μg of PB-CMV plasmid DNA was mixed with 15 mL Opti-MEM medium containing 400 μL 293fectin reagent (Thermo Fisher). The mixture was incubated for 5 minutes before being added to the shaker flask. Two days post transfection, each 300 mL culture was expanded to 3 shaker flasks containing a total of 900 mL medium, and the expression was continued for another 4 days.
Freestyle 293-F cells or a GnT1-knockout Freestyle 293-F cell line was used for generating the stable cell lines. The cells were transfected on 6-well plates. Each well contained 106 cells growing in 2 mL Freestyle 293 expression medium. For producing doxycycline-inducible cell lines, 2 μg of the PB-TRE expression plasmid, 0.5 μg of the PB-rtTA-neomycin helper plasmid (Li et al. (2013)) and 0.5 μg of the piggyBac transposase plasmid pCyL43 (Wang et al. (2008) Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A 105, 9290-9295.) were co-transfected to each well using the Lipofectamine 2000 reagent (Thermo Fisher). These cells were then selected using 2 μg/mL puromycin and 200 μg/mL G418 for two weeks. The ACE2-expressing cell line was constructed by co-transfecting the Freestyle 293-F cells with the PB-CMV plasmid encoding the full length human ACE2 and the pCyL43 plasmid. This cell line was selected using 2 μg/mL puromycin.
For stable expression, the cell lines were grown as suspension cultures in 1-L shaker flasks. Each flask contained 300 mL Freestyle 293 expression medium supplemented with 1 μg/mL doxycycline and 1 μg/mL aprotinin. Half of the culture was harvested and replaced by fresh medium every other day.
The proteins were purified from the harvested expression medium using Ni-NTA affinity chromatography. The proteins were eluted with phosphate buffered saline containing 300 mM imidazole and 0.1% (v/v) protease inhibitor cocktail (Sigma, P-8849). The proteins were further purified using size-exclusion chromatography. For the RBDs and ACE2, a Superdex 200 Increase (GE healthcare) column was used. For the S protein ectodomain, a Superose 6 Increase (GE healthcare) column was used.
Each biotinylation reaction contained 200 μM biotin, 500 μM ATP, 500 μM MgCl2, 30 μg/mL BirA, 0.1% (v/v) protease inhibitor cocktail and not more than 100 μM of the protein-AviTag substrate. The reactions were incubated at 30° C. for 2 hours. The biotinylated proteins were then purified by size-exclusion chromatography.
Fab-phage clones specific for the SARS-CoV-2 S protein were isolated from phage-displayed antibody libraries (Persson et al. (2013) J Mol Biol 22; 425(4):803-11) by multiple rounds of binding selections with the RBD immobilized in wells of neutravidin-coated microwell plates, as described (Persson et al. (2013)). Individual colonies were isolated from Escherichia coli infected with phage outputs from rounds 3 and 4, and individual Fab-phage clones were amplified. Antibody variable domains were sequenced by PCR amplification from phage supernatants for extraction of DNA sequences encoding antibody complementarity determining regions.
Phage clone variable domain DNA was amplified by PCR and subcloned into pSCSTa-hIg1 and pSCST1-hk vectors. Vectors for the heavy and light chains were transfected into HEK293F cells (Invitrogen, Grand Island, NY) using FectoPro according to the manufacturer's instructions (Polyplus Transfection, NY). Cell cultures were incubated at 37° C. for 4-5 days post-transfection. The cell cultures were centrifuged, and the supernatants were applied to a protein-A affinity column (˜2 mL packed beads per 600 mL culture) (Pierce, ThermoScientific, Rockford, IL). IgG proteins were eluted with 100 mM glycine, pH 2.0 and neutralized with 2 M Tris, pH 7.5. The eluent underwent buffer exchange and concentration in to PBS, pH 7.4 by centrifugation in a 50 kDa centrifugal concentrator.
For capture of biotinylated antigens, 384-well microplate wells were coated overnight at 4° C. with 2 μg/mL neutravidin in PBS pH 7.4. After coating, wells were blocked with 0.2% BSA in phosphate buffered saline (PBS) for one hour and washed 4 times with 0.05% TWEEN™ in PBS (PT buffer). Solutions of S protein (10 nM) or RBD (50 nM) or biotinylated control (50 nM) in PBS were used to immobilize target by incubating for 15 min at room temperature and washing with PT buffer. IgGs were diluted into PBT, applied to the wells, and incubated at room temperature for 30 min. The plates were washed with PBST (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05% TWEEN™ 20, pH 7.4.) and incubated for 30 min with anti-k-HRP (horse radish peroxidase) antibody conjugate (1:7500 dilution in PBT). The wells were washed 4-6 times with PBST and developed as described above. The absorbance at 450 nm was determined. To estimate EC50 values, data were fit to standard four-parameter logistic equations using Graphpad Prism (GraphPad Software, La Jolla, CA).
For competition ELISAs, S protein was immobilized in 384-well plates as above but at a concentration of 5 μg/well, then wells were quenched with 100 μg/mL biotin. Immobilized S protein was blocked with 100 nM IgG for 30 min and 100 nM biotinylated ACE2 was added to both IgG-blocked and non-blocked wells in parallel. Following a 30 min incubation, the plates were washed with PBST, and HRP/streptavidin conjugate (1:10000 dilution in PBST) was added and incubated for 30 min. The plates were washed with PBST, developed with TMB substrate, and quenched with 0.5 M H2SO4 before measuring absorbance at 450 nm.
The results of these ELISAs are presented in
To determine the affinity and binding kinetics of IgGs for the S protein, BLI experiments were performed on an Octet HTX instrument (ForteBio) at 1000 rpm and 25° C. Biotinylated S protein was first captured on SA biosensors (ForteBio) from a 2 μg/mL solution in PBT, in parallel with an identical concentration of an unrelated biotinylated control protein, followed by a 180 s quench step with 100 μg/mL biotin. After equilibrating with PBT, loaded biosensors were dipped for 600 s into wells containing serial 3-fold dilutions of IgG and subsequently were transferred back into assay buffer for 600 s dissociation. Binding response data were reference subtracted and were fitted with 1:1 binding model using ForteBio's Data Analysis software 9.0.
The binding kinetics for IgGs and SARS-CoV-2 S protein are presented in
For assays conducted in St Louis, SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was obtained from the Centers for Disease Control and Prevention (gift of Natalie Thornburg). Virus stocks were produced in Vero CCL81 cells (ATCC) and titrated by focus-forming assay on Vero E6 cells. Serial dilutions of mAbs were incubated with 102 focus-forming units (FFU) of SARS-CoV-2 for 1 h at 37° C. MAb-virus complexes were added to Vero E6 cell monolayers in 96-well plates and incubated at 37° C. for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 30 h later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with 1 μg/mL anti-S antibody CR3022 (Yan et al. (2020) A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science 368, 630-633) and HRP-conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% BSA. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies). Data were processed using Prism software (GraphPad Prism 8.0).
For assays conducted in Rome, SARS-CoV-2 strain 2019-nCoV/Italy-INMI was used. Antibodies were diluted to a concentration of 10 μg/ml in serum-free medium, and titrated in duplicate in two-fold serial dilutions. Equal volumes (50 p1) of approximately 100 TCID50/well virus and antibody dilutions, were mixed and incubated at 37° C. for 30 min. Subsequently, 96-well tissue culture plates with sub-confluent Vero E6 cell monolayers were infected with 100 μl/well of virus-serum mixtures in duplicate and incubated at 37° C. and 5% CO2 for two days. Then, the supernatant of each plate was carefully discarded and 120 p1 of a Crystal Violet solution containing 2% Formaldehyde was added to each well. After 30 min fixation, the fixing solution was removed and cell viability was measured by photometer at 595 nm (Synergy HTX Biotek).
The results of these neutralization assays are depicted in
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the inventions. Various substitutions, alterations and modifications may be made to the invention without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are within the scope of the invention.
The contents of all references, issued patents, and published patent applications cited through this application are hereby incorporated by reference. The appropriate component, process and methods of those patents, applications and other documents may be selected for the invention and embodiments thereof.
Described herein are multivalent binding molecules comprising two or more antibody binding fragments that bind a coronavirus, wherein the binding fragments are joined by a peptide linker. In an embodiment the multivalent binding molecule is at least bivalent or at least trivalent and does not comprise an Fc domain or a fragment thereof. In an embodiment of the invention at least two antibody fragments of the multivalent binding molecule are not identical.
Also described herein are multivalent binding molecules comprising an Fc domain, with or without effector function, a first and/or a second binding domain for a coronavirus polypeptide. In an embodiment of the multivalent binding molecules, the molecule comprises two binding domains, each binding domain comprises at least two coronavirus binding sites and the binding domains are attached to opposite termini of the Fc domain such that the two binding domains are not directly linked rather they are separated by the Fc domain.
Also described herein are multivalent binding molecules comprising an Fc domain, with or without effector function, and a binding domain for a coronavirus polypeptide comprising 2 or more, 3 or more, or 4 or more binding sites, wherein the binding domain is attached to the N-terminus of the Fc domain.
A subset of exemplary formats of the multivalent binding molecules of this invention is depicted in
The Fc domain in the multivalent binding molecules herein may alternatively be a fragment of the Fc domain comprising the constant heavy chain domain 3 (CH3 domain). In an embodiment of the invention the Fc domain of the multivalent molecule is engineered such that it does not target the cell that binds the multivalent molecule for ADCC or CDC-dependent death. In an embodiment of the invention the Fc domain of the multivalent binding molecule is a peptide dimer in a knob-in-hole configuration. The peptide dimer may be a heterodimer.
The binding domains may be attached directly to the Fc domain or attached to the Fc domain via a linker. The binding domains may be monospecific and bind to the same epitope on a covonavirus polypeptide, or to overlapping epitopes on a coronavirus polypeptide, or may be multispecific, binding to different epitopes on one or more coronavirus polypeptide. The coronavirus polypeptide may be, e.g., a coronavirus S protein, including the S1 and S2 subunits, or the receptor binding domain (RBD) of the S protein.
The coronavirus bound by the multivalent binding molecules may be an alpha or beta coronaviraus. In an embodiment, the coronavirus may be, e.g., HCoV-229E and HCoV-NL63, HCoV-HKU1, HCoV-OC43, MERS-CoV, SARS-CoV, or SARS-CoV-2, and the polypeptide may be e.g., a spike (S) protein, a membrane (M) protein, a nucleocapsid (N) protein, a hemagglutinin-esterase glycoprotein (HE) or small envelope (e) protein of the coronavirus. In a particular embodiment the polypeptide is a protein of SARS-CoV-2, e.g. a spike (S) protein and fragments thereof.
In an embodiment of the invention one or both binding domains comprise an antibody binding fragment, e.g., a scfv, a Fab, a VHH, or a diabody or combinations thereof that bind a coronavirus polypeptide. In an embodiment of the one binding domain is a diabody or two scFvs and the other binding domain comprises two Fabs.
An embodiment of this invention is a Diabody-Fc, a tetravalent binding molecule having (i) an Fc domain, (ii) one coronavirus binding domain attached to the N-terminus of the Fc domain that comprises a coronavirus-binding diabody.
An embodiment of this invention is a Diabody-Fc-Fab, a tetravalent binding molecule having (i) an Fc domain, (ii) one coronavirus binding domain, attached to the N-terminus of the Fc domain, that comprises a diabody that binds a coronavirus polypeptide, and (iii) a second coronavirus binding domain, attached to the carboxy terminus of the Fc domain, comprising two Fab fragments each binding an epitope on a coronavirus polypeptide.
An embodiment of this invention is a Diabody-Fc-scFv, a tetravalent binding molecule having (i) an Fc domain, (ii) one binding domain that comprises a coronavirus-binding diabody attached to the N-terminus of the Fc domain, and (iii) a second binding domain, attached to the carboxy terminus of the Fc domain comprising two coronavirus-binding scFv each binding an epitope on a coronavirus polypeptide.
An embodiment of this invention is an IgG-scFv, a tetravalent binding molecule having (i) an Fc domain, (ii) one coronavirus binding domain that comprises of two Fab fragments attached to the N-terminus of the Fc domain, each Fab binding a coronavirus polypeptide and (iii) one coronavirus binding domain composed of two scFvs that each bind a site on a coronavirus, wherein the scFvs are attached to the C-terminus of the Fe domain.
An embodiment of this invention is a an Fab-IgG, a tetravalent binding molecule having (i) an Fc domain, (ii) a first coronavirus binding domain that is composed of a first set of two Fabs that bind a coronavirus polypeptide, wherein the Fabs are linked in tandem, and a second set of two Fabs that bind a coronavirus polypeptide, wherein the Fabs are linked in tandem and wherein both sets of Fabs are attached to the same terminus of the Fc domain. For example, the Fab-IgG tetravalent binding molecule may comprise,
An embodiment of this invention is an IgG-Fab, a tetravalent binding molecule having (i) an Fe domain, (ii) a first coronavirus binding domain that comprises a two Fabs that bind a coronavirus polypeptide, and (iii) a second coronavirus binding domain that comprises a two Fabs that binds a coronavirus polypeptide, wherein the two binding domains are attached to opposite ends of the Fc domain. For example, the IgG-Fab may comprise,
Another embodiment of the multivalent binding molecule is an immunoglobulin-diabody, e.g., an IgG-diabody, a tetravalent binding molecule comprising a coronavirus-binding immunoglobulin, having a coronavirus-binding diabody attached to the carboxy terminal of the immunoglobulin Fe domain. For example, an IgG-diabody is a tetravalent binding molecule comprising (i) an Fc domain, (ii) an N-terminal coronavirus binding domain, comprising two coronavirus-binding Fabs and (ii) a C-terminal coronavirus binding domain comprising a coronavirus-binding diabody. The molecule comprises (1) a first set of a first and second monomer, wherein each monomer comprises a single-chain polypeptide comprising, from N-terminus to C-terminus: (a) a first heavy chain variable domain (VH) that binds a coronavirus polypeptide, (b) a heavy chain constant region domain 1 (CH1 domain) (c) an Fc region (or fragment thereof comprising a constant heavy chain domain 3 (CH3 domain)), (d) a peptide comprising a second VH that binds a coronavirus polypeptide, and a first light chain variable domain (VL) that binds a coronavirus polypeptide, and (2) second set of a third and fourth monomers, each comprising from N terminus to C terminus a second VL that binds a coronavirus polypeptide, and a constant light chain domain 1 (CL1 domain). The first set of monomers dimerize via their Fc regions, or fragments thereof, and the coronavirus polypeptide binding diabody is formed by the pairing of the second VH and first VL of one monomer with the first VL and second VH of the other monomer. The coronavirus polypeptide-binding Fabs are formed by the pairing of the first VH and CH1 of the first and second monomers, with the second VL and CL1 of the third and fourth monomers. In this format, the diabody forms the coronavirus polypeptide-binding domain on the C-terminus of the Fc domain and the Fabs form the coronavirus polypeptide-binding domain on the N-terminus of the Fc domain.
An embodiment of this invention is a tetravalent binding molecule in a Diabody-Fc-Fab format that binds a coronavirus polypeptide. This multivalent binding molecule comprises (1) a first set of a first and second monomer, wherein each monomer comprises a single-chain polypeptide comprising, from N-terminus to C-terminus: (a) a peptide comprising a first heavy chain variable (VH) domain, which binds coronavirus, and a first light chain variable (VL) domain, which binds coronavirus, (b) an Fc region (or fragment thereof comprising a constant heavy chain domain 3 (CH3 domain)), (c) a second VH domain, which binds coronavirus, and (d) a CH1 domain, and (2) a second set of a third and fourth monomer each monomer comprising from N-terminus to C-terminus a second VL domain, which binds coronavirus, and a constant light chain domain 1 (CL1). The first set of monomers dimerize via the Fc regions or fragments thereof and a bivalent coronavirus-binding diabody is formed by the pairing of the first VH domain and first VL domain of one monomer with the first VL domain and first VH domain of the other monomer, forming the first binding domain. The two coronavirus-binding Fabs are formed by the pairing of the second VL and CL1 with the second VH and CH1 thereby forming the second coronavirus binding domain. In this format, the diabody forms the first coronavirus binding domain on the amino terminus of the tetravalent molecule and the Fabs forms the second coronavirus binding domain on the C-terminus of the tetravalent binding molecule.
In an embodiment of this invention the binding moiety of the coronavirus binding domain is derived from an antibody that is monospecific binding specifically to one coronavirus polypeptide or is multispecific binding to more than one coronavirus polypeptide. In an embodiment of the invention the COVID-binding antibody binds to a RBD on the coronavirus S protein protein S1 subunit.
In an embodiment of this invention the multivalent binding molecule is monospecific binding to a single epitope on coronavirus. In an embodiment of this invention the multivalent binding molecule is multispecific binding to two or more epitopes on coronavirus.
In the multivalent binding molecules of this invention one or both of the binding domains may be multivalent, e.g., bi, tri, tetra or hexavalent and one or both of the multivalent binding domains may be multispecific, e.g., bi, tri, tetra or hexa-specific for the respective coronavirus polypeptide target. For example, the multivalent binding molecule may comprise a first coronavirus binding domain that is multivalent and monospecific (each binding site binding to the same epitopes) and a second coronavirus binding domain that is multivalent and multispecific, binding to different epitopes. In an embodiment of this invention the multivalent binding molecule comprises two binding domains that are bivalent and bispecific, each binding domain binding to two different epitopes on their respective target coronavirus polypeptides.
The VH and VL domains of the coronavirus binding domains may comprise the 3 light chain CDRs and 3 heavy chain CDRs of a coronavirus source antibody, or the three light chain CDRs and three heavy chain CDRs are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDRs of a coronavirus source antibody.
In an embodiment of this invention one or both coronavirus binding domain of the multivalent binding molecules comprises 3 light chain CDRs and 3 heavy chain CDRs of the antibodies of Table 2, or CDRs that are at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to 3 light chain CDRs and 3 heavy chain CDRs of the antibodies of Table 2. In some embodiments, the heavy chain and light chain CDRs of a VH and/or VL consist essentially of the heavy chain (hIg1) and light chain (hk) CDRs of an antibody of Table 2.
Also an embodiment of this invention are nucleic acid molecules encoding the multivalent binding molecules of this invention. An embodiment of this invention are nucleic acid molecules encoding the multivalent binding molecules described herein comprising the heavy chain and light chain CDRs set forth in Table 2. Also an embodiment of this invention are the nucleic acid molecules that encode VH and VL domains comprising respectively the heavy chain CDRs (CDR-H1 CDR-H2 and CDR-H3) and light chain CDRs (CDR-L1, CDR-L2 and CDR-L3) set forth in Table 2. In some embodiments, the nucleic acid molecule may comprise a nucleotide sequence encoding a multivalent binding molecule set forth in Table 3 or Table 4, e.g., Fab-VH, scFv-Fab, Fab-scFv, Fab-Diabody, VH(HC-CT)-Fab-scFv, VH(HC-NT)-Fab-scFv, VH(VL-NT)-Fab-scFv, VH-Fc, VH-VH-Fc, VH-Fc-VH, VH-Fc-scFv, VH-Fc-Fab, scFv-Fc, scFv-scFv-Fc, scFv-Fc-scFv, scFv-Fc-VH, scFv-Fc-Fab, Diabody-Fc, Diabody-Fc-Diabody, Diabody-Fc-VH, Diabody-Fc-scFv, Diabody-Fc-Fab, Fab-diabody-Fc, VH-hIgG1, VH-VH-hIgG1, scFv-hIgG1, scFv-scFv-hIgG1, Fab-hIgG1, Fab-Fab-hIgG1. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in Table 3 or Table 4.
The nucleic acid molecules can be inserted into a vector and expressed in an appropriate host cell and then the multivalent binding molecules may be isolated from the cells using methods well known in the art. As such, also an aspect of this invention are expression cassettes and vectors comprising the nucleic acid molecules that encode the polypeptides of the multivalent binding molecules described herein, as well as the VL and VH domains, the Fabs, scFvs and the diabodies comprising the CDRs of an antibody set forth in Table 2 and Table 4, and the Fc domains described herein. An aspect of this invention are the host cells expressing these expression cassettes and vectors.
The degeneracy of the genetic code allows for different nucleic acids to encode the same amino acid sequence as set forth herein. Accordingly, also included are nucleotide sequences that encode a multivalent binding molecule of this invention that binds coronavirus, e.g., a coronavirus S polypeptide.
Also included in another embodiment are codon degenerate or optimized sequences. In another embodiment, the nucleic acid sequences are at least 70%, at least 75%, at least 80%, at least 90% and at least 95% identical to nucleic acid sequences encoding the CDRs and the multivalent binding molecules set forth in Table 2 and Table 4.
In an embodiment, the nucleic acid is an isolated nucleic acid.
Another aspect is a vector comprising the nucleic acid herein disclosed. In an embodiment, the vector is an isolated vector.
The vector can be any vector suitable for producing an antibody and/or binding fragment thereof, or a coronavirus-binding multivalent binding molecule of this invention, including for example vectors described herein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses).
A further aspect is a recombinant cell producing a coronavirus-binding multivalent binding molecule of this invention or the vector herein disclosed.
The recombinant cell can be generated using any cell suitable for producing a polypeptide, for example suitable for producing an antibody and/or binding fragment thereof.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.
More particularly, bacterial host cells suitable for producing recombinant antibody producing cells include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genus Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art. Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the R-lactamase (penicillinase) and lactose promoter system, the trp promoter and the tac promoter. Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322, the pUC plasmids pUC18, pUC19, pUC118, pUC119, and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).
Suitable yeast and fungi host cells include, but are not limited to Saccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for the transformation of yeast and fungi are well known to those of ordinary skill in the art.
Suitable mammalian cells include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), NS-1 cells and any derivatives of these lines.
In an embodiment, the mammalian cells used to produce a recombinant antibody or a multivalent binding molecule of this invention are selected from CHO, HEK293 cells or FREESTYLE™ 293-F cells (Life technologies). FREESTYLE™ 293-F cell line is derived from the 293 cell line and can be used with the FREESTYLE™ MAX 293 Expression System, FREESTYLE™ 293 Expression System or other expression systems.
Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences.
In an embodiment, the vector is designed for production of light chain or heavy chain of the multivalent binding molecule.
Suitable insect cells include cells and cell lines from Bombyx or Spodotera species. Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series and the pVL series.
The recombinant expression vectors may also contain genes which encode a fusion moiety (i.e. a “fusion protein”) which provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including for example tags and labels described herein. Further, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
In an embodiment, expression of the multivalent binding molecules, or components of the multivalent binding molecules, is under the control of an inducible promoter. Examples of inducible non-fusion expression vectors include pTrc (28) and pET 11d.
The recombinant expression vectors may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein suchas—galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest. Other selectable markers include fluorescent proteins such as GFP which may be cotransduced with the nucleic acid of interest.
An embodiment of this invention is a pharmaceutical composition comprising a multivalent binding molecule or the nucleic acid molecules, expression cassettes and vectors encoding a multivalent binding molecule described herein and a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may further comprise an additional therapeutic agent, e.g., therapeutic small molecules, or a nucleic acid molecule, expression cassettes and vectors that encode the agent. The pharmaceutical composition may consist or consist essentially of a multivalent binding molecule of this invention, or the nucleic acid molecules expression cassettes and vectors encoding the multivalent binding molecule of this invention, and a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers, diluents and excipients, and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the multivalent binding molecule, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. Delivery systems for administering therapeutic agents are known in the art, see e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), Langer, 1990, Science 249:1527-1533; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574; Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides, poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of the multivalent binding molecule being administered.
The composition can be a lyophilized powder or aqueous or non-aqueous solution or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes. Other components that may be present in such compositions include water, surfactants (such as polysorbates, such as TWEEN™), alcohols, polyols, glycerin and vegetable oils, for example.
Suitable diluents for nucleic acids include but are not limited to water, saline solutions and ethanol. Suitable diluents for polypeptides, including antibodies or fragments thereof and/or cells include but are not limited to saline solutions, pH buffered solutions and glycerol solutions or other solutions suitable for freezing polypeptides and/or cells.
The composition can further comprise stabilizing agents, for example reducing agents, hydrophobic additives, and protease inhibitors which are added to physiological buffers.
Additional aspects of the invention are methods for using, producing and/or isolating the multivalent binding molecule described herein.
It has been reported the symptoms of coronavirus, e.g., COVID-19, infections include one or more of, e.g., fever, cough, shortness of breath, chills, repeated shaking with chills, muscle pain, headache, sore throat, loss of taste or smell, and can lead to physical complications, e.g., pneumonia and trouble breathing, organ failure in several organs, heart problems, severe lung condition, e.g., acute respiratory distress syndrome, blood clots, acute kidney injury and additional viral and bacterial infections. An embodiment of the invention relates to a method for treating, preventing, mitigating, or delaying the progression of a coronavirus, e.g., COVID-19, infection, or ameliorating a symptom of a coronavirus infection, comprising administering an effective amount of a multivalent binding molecule of this invention to a subject having or suspected of having a coronavirus infection. A method for treating, preventing, mitigating, or delaying the progression of a coronavirus infection includes reducing viral load in a subject in need thereof. The multivalent binding molecule of the present invention may be administered systemically or locally, e.g., by injection (e.g. subcutaneous, intravenous, intraperitoneal, intrathecal, intraocular, etc.), inhalation, implantation, topically, or orally. Depending on the route of administration, the multivalent binding molecule may be coated in a material to protect the molecules from the action of acids and other natural conditions which may inactivate them. The multivalent binding molecules described herein may be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition comprising the multivalent binding molecule can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. An extensive listing of excipients that can be used in such a composition, can be found in, for example, A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The multivalent binding molecules can also be administered in conjunction with other therapeutic regimens or agents for treatment of the infection and the associated symptoms and complications, e.g., convalescent serum therapy, small molecules, neutralizing monoclonal antibodies, recombinant antibodies, cytokines, or antivirals, e.g., LY-CoV555 (Eli Lilly), e.g., recombinant anti-CD47/PD-L1 bispecific antibody (IBI322)(Innovent Biologics, Inc.), protease inhibitors, e.g., nelfinavir mesylate or camostat mesylate, furin inhibitors, remdesivir, Actemra, Oluminant (baricitinib), merimepodib, dexamethaone (glucocorticoid), Aviptadil (RLF-100), hydroxycloropuine, Avigan (favipiravir, Avifavir), Bucillamine, niclosamide, aviptadil, PlKfyve kinase inhibitor, tyrosine kinase inhibitor efineptakin alfa, interferon beta, or SLV213 (Selva Therapeutics).
Effective dosages and schedules for administering the multivalent binding molecule of this invention and nucleic acids that encode them described herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of such multivalent binding molecule that must be administered will vary depending on, for example, the subject that will receive the multivalent binding molecule, the route of administration, the particular type of multivalent binding molecule used and other drugs being administered. Guidance in selecting appropriate doses for multivalent binding molecules of this invention is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith, Antibodies in Human Diagnosis and Therapy, Haber, eds., Raven Press, New York (1977) pp. 365-389. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the inflammation in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the multivalent binding molecules, nucleic acids and vectors is within the skill of the art.
An embodiment of the invention relates to a method for preventing or delaying the cleavage of a coronavirus S protein S2 subunit, or the fusion of the coronavirus, e.g., COVID-19, with a cell and internalization of the coronavirus by the cell, e.g., a cell having a receptor for the coronavirus, e.g. an ACE2 receptor, by contacting the cell with an effective amount of a multivalent binding molecule of this invention.
Yet another aspect relates to a method for screening or for diagnosing a subject having or suspected of having a coronavirus infection, the method comprising measuring the level of a coronavirus polypeptide or fragment thereof in a sample from a subject using a multivalent binding molecule or assay herein disclosed, wherein detecting a level of a coronavirus polypeptide or fragment thereof is indicative that the subject is infected with the coronavirus. The method may further comprise the step of comparing the level of a coronavirus polypeptide or fragment thereof in the sample bound by a multivalent binding molecule of this invention with a negative or positive control. An increased level of a coronavirus polypeptide or fragment thereof in the sample bound by the multivalent binding molecule as compared to a negative control, e.g., a value derived from a group of subjects without coronavirus infection, or convalescing from a coronavirus infection, is indicative that the subject is infected with coronavirus. A level of coronavirus similar or exceeding the level of coronavirus of the positive control, e.g., a value derived from a group of subjects known to have a coronavirus infection, is indicative that the subject is infected with coronavirus. The level of coronavirus polypeptide or fragment thereof bound by a multivalent binding molecule may be determined via an immunoassay comprising or using one or more multivalent binding molecule described herein. In an embodiment, the immunoassay is an enzyme linked immunosorbent assay (ELISA). In an embodiment the ELISA is a sandwich ELISA. The coronavirus polypeptide may be a coronavirus S protein or an S1 or S2 subunit or RBD of the S protein. If it is determined that the subject is infected with a coronavirus, therapeutic measures are administered to the subject.
An additional aspect of the invention is a method for prognosing coronavirus progression or lack thereof (e.g. recovery or worsening of disease). Accordingly an aspect is a method of prognosing a likelihood of recovery after coronavirus infection, the method comprising: a) measuring a level of a coronavirus polypeptide or fragment thereof in a sample from a subject by contacting the sample with the multivalent binding molecules of this invention; and b) comparing the level of a coronavirus polypeptide or fragment thereof bound by the multivalent binding molecules in the sample with a control, for example a control value derived from a group of subjects who recovered from or in whom the coronavirus disease symptoms regressed. A decreased level of coronavirus in the sample bound by the multivalent binding molecules compared to the control is indicative that the subject has an increased likelihood of recovery after coronavirus infection. An increased level of coronavirus in the sample bound by the multivalent binding molecules compared to the control is indicative that the subject has a reduced likelihood of recovery after coronavirus infection. The level of coronavirus may be measured by, e.g., measuring the amount of a coronavirus polypeptide or fragment thereof bound to the multivalent binding molecules of this invention by e.g., mass spectrometry, HPLC, immunoprecipitation or via an immunoassay, e.g. ELISA. The coronavirus polypeptide may be a COVID-19 S protein or an S1 or S2 subunit or RBD of the S protein. If it is determined that the subject has a decreased likelihood of recovery after coronavirus infection, therapeutic measures are administered to the subject.
A further embodiment of the invention is a method for prognosing the likelihood of progression of a coronavirus infection and associated adverse effects, the method comprising: a) measuring a level of a coronavirus polypeptide, or fragment thereof in a sample from a subject by detecting the level coronavirus polypeptide or fragment thereof with a multivalent binding molecule of this invention; and b) comparing the level of the coronavirus polypeptide or fragment thereof in the sample with a control, wherein an increased level of the coronavirus polypeptide or fragment thereof in the sample compared to the control is indicative of the level of coronavirus. In an embodiment where the control is a control value derived from a group of subjects who did not recover or progressed an increased level of the coronavirus polypeptide or fragment thereof is indicative that the subject has a decreased likelihood of recovering from a coronavirus infection. In an embodiment, the control is a control value derived from a group of subjects who recovered from coronavirus infection and an increased level of a coronavirus polypeptide or fragment thereof in the subject's sample bound by a multivalent binding molecule of this invention compared to the control is indicative that the subject has a decreased likelihood of recovery after coronavirus infection and/or an increased likelihood of coronavirus infection and associated adverse effects progressing. The level of coronavirus may be determined by immunoprecipitation of coronavirus or a coronavirus polypeptide or fragment thereof with the multivalent binding molecules of this invention, or via an immunoassay, e.g. ELISA, immunoblot, mass spectrometry, or HPLC, using the multivalent binding molecules of this invention as the capture or detection antibody. The coronavirus polypeptide may be a COVID-19 S protein or an S1 or S2 subunit or RBD of the S protein.
The sample may be taken from a subject after receiving a treatment for a coronavirus infection and compared to a pre-treatment sample. Alternatively, the patient can be monitored after a repeating interval to assess whether a treatment with the multivalent molecules of this invention or other intervention is necessary. In an embodiment, the test is repeated and plotted to assess the subject's progression.
In an embodiment, the sample used in the methods described herein is a tissue sample, e.g. lung tissue, or a biological fluid such as blood, or a fraction thereof such as plasma or serum, or a lung secretion or a pulmonary lavage, or a nasal secretion. In an embodiment the biological fluid is saliva, a lung secretion, or a nasal secretion and can be obtained as, e.g., oropharyngeal (throat) swab, nasopharyngeal swab, anterior nasal swab, and mid-turbinate nasal swab.
In another embodiment, the sample used in the methods described herein is selected from a fresh sample such as a fresh biological fluid sample or tissue sample (e.g. including not frozen), one-time frozen biological fluid sample or tissue sample (e.g. frozen a single time at the time of obtaining the sample)) or a repeat frozen sample (e.g. frozen and thawed and frozen biological fluid sample or repeat frozen tissue sample). In an embodiment, the sample comprises native or natively folded coronavirus polypeptide. In an embodiment, the sample is a fixed sample such as a mildly fixed sample wherein the fixation induces limited denaturation and/or unfolding of a coronavirus polypeptide.
Also an aspect of this invention is a method for making the multivalent binding molecules described herein. The amino acid sequences of coronaviruses, e.g., COVID-19, and its polypeptides, e.g. the S protein, are known, and methods to produce libraries of antibodies that bind to a selected protein or nucleic acid, or fragments thereof, are well known in the art (see e.g., U.S. publication no. 2015/0232554, inventors Gurney et al. and US publication no. 2016/0194394, inventors Sidhu et al. and US 20190040144, inventors Pan et al.; U.S. publication no. 2017/0166636, inventors Wu et al.; U.S. publication no. 2016/0208018, inventors Chen et al.; U.S. publication no. 2016/0053022, inventors Macheda et al.). And a variety of methods are known in the art for generating and screening phage display libraries for antibodies, and antibody fragments, scFv, Fab, VL, and VH possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004), all incorporated herein by reference. In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360, all incorporated herein by reference. The multivalent binding molecules/synthetic antibodies of this invention may be made recombinantly, e.g., by Gibson assembly (see Gibson et al. (2009) Nature Methods. 6 (5): 343-345 and Gibson DG. (2011) Methods in Enzymology. 498: 349-361), or the molecules may be made synthetically e.g., using a commercial synthetic apparatus, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. As such libraries of antibodies and antibody fragments and multivalent binding molecules that bind a selected coronavirus polypeptide can be readily generated using methods known in the art.
In another embodiment, a nucleic acid encoding a multivalent binding molecule described herein is expressed in a host cell to make the multivalent binding molecule. In an embodiment, the method comprises: a) expressing in a host cell a nucleic acid encoding a multivalent binding molecule thereof herein disclosed; b) culturing the host cell to produce the multivalent binding molecule; and c) isolating and/or purifying the multivalent binding molecule from the host cell.
In some embodiments, a nucleic acid encoding a heavy chain or light chain of the multivalent binding molecule is expressed. In other embodiments, multiple nucleic acids are expressed, for example a nucleic acid encoding a heavy chain and a nucleic acid encoding a light chain of the multivalent binding molecule.
Suitable host cells and vectors are described above. Vectors and nucleic acids encoding a multivalent binding molecule described herein may be introduced into cells, e.g., mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection and other liposome-based transfection agents, electroporation or microinjection.
Nucleic acid encoding a multivalent binding molecule described herein may be directly introduced into mammalian cells using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors.
In an embodiment of this invention, a multivalent binding molecule comprising an Fc domain and one coronavirus binding domain attached to the N-terminus of the Fc domain comprised of a diabody is generated by,
In an embodiment of this invention a multivalent binding molecule of this invention, comprising a first coronavirus binding domain comprised of a diabody and a second coronavirus binding domain comprised of two coronavirus-binding Fabs, is generated by,
In an embodiment of this invention the multivalent binding molecule having two coronavirus binding Fabs linked to one terminus of the Fc domain and two coronavirus-binding scFvs or a coronavirus-binding diabody linked to the other terminus of the Fc domain is generated by,
The coronavirus-binding source antibody in the methods described herein may be an antibody that binds an epitope of any coronavirus polypeptide, but preferably to an epitope of a coronavirus S protein, e.g., the RBD or N-terminal domain (NTD) of the S1 subunit or the fusion peptide (FP), heptapeptide repeat sequences HR1 and HR2, TM domain, and cytoplasm domain of the S2 domain.
The coronavirus-binding source antibody may be an antibody that is monospecific binding specifically to one coronavirus polypeptide or multispecific binding to more than one coronavirus polypeptides.
The source antibody may be a full-length antibody or an antibody fragment that binds a coronavirus polypeptide, e.g., a Fab, a VL or VH. The light chain and heavy chain CDRs, the VH and/or VL in one or both of the coronavirus binding domains of the multivalent binding molecule may be identical to the CDRs, the VH and/or VL of the coronavirus source antibody or may be at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDRs, VH or VL of the source antibody and still retain binding to the coronavirus polypeptide. The CDRs, the VH and/or VL in the coronavirus-binding domain of the multivalent binding molecule may be identical to the CDRs, the VH and/or VL of a COVID-19-binding antibody of Table 2 or Table 4, or may be at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the CDRs, VH or VL of a COVID-19-binding antibody of Table 2 or Table 4 and still retain binding to the COVID-19 polypeptide. In some embodiments, the amino acid sequence of the CDRs, the VH and/or VL in the coronavirus-binding domain of the multivalent binding molecule consist essentially of the amino acid sequence of a CDR, VH, and/or VL of Table 2 or Table 4.
In an embodiment of this invention, two heavy chain polypeptide monomers of the multivalent binding molecule dimerize via knob in hole configuration of their Fc sequences. The multivalent molecules of this invention may be generated by dimerizing two polypeptides in a “knob-in-hole” configuration. The knob-in-hole configuration increases the modularity of this invention by facilitating the association of peptides that comprise binding moieties that bind different epitopes on a coronavirus polypeptide. Methods for engineering Fc molecules via the knobs into holes design are well known in the art, see e.g., WO2018/026942, inventors Van Dyk et al., Carter P. (2001) J. Immunol. Methods 248, 7-15; Ridgway et al. (1996) Protein Eng. 9, 617-621; Merchant, et al. (1998) Nat. Biotechnol. 16, 677-681, and; Atwell et al., (1997) J. Mol. Biol. 270, 26-35.
The binding domains of the multivalent binding molecules may be linked to the Fc domain via a linker. In some embodiments, adjacent VH and VL domains may be attached to each other via a peptide linker. In some embodiments, adjacent constant domains and variable domains are attached via a linker. The linker may be, e.g. a polypeptide linker, or a non-peptidic linker. In some embodiments the constant domains and variable domains of the multivalent binding molecules are attached to the Fc domain via a peptide linker. Suitable linkers are well known in the art, e.g., an XTEN linker (see WO2013120683, inventors Schellenberger et al.) In some embodiments, the peptide linker comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or at least 100 amino acids. In some embodiments, the peptide linker is between 5 to 75, 5 to 50, 5 to 25, 5 to 20, 5 to 17, 5 to 16, 5 to 15, or 5 to 10, 1-10, 1-5, or 2-4 amino acids in length. Examples of peptide linkers are set forth in Table 4.
The modular aspects of this invention allow for combining antigen binding fragments derived from antibodies that bind to the same or different coronavirus epitopes or coronavirus polypeptides, to generate various binding domains and in some embodiments attaching different binding domains on the opposite termini of the Fe domain, to generate a multivalent binding molecule that binds selected coronavirus polypeptides. The modular aspects of this invention also allow for attaching a scFv by its VL or VH domain to the C-terminus or N-terminus of the Fc domain or the C-terminus or N-terminus another antibody fragment. As such the scFv orientation in the multivalent molecules from its amino to carboxy terminus may be either VL-linker-VH or VH-linker-VL.
The Fc domain of the multivalent binding molecules, with or without the linker, is greater than 100 amino acids, greater than 125 amino acids greater than 150 amino acids, greater than 175 amino acids or greater than 200 amino acids. The Fc domain may be about 100 amino acids to about 250 amino acids in length, about 125 amino acids to about 250 amino acids in length or about 150 amino acids to about 220 amino acids in length.
The multivalent binding molecules of this invention can be used in a variety of assays for binding, detecting and measuring coronavirus or a coronavirus polypeptide in a sample. For example, the multivalent binding molecules can be used in an ELISA, as well as immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA)(Muhammad S. Alam, November 2018) Current Protocols in Immunology Vol 123, Issue 1.), mass spectroscopy-based techniques, particle-based flow cytometric assay, fluorescence-activated cell sorting (FACS) and ELISA.
Immunodetection methods as described herein generally involve the detection or measuring complexes of multivalent binding molecules and a coronavirus polypeptide, e.g. an S1 of S2 subunit or RBD, using multivalent binding molecules disclosed herein. The detection of such complexes may be achieved through different methods, for example by using a detectable label or marker, such as a radioactive, fluorescent or enzymatic tag. Detection of these complexes may also involve the use of a ligand such as a secondary antibody and/or binding fragment thereof specific for a coronavirus polypeptide or for the multivalent binding molecules:coronavirus polypeptide complex.
They can also be used to make detection assays, for example a sandwich ELISA, as an antibody or the multivalent binding molecules can be used as a capture reagent to isolate a coronavirus or a coronavirus polypeptide while another antibody or multivalent binding molecule binding a distinct epitope can be used as a detection reagent.
Accordingly, another aspect is an immunoassay comprising one or more multivalent binding molecules described herein.
In an embodiment, the immunoassay is an enzyme linked immunosorbent assay (ELISA). The multivalent binding molecules may be used in the context of detection assays such as ELISAs, for example sandwich ELISAs. The multivalent binding molecules of this invention may be used as capture and detection antibodies for the detection of coronavirus or a coronavirus polypeptide in solution. For example, full length IgGs recognizing the coronavirus protein, e.g., the S1 subunit, are immobilized and incubated with coronavirus or a coronavirus polypeptide or subunit. The samples are then incubated with biotinylated IgGs recognizing epitopes on the virus, polypeptide, or subunit.
In an embodiment, the ELISA is a sandwich ELISA comprising a capture agent and a detection agent, wherein either the capture agent or the detection agent is a multivalent binding molecule that has CDRs identified herein and which specifically binds a COVID-19 polypeptide, for example which specifically binds S1 subunit, e.g., the RBD, wherein the capture agent and the detection agent bind different epitopes. In another embodiment, only one of the capture and detection antibodies is a multivalent binding molecule of this invention and the other capture or detection agent is an antibody and/or binding fragment thereof that binds a COVID-19 polypeptide, but is not a multivalent binding molecule of this invention.
In one embodiment, the CDRs of the capture agent and/or detection agent are the CDRs of an antibody in Table 2.
In one embodiment, the immunoassay is for the detection and/or measuring of a coronavirus or coronavirus polypeptide, e.g., the S1 or S2 subunit of the COVID-19 S polypeptide, in a sample, wherein the method of making the immunoassay comprises: a) coating a solid support with the capture agent; b) contacting the capture agent with the sample under conditions to form a capture agent:coronavirus complex and/or a capture agent:coronavirus polypeptide complex; c) removing unbound sample; d) contacting the complex with the detection agent; e) removing unbound detection agent; and f) detecting and/or measuring the amount of complex, wherein the amount of complex is indicative of the amount of coronavirus or coronavirus polypeptide in the sample.
In an embodiment, the ELISA is a competitive ELISA. In an embodiment, the ELISA is a direct ELISA. In an embodiment, the ELISA is an indirect ELISA.
As used herein, “solid supports” include any material to which coronavirus or a coronavirus polypeptide and multivalent binding molecules are capable of binding to. For example, the solid support can include plastic, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses and polyacrylamides. For example, the solid support is a microtiter plate, magnetic beads, latex beads or array surfaces.
In embodiments of the immunoassays described herein, the sample may be contacted with an multivalent binding molecules under appropriate conditions, for example, at a given temperature and for a sufficient period of time, to allow effective binding of a coronavirus polypeptide to the multivalent binding molecules, thus forming a multivalent binding molecules:coronavirus polypeptide complex, such as a capture multivalent binding molecules:coronavirus polypeptide complex. For example, the contacting step is carried out at room temperature for about 30 minutes, about 60 minutes, about 2 hours or about 4 hours. For example, the contacting step is carried out at about 4° C. overnight.
The multivalent binding molecules as described herein may be, e.g., complexed with a coronavirus polypeptide in a suitable buffer. For example, the buffer has a pH of about 5.0 to about 10.0. For example, the buffer has a pH of 4.5, 6.5 or 7.4. For example, the buffer is a HBS-EP buffer, a KRH buffer or Tris-buffered saline. For example, the buffer comprises BSA and/or TWEEN™ 20.
Any unbound material in a sample may be removed by washing so that only the formed multivalent binding molecules:coronavirus polypeptide complex remains on the solid support. For example, the unbound sample is washed with phosphate-buffered saline, optionally comprising bovine serum albumin (BSA).
In an embodiment, the detection agent is labelled and/or conjugated to a tag.
For example, the detection agent is directly labelled and/or conjugated. For example, the detection agent is indirectly labelled and/or conjugated. Indirect labels include for example fluorescent or chemiluminescent tags, metals, dyes or radionuclides attached to the antibody. Indirect labels include for example horseradish peroxidase, alkaline phosphatase (AP), beta-galactosidase and urease. For example, HRP can be used with a chromogenic substrate, for example tetramethybenzidine, which produces a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
An embodiment of the invention is an assay for detecting and/or measuring level of coronavirus or a coronavirus polypeptide in a sample, the assay comprising: a) contacting a sample with the multivalent binding molecules of this invention under conditions to form a multivalent binding molecule:coronavirus or a coronavirus polypeptide complex; and b) detecting and/or measuring the multivalent binding molecule:coronavirus or a coronavirus polypeptide complex. A further embodiment is an assay for detecting and/or measuring coronavirus or a coronavirus polypeptide the method comprising: a) contacting a sample, the sample being a biological fluid, with the multivalent binding molecules of this invention under conditions to form a multivalent binding molecule:coronavirus or a coronavirus polypeptide complex; and b) detecting and/or measuring the multivalent binding molecules:coronavirus or a coronavirus polypeptide complex.
In an embodiment, the assay is for detecting coronavirus or a coronavirus polypeptide and the assay is performed under non-denaturing or mildly denaturing conditions.
In an embodiment, the multivalent binding molecule:coronavirus or a coronavirus polypeptide complex is detected directly for example wherein the multivalent binding molecule is labeled with a detectable tag or fusion moiety. In an embodiment, the complex is detected indirectly using a secondary multivalent binding molecules or antibody specific for the multivalent binding molecule:coronavirus or a coronavirus polypeptide complex.
In an embodiment, the assay for detecting the multivalent binding molecule in complex with a coronavirus or coronavirus polypeptide is an immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA), mass spectroscopy-based techniques (CyTof) and fluorescence-activated cell sorting (FACS), and mass spectroscopy-based technique.
Detecting can be performed using methods that are qualitative or measured using quantitative methods, for example by comparing to a standard or standard curve.
In an embodiment, sample may be a biological sample from a subject, e.g., a human or other primate, or the sample may be from another source. The biological sample from a subject may be a tissue or a fluid, e.g., blood and its component parts, serum or plasma, saliva, a lung secretion or a lung lavage, nasal secretion, or urine etc. Other samples may be, e.g., wastewater or a body of water where wastewater drains, e.g., a river, canal, pond, bay etc.
A further aspect relates to a kit comprising i) a multivalent binding molecule of this invention, ii) a nucleic acid, iii) a composition or iv) a recombinant cell of this invention, comprised in a vial such as a sterile vial or other housing and optionally a reference agent and/or instructions for use thereof.
In an embodiment, the kit further comprises an additional binding agent, e.g., another multivalent binding molecule of this invention comprising the light chain CDRS having amino acid sequences of the hk CDR1, CDR2 and CDR3 of an antibody of Table 2 and heavy chain CDRs of the hIg1 CDR1, CDR2 and CDR3 of an antibody in Table 2, or of an antibody that binds a coronavirus polypeptide.
In an embodiment, the kit comprises components and/or is for use in performing an assay described herein.
For example, the kit is an ELISA kit and can comprise a capture agent that binds coronavirus, e.g., an antibody or multivalent antibody of this invention, for example attached to a solid support, and a second detection agent, e.g., an antibody or multivalent binding molecule of this invention that binds to (i) coronavirus or a coronavirus polypeptide, and/or (ii) a complex of the capture agent:coronavirus or a coronavirus polypeptide, and that is conjugated to a detectable label. Any combination of antibodies or antibody fragments or multivalent binding molecules described herein can be used with the provision that at least one of the capture or detection agent is a coronavirus binding multivalent binding molecule of this invention.
In an embodiment, the kit is a diagnostic kit and the instructions are directed to a method described herein.
General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., CSH Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998).
Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
Vero E6 (CRL-1586, American Type Culture (ATCC)), HEK293T (CRL-3216 ATCC) and HEK293T cells stably overexpressing ACE2 were maintained at 37° C. in 5% CO2 in DMEM containing 10% (vol/vol) FBS. Expi293F cells (Thermo Fisher Scientific, A1452) were maintained at 37° C. in 8% CO2 in Expi293F Expression Medium (Thermo Fisher Scientific, A1435102).
The previously reported piggyBac transposase-based expression plasmid PB-T-PAF (PMID: 23476064) containing a CMV promotor (PB-CMV) and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was used for large-scale transient expression. cDNA encoding the SARS-CoV-2 S protein ectodomain trimer (residues 1-Attorney Docket No. 17344P-000035-US-NP 1211), followed by a foldon trimerization motif (PMID: 9261070), a 6×His tag and an AviTag biotinylation motif (PMID: 25560075) was cloned in to the PB-CMV vector using standard molecular biology techniques and residues 682-685 (RRAR) and 986-987 (KV) were mutated to SSAS and two proline residues to remove the furin cleavage site on the SARS-CoV-2 S protein and stabilize the pre-fusion form (PMID: 28807998), respectively. The SARS-CoV-2 receptor binding domain (RBD, residues 328-528), the soluble human ACE2 construct (residues 19-615), and the SARS-CoV RBD (residues 315-514), each followed by a 6×His tag and an AviTag, were similarly cloned in to the same vector. For expression, PB-CMV expression constructs were mixed with Opti-MEM medium containing 293fectin reagent (Thermo Fisher) and the mixture incubated for 5 minutes before addition to the shaker flask containing 106 Freestyle 293-F cells/mL grown in suspension in Freestyle 293 expression medium (Thermo Fisher). Expression was allowed to continue for 6 days before purification.
Protein purification and in vitro biotinylation
Expressed proteins were harvested from expression medium by binding to Ni-NTA affinity resin, eluted with IX PBS containing 300 mM imidazole and 0.1% (v/v) protease inhibitor cocktail (Sigma, P-8849), then further purified by size-exclusion chromatography. For the RBDs and ACE2, a Superdex 200 Increase (GE healthcare) column was used. For the S protein ectodomain, a Superose 6 Increase (GE healthcare) column was used. Purified proteins were site-specifically biotinylated in a reaction with 200 μM biotin, 500 μM ATP, 500 μM MgCl2, 30 μg/mL BirA, 0.1% (v/v) protease inhibitor cocktail and not more than 100 μM of the protein-AviTag substrate. The reactions were incubated at 30° C. for 2 hours and biotinylated proteins then purified by size-exclusion chromatography.
A highly validated, synthetic, phage-displayed antibody library (Persson et al. CDR-H3 diversity is not required for antigen recognition by synthetic antibodies. J Mol Biol 2013; 425:803-11) was panned on SARS-CoV-2 RBD in solution. In each round, phage library was first depleted on neutravidin immobilized in wells of a 96 Maxisorp plate from a 2 μg/mL solution incubated with shaking overnight at 4° C., then incubated with 50 nM biotinylated RBD in solution for two hours at room temperature. Protein-phage complexes were captured in wells coated with neutravidin as above for 15 min at RT, then washed with IX PBS pH7.4 containing 0.05% TWEEN™ and phage eluted for 5 min with 0.1 M HCl before neutralizing with IM Tris pH 8.0. Eluted phage were amplified, purified, then panned against target for a total of 5 rounds, before isolation, amplification and sequencing of individual phage clones as previously described (Persson).
For ELISAs, plates were coated with neutravidin, as above, then blocked with 0.2% BSA in PBS for 1 hr. Biotinylated target protein was captured from solution by incubation in neutravidin-coated and BSA-blocked wells for 15 min with shaking at RT before addition and 30 min incubation of binding phage or antibody. Plates were washed then incubated with an appropriate secondary antibody before development with TB substrate as described (Sidhu et al., Journal of Molecular Biology Vol. 338(2): 299-310 (2004)).
DNA fragments encoding heavy chain FAb regions (VH-CH1; terminating at threonine 238, Kabat numbering) were amplified by PCR from the IgG expression constructs. Tetravalent antibody constructs were generated by fusing these fragments with their respective IgG heavy chain in the pSCSTa mammalian expression vector using Gibson assembly (NEBuilder HiFi DNA Assembly Cloning Kit; NEB. Fab-IgG constructs were arranged by fusing a heavy chain FAb domain to the N-terminus of the IgG via a S(G4S)3 linker. IgG-Fab constructs were arranged by fusing a heavy chain FAb domain to the C-terminus of using a G(G4S)2GGGTG linker. For both formats, the Fc region terminated at glycine 447 (Kabat numbering).
IgG and tetravalent antibodies were produced in Expi293F (ThermoFisher) by transient transfection, as described (Miersch, BioRxiv, 2020 https://doi.org/10.1101/2020.06.05.137349). For IgG expression, equivalent amounts of plasmids encoding heavy chain and light chains were transfected, whereas for tetravalent modalities, a ratio of 2:1 light chain to heavy chain plasmids was used. After a 5-day expression period, antibodies were purified using rProtein A Sepharose (GE Healthcare), then buffer exchanged and concentrated using Amicon Ultra-15 Centrifugal Filter devices (Millipore). IgGs were stored in PBS (Gibco), while tetravalent antibodies were stored in a buffer consisting of 10 mM L-Histidine, 0.9% sucrose, 140 mM NaCl, pH 6.0.
Fifty micrograms of protein were injected onto a TSKgel BioAssist G3SWxl (Tosoh) fitted with a guard column using an NGC chromatography system and a C96 autosampler (Biorad). The column was preequilibrated in a PBS mobile phase and protein retention was monitored by absorbance at 215 nm during a 1.5 CV isocratic elution in PBS.
The binding kinetics and estimation of apparent affinity (KD) of antibodies against SARS-CoV-2 RBD we determined by biolayer interferometry experiments performed on an Octet HTX instrument (ForteBio) at 1000 rpm and 25° C. Biotinylated CoV2 S protein was first captured on streptavidin biosensors from a 2 μg/mL solution to achieve a binding response of 0.4-0.6 nm and unoccupied sites quenched with 100 μg/mL biotin. Antibodies were diluted with assay buffer (PBS, 1% BSA, 0.05% TWEEN™ 20) and 67 nM of an unrelated biotinylated protein of similar size was used as negative control. Following equilibration with assay buffer, loaded biosensors were dipped for 600 s into wells containing 3-fold serial dilutions of each antibody from 67 nM and subsequently were transferred for 600 s back into assay buffer. Binding response data were reference subtracted and were fitted with 1:1 binding model using ForteBio's Data Analysis software 9.0.
Thermostability of expressed antibodies were determined by differential scanning fluorimetry (DSF) using Sypro Orange as described (Majka, J Biomol Screen, 2015) instead using a 1 μM solution of antibody and temperature range from 25-100° C. in 0.5° C. increments.
To generate SARS-Cov-2 pseudovirus, human embryonic kidney 293 (HEK 293, ATCC® CRL-1573™) cells were seeded at 0.3×106 cells/well in DMEM (ThermoFisher Scientific, Cat. No. 11995073) supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco, Cat. No. 15070063) and grown overnight at 37° C., 5% CO2. HEK 293 cells were then co-transfected with 1 μg of pNL4-3.luc.R-E-plasmid (luciferase expressing HIV-1 with defective envelop protein) (NIH AIDS Reagent Program, Cat. No. 3418) and 0.06 μg of CMV-promoter driven plasmid encoding SARS-CoV-2 wild type or mutant S protein variants using Lipofectamine™ 2000 transfection reagent (ThermoFisher Scientific, Cat. No. 11668027). Pseudovirus was harvested by collecting supernatant 48 h after transfection and filter sterilized (0.44 μm, Millipore Sigma, Cat. No. SLHA033SS).
HEK 293 cells (ATCC® CRL-1573) stably over-expressing full-length human ACE2 protein were seeded in 96 well white polystyrene microplates (Corning, Cat. No. CLS3610) at 0.03×106 cells/well in DMEM (10% FBS and 1% Pen-Strep), and grown overnight at 37° C., 5% CO2. To test the inhibition of pseudovirus entry of antibodies, the desired concentrations of antibodies were mixed with pseudoviruses, incubated at room temperature for 10 m, and were used to infect cells. The cells were incubated at 37° C., 5% CO2 for 6 hours, then the medium was replaced with fresh DMEM (10% FBS and 1% Pen-Strep), and again every 24 hours up to 72 hours. To measure the luciferase signal (virus uptake), DMEM was removed and cells were replaced in DPBS (ThermoFisher, Cat. No. 14190250) and mixed with an equal volume of ONE-Glo™ EX Luciferase Assay System (Promega, Cat. No. 8130). Relative luciferase units were measured using a BioTek Synergy Neo plate reader (BioTek Instruments Inc.). The data was then analyzed by GraphPad Prism Version 8.4.3 (GraphPad Software, LLC.).
SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was obtained from the Centers for Disease Control and Prevention (gift of Natalie Thornburg). Virus stocks were produced in Vero CCL81 cells (ATCC) and titrated by focus-forming assay on Vero E6 cells. Serial dilutions of mAbs were incubated with 102 focus-forming units (FFU) of SARS-CoV-2 for 1 h at 37° C. MAb-virus complexes were added to Vero E6 cell monolayers in 96-well plates and incubated at 37° C. for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 30 h later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with 1 μg/mL of CR3022 (Yuan et al, Science, 2020,) anti-S antibody and HRP-conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% BSA. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies). Data were processed using Prism software (GraphPad Prism 8.0).
Using a phage-displayed human antigen-binding fragment (Fab) library similar to the highly validated library F (Persson et al., J Mol Biol 2013; 425:803-11), we performed four rounds of selection for binding to the biotinylated RBD of SARS-CoV-2 immobilized on streptavidin-coated plates. Screening of 384 clones for binding to CoV-2 RBD, revealed 358 clones that bound to the RBD but not to streptavidin, and for 66 of these clones, binding to the RBD was blocked by ACE2 (
To estimate affinities, ELISAs were performed with serial dilutions of IgG protein binding to biotinylated S protein trimer captured with immobilized streptavidin, and these assays showed that three synthetic IgGs 15031, 15032, and 15033 bound with EC50 values in the sub-nanomolar range (
To understand the molecular basis for high affinity binding and antagonism of ACE2 binding, we solved the crystal structure of an affinity matured variant of Fab 15033 in complex with the SARS-CoV2 RBD at 3.0 Å (
Based on our structural analysis and the rapid combinatorial protein engineering and our evaluation of binding kinetics described herein we improved the affinity of IgG 15033 and contemplate that the multivalent binding molecules described herein based on this IgG will likewise show improved affinity. The synthetic library was designed with tailored diversification of key positions in all three heavy chain complementarity-determining regions (CDRs) and the third CDR of the light chain (CDR-L3). Notably, CDR-L3 is substantially buried at the interface with the RBD (
We further enhanced the binding and neutralization of our best Abs by generating tetravalent antibody formats that do not exist in nature. Each SARS-CoV2 particle displays multiple S protein trimers, suggesting that multivalent Fab binding could significantly enhance apparent affinity, especially since at least two Fab 15033 molecules could potentially bind to even a single S protein trimer (
Our ultimate aim was to produce therapeutic antibodies that could be used to treat COVID-19 in patients, and aside from high affinity and specificity, effective antibody drugs must also possess favorable biophysical properties including high yields from recombinant expression in mammalian cells, high thermodynamic stability, and lack of aggregation and excessive hydrophobic surface area. All synthetic IgGs and Fab-IgG and IgG-Fabs comprising the CDRs of 15031, 15032, 15033 and 15033-7 were produced in very high yields by transient expression in 293T cells (160-200 mg/L, Table 5). All proteins were also highly thermostable with melting temperatures (Tm2) of the CH3/Fab domain (Ionescu et al. Contribution of Variable Domains to the Stability of Humanized IgG1 Monoclonal Antibodies. J. Pharm. Sci. 2008, 97, 1414-1426. 10. Garber, E.; Demarest, S. J. A Broad Range of Fab Stabilities within a Host of Therapeutic IgGs. Biochem. Biophys. Res. Commun. 2007, 355, 751-757.) ranging from 81-87° C., which was comparable to the melting temperature of the trastuzumab Fab (79.5° C., Table 5,
We assessed the effects of the synthetic IgGs on virus infection in a focus reduction neutralization test that measured the infection of ACE2-expressing VeroE6 cells with the clinically isolated SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020. All three synthetic IgGs from the naïve library exhibited dose-dependent neutralization of viral infection, confirming their neutralization capacity and the absence of cytopathic effects (
To explore antibody-mediated neutralization of potential escape mutants, we generated an HIV-gag-based virus-like particle pseudo-typed with SARS-CoV-2 S protein and assessed uptake in HEK-293 cells stably over-expressing ACE2 cells. After confirming ACE2-dependent uptake, RBD-mediated inhibition (
Most natural neutralizing antibodies (nAbs) target the S1 subunit, and bind to the RBD and compete with ACE2. A distinct subset of nAbs, however, have been shown to target a neutralizing epitope on the NTD (Liu, L., et al., (2020) Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 S protein. Nature 584, 450-456.). Several natural nAbs have been produced recombinantly and engineered further to develop drugs that have been successful for inhibiting SARS-CoV-2 infection in patients (Taylor, P. C., et al., (2021) Neutralizing monoclonal antibodies for treatment of COVID-19. Nat Rev Immunol 21, 382-393.). However, current approved antibody drugs must be administered at very high doses and have proven to be ineffective against many variants of concern (VOCs) that have arisen since the original COVID-19 outbreak. Indeed, most VOCs that resist current therapeutic nAbs contain mutations within the RBD that disrupt binding to the nAbs (Wang, P., et al., (2021) Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135.) but not to ACE2 (Zhu, X., et al., (2021) Cryo-electron microscopy structures of the N501Y SARS-CoV-2 S protein in complex with ACE2 and 2 potent neutralizing antibodies. PLoS Biol 19, e3001237.).
To address these limitations on the potency and breadth of coverage of current bivalent IgG therapies, several groups have developed higher valence protein-based inhibitors (De Gasparo, R., et al., (2021) Cryo-electron microscopy structures of the N501Y SARS-CoV-2 S protein in complex with ACE2 and 2 potent neutralizing antibodies. PLoS Biol 19, e3001237.). These include small modular Ab domains or non-antibody scaffolds that can be assembled as multimers with enhanced potency due to simultaneous engagement with all three RBDs on an S protein trimer (Rujas, E., et al., (2021) Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers. Nat Commun 12, 3661-12.) (Cao, L., et al., (2020) De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science 370, 426-431.). Alternatively, we have shown that the fusion of additional Fab arms to either the N- or C-terminus of an IgG heavy chain results in tetravalent IgG-like molecules with enhanced potency and effectiveness against VOCs that resist bivalent IgGs (Miersch, et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.).
Described herein are synthetic peptides that bind to the S protein to augment neutralization potency of IgGs in the form of tetravalent peptide-IgG fusions comprising binding sites of potent nAbs combined with the small, modular binding sites of peptides using naïve phage-displayed peptide libraries to derive synthetic peptides that bind to neutralizing epitopes on the RBD or the NTD. We showed that these small peptides could be fused to a moderate affinity nAb to develop peptide-IgG fusions with affinities enhanced by over two orders of magnitude. Most importantly, one such peptide fusion was able to greatly enhance the neutralization potency against SARS-CoV-2 and VOCs for a high affinity nAb we had engineered earlier (Miersch, et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.), and also, for a clinically approved therapeutic nAb developed by others (Hansen, J., et al. (2020) Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail. Science 369, 1010-1014.).
The trimeric structure of the SARS-CoV-2 S protein can be exploited to engineer Ab-based inhibitors with enhanced neutralization, by engagement of three neutralizing epitopes on a single trimer. Structural studies from us and others have shown that potent neutralizing IgGs engage two RBDs on an S protein trimer (Yan, R., et al., (2021) Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies. Cell Res. 31, 517-525.), and we have shown that the addition of Fab arms to either end of an IgG can further enhance neutralization by enabling engagement to the third RBD (Miersch, S., et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.).
Described herein are small peptides that greatly enhance the affinities and potencies of tetravalent peptide-IgG fusions compared with our bivalent IgG 15033-7. Most importantly, peptide-IgG 33-7Hp102 acted as a potent inhibitor of virus variants that resisted IgG 15033-7. Indeed, we showed further that a peptide-IgG version of the approved drug RGN10933 was able to potently neutralize a virus variant against which REGN10933 was completely ineffective. To gain insight into the structural basis for how a peptide within a peptide-IgG fusion could enhance affinity, we examined our published model of two 15033-7 Fabs bound to the S protein trimer, reasoning that this likely provides an accurate view of how the two Fab arms of a bivalent IgG would bind (
Next, an analogous structural model of two RGN10933 Fabs bound to an S protein trimer (
We also developed peptides that bound to the NTD, likely at a site that overlaps with epitopes for other neutralizing nAbs (
The ultimate value of peptide-IgG fusions targeting SARS-CoV-2 and its variants resides in the potential for the development of superior therapeutics for the treatment of COVID-19 and other viral diseases. Currently the biophysical properties of the peptide-IgG fusions are being assessed and optimized with the aim of developing drug-grade biologics. However, there is already strong precedent for peptide-IgG fusions as clinical drugs in the form of biologics that have reached phase 2 clinical trials as cancer therapeutics (Autio, K. A., et al., (2020) Probody Therapeutics: An Emerging Class of Therapies Designed to Enhance On-Target Effects with Reduced Off-Tumor Toxicity for Use in Immuno-Oncology. Clin Cancer Res 26, 984-989.). These anti-cancer biologics are very similar in format to the anti-viral peptide-IgG fusions we report here (Polu, K. R., and Lowman, H. B. (2014) Probody therapeutics for targeting antibodies to diseased tissue. Expert Opin Biol Ther 14, 1049-1053.). Additionally, anti-cancer peptide-IgG fusions have exhibited good pharmacokinetic profiles (Stroh, M., et al., (2019) Quantitative Systems Pharmacology Model of a Masked, Tumor-Activated Antibody. CPT Pharmacometrics Syst Pharmacol 8, 676-684.) and tissue penetration (Desnoyers, L. R., et al., (2013) Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index. Sci Transl Med 5, 207ra144-207ra144.), and they have been well tolerated in the clinic (Sanbom, R. E., et al., (2021) CX-072 (pacmilimab), a Probody PD-L1 inhibitor, in combination with ipilimumab in patients with advanced solid tumors (PROCLAIM-CX-072): a first-in-human, dose-finding study. J Immunother Cancer 9, e002446.). Taken together, our results establish peptide-IgG fusions as a powerful means for greatly enhancing the potency and coverage of next-generation biologics for the treatment of disease caused by SARS-CoV-2 and its variants.
In a first embodiment of the invention, at least one peptide that binds the SARS-CoV-2-S protein is fused to an anti-SARS-CoV-2 antibody. In another embodiment, the fusion includes at least one peptide fused to the N-terminus of at least one of the light chains of the anti-SARS-CoV-2 antibody. In another embodiment, the fusion includes at least one peptide fused to the N-terminus of at least one of the heavy chains of the anti-SARS-CoV-2 antibody. In another embodiment, the fusion molecule comprises at least one peptide fused to the N-terminus of anti-SARS-CoV-2 antibody IgG 33-7. In another embodiment, the fusion molecule comprises at least one peptide fused to the N-terminus of anti-SARS-CoV-2 antibody that engages at least one neutralizing epitopes on a single S protein trimer. In another embodiment, the fusion molecule comprises at least one peptide fused to the N-terminus of anti-SARS-CoV-2 antibody that engages three neutralizing epitopes on a single S protein trimer.
In another embodiment, the fusion molecule comprises peptide p102 and/or p16 fused to the N-terminus of anti-SARS-CoV-2 antibody IgG 33-7. In another embodiment, the fusion molecule comprises peptide p102 and/or p16 fused to the N-terminus of anti-SARS-CoV-2 antibody IgG 33-7.
In another embodiment, the fusion includes at least one peptide fused to the N-terminus of each of the heavy chains of the anti-SARS-CoV-2 antibody. In another embodiment, the fusion includes at least one peptide fused to the N-terminus of each of the light chains of the anti-SARS-CoV-2 antibody. In another embodiment, the fusion molecule comprises an anti-SARS-CoV-2 antibody and peptides p102 and/or p16. In another embodiment, the fusion molecule comprises an anti-SARS-CoV-2 antibody and at least one peptide fused on each of the heavy chain and the light chains, where the peptide fused to the heavy chain can be the same or different than the peptide fused to the light chain. In another embodiment, the fusion molecule comprises an anti-SARS-CoV-2 antibody IgG 33-7 and at least one peptide fused on each of the heavy chain and the light chains, where the peptide fused to the heavy chain can be the same or different than the peptide fused to the light chain. In another embodiment, the fusion molecule comprises an anti-SARS-CoV-2 antibody IgG 33-7 and peptide p102 and/or p16 fused on each of the heavy chain and the light chains, where the peptide fused to the heavy chain can be the same or different than the peptide fused to the light chain.
The SARS-CoV-2 S protein ectodomain and RBD were produced and purified as described (Miersch, S., et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.) Purified proteins were site-specifically biotinylated in a reaction with 200 μM biotin, 500 μM ATP, 500 μM MgCl2, 30 μg/mL BirA, 0.1% (v/v) protease inhibitor cocktail and not more than 100 μM of the protein-AviTag substrate. The reactions were incubated at 30° C. for 2 hours and biotinylated proteins were purified by size-exclusion chromatography.
IgG and peptide-IgG fusion proteins were produced as described (Miersch, S., et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.). S protein-binding peptides were fused to the N-terminus of the heavy or light chain through a 20-residue linker (sequence: GGGGSGGGGSGGGGSGGGGS) using standard molecular biology techniques.
Naïve libraries were constructed as described (Arita, Y., et al., (2016) Rapid isolation of peptidic inhibitors of the solute carrier family transporters OATP1B1 and OATP1B3 by cell-based phage display selections. Biochem. Biophys. Res. Commun. 473, 370-376.). For libraries for peptide optimization, oligonucleotides were synthesized using degenerate codons encoding for the amino acids at each position indicated in
Phage ELISAs were performed, as described (Miersch, S., et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.), but with the following modifications; 384-well maxisorp plates (Sigma) were coated with NAV, or left uncoated as negative control, and blocked with PBS, 0.5% Bovine serum albumin (BSA). Biotinylated target protein was captured by incubation in NAV-coated and BSA-blocked wells, or with buffer solution alone as negative control, at room temperature. For competition ELISAs, blocking IgG was incubated with coated and blocked wells for 1 hour at room temperature. Wells were incubated with peptide-phage in PBS, 0.5% BSA for 1 hour. Plates were washed, incubated with anti-M13-HRP antibody (Sino Biological, catalog number 11973-MM05T-H) and developed with TMB substrate (Mandel, catalog number KP-50-76-03).
For IgGs and peptide-IgG fusions, binding kinetics for the S protein were determined by BLI with an Octet HTX instrument (ForteBio), as described (Miersch, S., et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.). For peptides, biotinylated peptides (Table 6) were immobilized on streptavidin-coated sensors that were subsequently blocked with excess biotin. Following equilibration with assay buffer, loaded biosensors were dipped for 600 seconds into wells containing 3-fold serial dilutions of S protein ECD, and subsequently, were transferred back into assay buffer for 600 seconds. Binding response data were corrected by subtraction of response from a reference and were fitted with a 1:1 binding model using the ForteBio Octet Systems software 9.0. We determined KD values at various peptide loading densities to ensure that high densities were not impacting kinetic measurements at the sensor.
Sequences of binding peptides were imported into Geneious R9 software (Biomatters Ltd.). Peptides were sorted based on the library from which they originated and aligned separately using the MUSCLE algorithm. In the case of peptides lacking cysteines, a strict penalty was imposed on the formation of gaps during alignment. Sequence logos were created using the weblogo server (https://weblogo.berkeley.edu/logo.cgi).
Serial dilutions of IgGs were incubated with the virus at 37° C. for 1 hour. The IgG-virus mixture was transferred to 96-well tissue culture plates containing sub-confluent monolayers of Vero E6 cells in duplicate and incubated at 37° C. and 5% CO2. Infection was monitored in one of two ways. The first used a crystal violet-based method as follows. Supernatants were carefully discarded and 100 μl Crystal Violet solution containing 4% formaldehyde was added to each well. Cells were washed and 100 μl containing 50 parts of absolute ethanol (Sigma, St. Louis, MO), 49 parts of MilliQ water and 1 part of glacial acetic acid (Sigma catalog number 64-19-7) were added to each well. Plates were incubated for 15 minutes at room temperature and read by a spectrophotometer at 590 nm. Alternatively, infection was monitored using an ELISA-based method with the anti-S protein antibody CR3022, followed by incubation with HRP-conjugated goat anti-Fc antibody (Invitrogen, catalog number A18817). Stained cells were visualized using KPL TrueBlue peroxidase (SeraCare, catalog number 5510) and quantified on an ImmunoSpot microanalyzer (Cellular Technologies). All data were analyzed using Prism software (GraphPad Prism 8.0).
The model of two 15033-7 Fabs bound to the SARS-CoV-2 S protein (PDB entry 7KMK) was imported into PyMol (DeLano Scientific, LLC). Distances between the HC N-termini and the 15033-7 epitope on the unbound RBD were measured as the distance between the Ca of the first residue of the HC and the CP of Tyr489 of the S protein. To build the model of two RGN10933 Fabs bound to the S protein, the model of RGN10933 and RGN10987 bound to the RBD (PDB entry 6XDG) was imported into PyMol along with the data from PDB entry 7KMK. The data from PDB entry 6XDG were duplicated and the RBDs of the model were superposed with the two RBDs in the “up” position in the model from PDB entry 7KMK. The RBDs in the model from PDB entry 6XDG, RGN10987 Fabs, and 15033-7 Fabs were then eliminated from the model, leaving only the two RNG10933 from PDB entry 6XDG bound to the S protein from PDB entry 7KMK. Distances were measured the same way as for 15033-7.
In order to isolate novel peptides that bound to the S protein of SARS-CoV-2, we used phage-displayed libraries of 16-residue peptides. Next, phage representing a panel of 10 peptide libraries in which diversified positions were encoded by an equimolar mixture of 19 codons representing all genetically-encoded amino acids except cysteine, and an “unconstrained” library (X16) containing 16 diversified positions with no fixed positions (Arita, Y., et al., (2016) Rapid isolation of peptidic inhibitors of the solute carrier family transporters OATP1B1 and OATP1B3 by cell-based phage display selections. Biochem. Biophys. Res. Commun. 473, 370-376.) were pooled together. Other libraries contained 14 diversified positions and two fixed cysteine residues separated by 4-12 diversified positions. These “constrained” libraries were designed to display peptides containing disulfide-bonded loops, which have been found to promote tertiary structures that can enhance binding to proteins (Bozovicar, K., and Bratkovie, T. (2021) Small and Simple, yet Sturdy: Conformationally Constrained Peptides with Remarkable Properties. Int J Mol Sci 22, 1611.).
Phage representing the library pool were cycled through five rounds of binding selections with immobilized S protein ECD or RBD, and several hundred clones were analyzed for binding to the ECD. Clones that exhibited strong binding signals in phage ELISAs with the ECD and negligible signals with bovine serum albumin (BSA) and neutravidin (NAV) were subjected to DNA sequence analysis. This process yielded 160 and 128 unique peptide sequences from the ECD and RBD selections, respectively. Alignment of the sequences revealed that most (85%) of the ECD-selected peptides were derived from two libraries: 63% and 22% were from the X16 or C-X4-C library, respectively (
For more detailed characterization, we chose four peptides that closely matched the consensus motifs for their respective families. These included a peptide from the X16 library (p16) and a peptide from the C-X4-C library (p62), which were selected for binding to the S protein ectodomain, and two peptides (p102 and p103) from the C-X12-C library, which were selected for binding to the RBD (
Biased peptide-phage libraries were designed to further optimize peptides p16 and p102 (
Based on this process, we identified four variants of peptide p16 (p16a, p16b, p16c, p16d) and two variants of peptide p102 (p102a, p102b) for chemical synthesis (
We next explored whether peptide fusions could enhance the affinity of a neutralizing IgG targeting the ACE2-binding site of the RBD. For this purpose, we used the moderate affinity IgG 15033 that we had selected from a naïve phage-displayed synthetic antibody library (Miersch, S., et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.), in order to accurately discern affinity differences. Peptide p16 or p102 was fused to the N-terminus of either the light chain (LC) or heavy chain (HC) of IgG 15033 with an intervening 20-residue Gly/Ser linker. The resulting peptide-IgG fusion proteins and IgG 15033 were purified by transient transfection in mammalian Expi293F cells (Miersch, S., et al., (2021) Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Journal of Molecular Biology 433, 167177.). Peptide-IgG fusions can be purified to homogeneity with a single Protein A affinity chromatography step as evidenced by Coomassie-stained SDS-PAGE gels, that resolve differences in molecular weight between peptide-fused heavy or light chain under reducing conditions (
We explored the critical question of whether peptide fusions could enhance the potency of nAbs in cell-based assays of SARS-CoV-2 infection. For this purpose, we fused peptide p102 to the N-terminus of the HC of IgG 15033-7, a more potent variant of IgG 15033 with an optimized LC20. The resulting peptide-IgG fusion protein (33-7Hp102) was compared to IgG 15033-7 in assays that measured the infection of ACE2-expressing Vero E6 cells with a panel of six authentic SARS-CoV-2 variants including the original Wuhan virus (B.1) and later emerging VoC, including variants isolated in Italy (B.1.1), the United Kingdom (B.1.1.7), South Africa (B.1.351 and B.1.529), Nigeria (B.1.525), and Brazil (P.1). In every case, the potency of the peptide-IgG fusion greatly exceeded that of the IgG (
We also explored whether the modularity of the peptides could be exploited to enhance the potency of a clinically approved therapeutic nAb (REGN10933,
In a pseudovirus infection assay, IgG 15033-7 was evaluated in parallel against both the B.1 and B.1.1.529 pseudovariants and while it potently neutralized the B.1 variant (IC50<100 ng/mL), it was inactive against B.1.1.529 up to the maximum antibody concentration tested (250 nM) (
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the inventions. Various substitutions, alterations and modifications may be made to the invention without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are within the scope of the invention.
The contents of all references, issued patents, and published patent applications cited through this application are hereby incorporated by reference. The appropriate component, process and methods of those patents, applications and other documents may be selected for the invention and embodiments thereof.
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
ACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGGAGGCGCTAC
TGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGA
TTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GTGGAGGTGGCAGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCAT
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GTGGAGGTGGCTCCGGAGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGG
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
GGSGGGGS
EVQLVESGGGLVQPGRSLRLSCAASGFDLTGSYMHWVRRAPGKGEELVAGISASGGATAYADSVKGRATIS
GCGACCTGTACAGCGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTC
TACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGGAGGCGCTA
CTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATG
GTTACTACTCTGGTGCTTTTGATTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGCGGCGGAGGCTCCGGC
GGCGGAGGATCC
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCAC
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
GGTGGCAGTAGCAGTGGTGCC
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTC
SVSSAVAWYQQKPGKAPKLLYSASDLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATVYCQQGSYLFTFGQGTKVEIKGTTA
ASGSSGGSSSGA
EVQLVESGGGLVQPGGSLRISCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKG
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
CCGACCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
SGWRYYSGAFDYWGQGTLVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSA
SDLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGSYLFTFGQGTKVEIK
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
SGG
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKGRFTISADTS
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
GGTGGCAGTAGCAGTGGTGCC
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTC
SVSSAVAWYQQKPGKAPKLLIYSASDLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGSYLFTFGQGTKVEIKGTTA
ASGSSGGSSSGA
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKG
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCT
ACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTCTCGTTCTTCTTCTTACTCTTCTTCT
GGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
GGGSGGGGSGGGTG
EVQLVESGGGLVQPGRSLRLSCAASGFDLTGSYMHWVRRAPGKGEELVAGISASGGATAYADS
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
GGTGGCAGTAGCAGTGGTGCC
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTC
SVSSAVAWYQQKPGKAPKLLIYSASDLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQGSYLETFGQGTKVEIKGTTA
ASGSSGGSSSGA
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKG
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GTGGAGGTGGCTCCGGAGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGG
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GTGGAGGTGGCTCCGGAGGAGGTGGCAGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGG
GTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT
TGTCCAGCGCCGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACAGCGCCAGCGACCTG
CTGGTGCTTTTGATTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACAC
TCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTT
GACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGT
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACAC
TCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGT
GGAGGTGGCTCCGGAGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGG
SSYSSSGWRYYSGAFDYWGQGTLVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKOTLMISRTPEVTCV
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACAC
TCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGT
GGAGGTGGCTCCGGAGGAGGTGGCAGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
CTCACTGGTTCTTACATGTGGGTGCGTCGTGCCCCGGGTAAGGGCGAGGAACTGGTTGCAGGTATTTCCGCTTCTGG
AGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACC
GGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACAC
TCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGT
GGAGGTGGCTCCGGAGGAGGTGGCAGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
TGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCCAGCCTC
CTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
GGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSRSSSYSSSGWRYYSGAFDYWGQGTLVTVSS
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACAC
TCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTT
GACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGT
CTCACTGGTTCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCOGCTTC
TGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCT
TTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCAC
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
ACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGGAGGCGCTAC
TGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGA
TTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTGGCTCCGGAG
GAGGTGGCAGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCT
ACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGGAGGCGCTAC
TGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGA
TTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTGGCTCCGGAG
GAGGTGGCAGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACC
CACTGGTTCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTG
GAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTAC
TGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTG
GCTCCGGAGGAGGTGGCAGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTC
SRSSSYSSSGWRYYSGAFDYWGQGTLVTVSSGGGGGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV
ACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGGAGGCGCTAC
TGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAAATGA
TTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTGGCTCCGGAG
GAGGTGGCAGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC
GGTAGTAGTGGTGGCAGTAGCAGTGGTGCC
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGG
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCT
GGGGGCTCTGGAGGCGGAACCGGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGCGT
YSGAFDYWGQGTLVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
GAFDYWGQGTLVTVSS
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
ACCTCACTGGTTCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCC
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCT
GGGGGCTCTGGAGGCGGAACCGGTGAGGT
TCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGG
YSGAFDYWGQGTLVTVSSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
YSGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
TTCTTACCTGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGTGGAGGTGGCTCCGGAGGAGGTGGC
AGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTATGGACTACTGGGGTCAAGGAACCCTG
GGTTCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGG
AGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCT
TCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGG
GTGGAGGTGGCAGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACC
GTGGCTCCGGAGGAGGTGGCAGT
GACAAGACACATACCTGTCCACCTTGTCCGGCACCTGAGCTGCTTGGAG
GCTCTGGAGGCGGAACCGGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGCGTTCA
TTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
SYLFTFGQGTKVEIKGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
HWVRRAPGKGEELVAGISASGGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSRSSSYSSSGW
RYYSGAFDYWGQGTLVTVSS
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
TTCTTACCTGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGTGGAGGTGGCTCCGGAGGAGGTGGC
AGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
TCCGTGTCCAGCGCTGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACTCGGCATCC
CTGGTTCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCT
GGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGC
CTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
TTCTTACCTGTTCACGTTCGGACAGGGTACCAAGGTGGAGATCAAAGGTGGAGGTGGCTCCGGAGGAGGTGGC
AGT
GACAAGACACATACCTGTCCACCTTGTCCGGCACCTGAGCTGCTTGGAGGCCCTAGCGTTTTTCTCTTCCCGC
TCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGGAGG
CGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCT
CTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCA
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GTGGAGGTGGAAGTGGAGGAGGTGGCAGTGGTGGAGGA
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCT
CTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTGGCAGTGATA
CCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAG
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
ACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCAT
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
ACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTGGCTCCGGA
GGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGT
YSSSGWRYYSGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCT
CTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCT
YSGAFDYWGQGTLVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLE
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCT
TAGTAGTGGTGGCAGTAGCAGTGGTGCC
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGG
TTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGGTGGAGGTGGCTCCGGAGGAGGTGGCAGTGA
ATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCC
TCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCAC
YSGAFDYWGQGTLVTVSSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLI
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGSTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GTGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTT
GGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSRSSSYSSSGWRYYSGAFDYWGQGTLVTVSSAS
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GTGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTT
CGGAGGTGGCAGCGGTGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAG
CTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCC
GGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSRSSSYSSSGWRYYSGAFDYWGQGTLVTVSSAS
MHWVRQAPGKGLEWVAGISASGGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSRSSSYSSSGW
RYYSGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GCGGAACCGGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGCGTTCACTCCGTTTGTC
GGGSGGGTG
EVQLVESGGGLVQPGRSLRLSCAASGFDLTGSYMHWVRRAPGKGEELVAGISASGGATAYADSVKG
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GCGGAACCGGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGCGTTCACTCCGTTTGTC
TCTTACATGCACTGGGTGCGTCGTGCCCCGGGTAAGGGCGAGGAACTGGTTGCAGGTATTTCCGCTTCTGGAGG
CGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCT
CTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
GGGSGGGTG
EVQLVESGGGLVQPGRSLRLSCAASGFDLTGSYMHWVRRAPGKGEELVAGISASGGATAYADSVKG
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GCGGAACCGGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATC
GTGCC
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCA
GGGSGGGTG
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLYSASDLYSGVPSRFSGSRSGT
SYSSSGWRYYSGAFDYWGQGTLVTVSS
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GCGGAACCGGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATC
GTGCCGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCA
TGCCGCTAGTGGTAGTAGTGGTGGCAGTAGCAGTGGTGCC
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCT
CTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG
GGGSGGG
T
G
DIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQKPGKAPKLLIYSASDLYSGVPSRFSGSRSGT
SYSSSGWRYYSGAFDYWGQGTLVTVSSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQSVSSAVAWYQQ
SGAEVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKGRFTISAD
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GAACCGGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTG
GGSGGGTG
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKG
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GCGGAACCGGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTC
GGGSGGGTG
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVK
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GCGGAACCGGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTC
TGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGT
GGGSGGGTG
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVK
GISASGGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSRSSSYSSSGWRYYSGAFDYWGQGTLVT
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GGTGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGT
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGGGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
GTGGAGGTGGCAGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACC
TATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATC
TTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCA
TGGAGGTGGCAGT
GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCA
SCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
AGCGGTGGAGGTGGCAGT
GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTC
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
ACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCAT
GGTTCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGG
AGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCT
TCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTA
GGSGGGGS
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKG
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAA
TCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCT
CTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCT
ACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTTCTGGAGGCGCT
ACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACAGCCTACCTACAA
TTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAA
SSYSSSGWRYYSGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
TCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAA
CTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGT
TCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTC
GS
EVQLVESGGGLVQPGGSLRLSCAASGFDLTGSYMHWVRQAPGKGLEWVAGISASGGATAYADSVKGRFTISADT
GCATCCGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCA
GTATTTCCGCTTCTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACAT
GTTCTTCTTCTTACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTC
CGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGT
CACTGGTTCTTACATGCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAGGTATTTCCGCTT
CTGGAGGCGCTACTGCTTATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAAAAACACA
ACTCTTCTTCTGGTTGGCGTTACTACTCTGGTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTC
GTGCTTTTGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCGGCTAGCACCAAGGGCCCATCGGTCTTCC
MHWVRQAPGKGLEWVAGISASGGATAYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSRSSSYSSSGW
RYYSGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
This application is a U.S. national phase entry of PCT International Application Serial No. PCT/IB2022/053050, filed on Mar. 31, 2022, which claims priority to U.S. Provisional Application Ser. No. 63/168,916, filed on Mar. 31, 2021, and U.S. Provisional Application Ser. No. 63/264,598, filed on Nov. 26, 2021, all of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2022/053050 | 3/31/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63264598 | Nov 2021 | US | |
| 63168916 | Mar 2021 | US |
