WO2022235960A1 - Anticorps neutralisants qui se lient à des protéines de spicule du sars-cov-2 - Google Patents
Anticorps neutralisants qui se lient à des protéines de spicule du sars-cov-2 Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present disclosure provides antigen binding proteins that specifically bind the spike protein (S protein) of SARS-CoV-2 and nucleic acids that encode the antigen binding proteins, vectors comprising the nucleic acids, host cells harboring the vectors, and methods of use thereof, including methods of treating SARS-CoV-2 infection and methods of preventing infection with SARS-CoV-2.
- S protein spike protein
- SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavims 2
- 2019-nCoV Severe Acute Respiratory Syndrome Coronavims 2
- SARS-CoV-2 gains entry to human cells by using the angiotensin-converting enzyme 2 (ACE2) protein as a receptor.
- ACE2 angiotensin-converting enzyme 2
- S spike proteins of both SARS-CoV and SARS-CoV-2 are transmembrane glycoproteins that form homotrimers. Binding of ACE2 on host cells by the S protein leads to internalization of the vims.
- the SARS-CoV-2 spike protein (S protein, NCBI Accession QHU79204.1, isolate "WA1/2020”) includes two regions or domains known as SI (the N-terminus to amino acid 685) and S2 (amino acids 686 to 1273) that are cleaved into subunits by a cellular protease during the infection process.
- SI the N-terminus to amino acid 685
- S2 amino acids 686 to 1273
- the SI subunit which mediates the interaction between the Spike (S) protein and ACE2
- NTD N-terminal domain
- RBD receptor binding domain
- the S2 subunit which includes an extracellular domain, a transmembrane domain, and a cytoplasmic tail, mediates virus-host membrane fusion that results in entry of the virus into the host cell.
- WA1/2020 is the SARS-CoV-2 strain isolated from a patient in Washington state
- VOCs Variants of Concern
- the neutralizing antibodies disclosed herein are disclosed to bind mutants of the SARS-CoV-2 SI protein and reduce infection of cells by the SARS-CoV-2 B.1.351 variant and reduce pathology due to SARS-CoV-2 B.1.351 infection in animals.
- these antibodies are further engineered to include mutations of the Fc region, such as mutations to reduce antibody- dependent-enhancement (ADE) of infection.
- AD antibody- dependent-enhancement
- antigen-binding proteins that specifically bind the spike (S) protein of the SARS-CoV-2 coronavims (Genbank Accession QHD43416), wherein the antigen-binding proteins comprise a heavy chain variable domain sequence having a heavy chain complimentary determining region (“CDRH”) 1 (CDRH1) , and/or a CDRH2, and/or CDRH3 that independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1, and/or a CDRH2, and/or CDRH3 as set forth in SEQ ID NO:6.
- CDRH heavy chain complimentary determining region
- antigen-binding proteins that specifically bind the spike (S) protein of the SARS-CoV-2 coronavims (Genbank Accession QHD43416), wherein the antigen-binding proteins comprise a light chain complimentary determining region (“CDRL”) 1 (CDRL1), and/or a CDRL2, and/or a CDRL3 that independently has at light chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1, and/or a CDRL2, and/or CDRL3 set forth in SEQ ID NO:7.
- CDRL light chain complimentary determining region
- antigen-binding proteins comprising a heavy chain variable domain comprising a CDR having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1 as set forth in SEQ ID NO:6, and/or a CDRH2 as set forth in SEQ ID NO:6, and/or a CDRH3 as set forth in SEQ ID NO:6.
- antigen-binding proteins comprising a light chain variable domain comprising at least one CDR at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1 as set forth in SEQ ID NO:7, and/or a CDRL2 as set forth in SEQ ID NO:7, and/or a CDRL3 as set forth in SEQ ID NO:7.
- antigen-binding proteins comprising a CDRH1, a CDRH2, and a CDRH3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1, a CDRH2, and a CDRH3 as set forth in SEQ ID NO:6.
- antigen-binding proteins comprising a CDRL1, a CDRL2, and a CDRL3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1, a CDRL2, and a CDRL3 as set forth in SEQ ID NO:7.
- antigen-binding proteins comprising a CDRH1, a CDRH2, and a CDRH3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1, CDRH2, and/or CDRH3 set forth in SEQ ID NO:6; and a CDRL1, a CDRL2, and a CDRL3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NO:7.
- antigen-binding proteins comprising a heavy chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:6.
- antigen-binding proteins comprising a light chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:7.
- antigen-binding proteins comprising a heavy chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:6 and a light chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:7.
- antigen-binding proteins comprising a CDRH1 having the amino acid sequence of SEQ ID NO:8, a CDRH2 having the amino acid sequence of SEQ ID NO:9, and a CDRH3 having the amino acid sequence of SEQ ID NO: 10.
- antigen-binding proteins comprising a light chain variable region comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 11, a CDRL2 having the amino acid sequence of SEQ ID NO: 12, and a CDRL3 having the amino acid sequence of SEQ ID NO:13.
- antigen-binding proteins comprising a CDRH1 having the amino acid sequence of SEQ ID NO:8, a CDRH2 having the amino acid sequence of SEQ ID NO:9, and a CDRH3 having the amino acid sequence of SEQ ID NO: 10 and a CDRL1 having the amino acid sequence of SEQ ID NO: 11, a CDRL2 having the amino acid sequence of SEQ ID NO: 12, and a CDRL3 having the amino acid sequence of SEQ ID NO: 13.
- antigen-binding proteins comprising a heavy chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6 and a light chain variable domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:7
- antigen-binding proteins comprising heavy chain variable domain having the sequence of SEQ ID NO:6 and a light chain variable domain having the sequence of SEQ ID NO:7.
- antigen-binding proteins that bind the SI protein of SARS-CoV-2 WA1/2020 with a binding affinity of (KD) of between about 1 nM and about 5 nM.
- antigen-binding proteins that bind an SI protein having the mutations HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, and P681H with respect to SEQ ID NO:4 with a binding affinity of (KD) of between about 1 nM and about 5 nM.
- antigen binding proteins that bind the SI protein having the mutations K417N, E484K, N501Y, and D614G with respect to SEQ ID NO:4 with a binding affinity of (KD) of between about 1 nM and about 5 nM.
- antigen-binding proteins that bind the S 1 protein having having the mutations K417N, E484K, N501Y, and D614G with a binding affinity of (KD) of between about 1 nM and about 5 nM
- antigen-binding proteins comprising an antibody or antibody fragment.
- antigen-binding proteins comprising a fully human antibody, a heavy chain variable region and a light chain variable region of a fully human antibody, or an antibody fragment derived from a fully human antibody.
- antigen-binding proteins comprising an IgG antibody, which is optionally an IgGl, IgG2, IgG3, or IgG4 antibody.
- antigen-binding proteins comprising an IgG antibody, which is optionally an IgGl or an IgG4 antibody.
- antigen-binding proteins comprising a heavy chain variable domain sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:6 and a light chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:7 and/or having the heavy chain CDR sequences of SEQ ID NOs:8, 9, and 10 and the light chain CDR sequences of SEQ ID NOs:ll, 12, and 13 can be or comprise an antibody fragment, such as for example a Fab fragment, a Fab’ fragment, or F(ab’)2 fragment.
- antigen-binding proteins with at least 95% identity to SEQ ID NO:6 and an amino acid sequence with at least 95% identity to SEQ ID NO:7 and/or having the heavy chain CDR sequences of SEQ ID NOs:8, 9, and 10 and the light chain CDR sequences of SEQ ID NOs:ll, 12, and 13 can be or comprise a single chain antibody (e.g ., an ScFv).
- antigen-binding proteins are or are derived from a fully human antibody or a fully human antibody fragment, for example, a fully human IgGl, IgG2, IgG3, IgG4,
- IgA, IgD, IgE, or IgM or a fully human single chain antibody, fully human Fab fragment, a single chain antibody, or is an antigen binding protein derived from or comprising any of these.
- antigen-binding proteins having one or more mutations in the Fc region.
- antigen-binding proteins comprising one or more mutations that decreases antibody dependent enhancement (ADE) and/or one or more mutations that increases antibody half-life.
- antigen-binding proteins comprising one or more mutations in the Fc region that reduce ADE selected from the group consisting of L234A, L235A, L235E, N297A, N297Q, N297D, P329A and P329G.
- antigen-binding proteins comprising mutations L234A and L235A (referred to as a LALA mutant).
- antigen-binding proteins comprising the sequence of SEQ ID NO: 14 or 16.
- antigen-binding proteins comprising one or more mutations in the Fc region that increase the half-life of the antibody in serum.
- antigen-binding proteins comprising one or more mutations selected from the group consisting of M252Y; S254T; T256D or T256E; T307Q; T307W; M428L; and N434S.
- antigen-binding proteins comprising the mutations M252Y; S254T; and T256E (referred to as a YTE mutant).
- antigen-binding proteins comprising a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment or a single chain antibody.
- antigen-binding proteins comprising an scFv.
- antigen-binding proteins comprising or derived from any thereof, specifically binds a coronavirus spike protein (e.g., a spike protein comprising SEQ ID NO:l or SEQ ID NO:2, or a spike protein of a coronavirus comprising a sequence having at least least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2) with a KD of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, or less than 0.01 nM.
- a coronavirus spike protein e.g., a spike protein comprising SEQ ID NO:l or SEQ ID NO:2, or a spike protein of a coronavirus comprising a sequence having at least least 90%, at least 95%, at least 96%, at least 97%, at least 98%,
- the antigen-binding protein can bind the S protein of a coronavirus (e.g., the S protein of SARS-CoV-2) with a KD of between of between about 200 nM and about 0.01 nM, or between 100 nM and 0.1 nM, or between 100 nM and 1 nM.
- a coronavirus e.g., the S protein of SARS-CoV-2
- an antibody S7E5041 antibody having a heavy chain variable sequence of SEQ ID NO:6 and a light chain variable sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, having a KD for binding the S protein of SARS-CoV-2 of between about 100 nM and about 1 nM or between about 10 nM and about 1 nM.
- any KD described herein for binding the S protein is determined by surface plasmon resonance, e.g., as described in Example 2.
- antigen-binding proteins which can be derived from, comprise, or be an antibody, such as a fully human antibody, and may be, as nonlimiting examples, an IgG, a Fab fragment, or a single chain antibody, or can be an antigen-binding protein derived from any thereof, specifically binds the SI subunit of a coronavirus S protein (e.g., SEQ ID NO:4 or an SI subunit of a spike protein of a coronavirus comprising a sequence having at least least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4) with a KD of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, or less than 0.01 nM.
- a coronavirus S protein e.g., SEQ ID NO:4 or an SI subunit of a spike protein
- antigen-binding proteins that can bind the SI subunit of a coronavirus (e.g., the SI subunit of SARS-CoV-2) with a KD of between of between about 200 nM and about 0.01 nM, or between 100 nM and 0.1 nM, or between 100 nM and 1 nM.
- the antibody is the fully human S7E5041 antibody or antibody fragment, or an antigen-binding protein derived therefrom, having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the SI subunit of the S protein of SARS-CoV-2 with a KD of between about 10 nM and about 1 nM or between about 5 nM and about 1 nM.
- antigen-binding proteins which can be derived from, comprise, or be an antibody, such as a fully human antibody, and may be, as nonlimiting examples, an IgG, a Fab fragment, or a single chain antibody, or can be an antigen-binding protein derived from any thereof, can specifically bind the SI protein of a variant SARS-CoV-2 such as a B.1.1.7 variant or a B.1.351 variant with a KD of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, or less than 0.01 nM.
- antigen-binding proteins that can bind the SI subunit of a coronavirus (e.g., the SI subunit of SARS-CoV-2) with a KD of between of between about 200 nM and about 0.01 nM, or between 100 nM and 0.1 nM, or between 100 nM and 1 nM.
- fully human S7E5041 antibody or an antibody fragment, or an antigen-binding protein derived therefrom having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the SI subunit of the S protein of SARS-CoV-2 with a KD of between about 10 nM and about 1 nM or between about 5 nM and about 1 nM.
- antigen-binding proteins which can be derived from, comprise, or be an antibody, such as a fully human antibody, and may be, as nonlimiting examples, an IgG, a Fab fragment, or a single chain antibody, or can be an antigen-binding protein derived from any thereof, can specifically bind the SI protein of a variant SARS-CoV-2 having any of the mutations as provided in Figure 2 or Figure 6, with or without the D614G mutation with a KD of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, or less than 0.01 nM.
- antigen-binding proteins that can bind the SI subunit of a coronavirus (e.g., the SI subunit of SARS-CoV-2) with a KD of between of between about 200 nM and about 0.01 nM, or between 100 nM and 0.1 nM, or between 100 nM and 1 nM.
- the fully human S7E5041 antibody or antibody fragment, or an antigen-binding protein derived therefrom having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the SI subunit of the S protein of SARS-CoV- 2 with a KD of between about 10 nM and about 1 nM or between about 5 nM and about 1 nM.
- an antigen-binding protein provided herein can specifically bind the S 1 protein of the WA1/2020 strain, can specifically bind an SI protein having the D614G mutation, and can also specifically bind the SI protein of variants of SARS-CoV-2 having any of the mutations as provided in Figure 2 or Figure 6, with or without the D614G mutation, with a KD of less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, or less than 0.01 nM.
- the antigen-binding protein can bind the S 1 subunit of a coronavirus, e.g., the SI subunit of SARS-CoV-2 WA1/2020 (SEQ ID NO:4) having any of the mutations of provided in Figure 2 or Figure 6 with a KD of between of between about 200 nM and about 0.01 nM, or between 100 nM and 0.1 nM, or between 100 nM and 1 nM.
- a coronavirus e.g., the SI subunit of SARS-CoV-2 WA1/2020 (SEQ ID NO:4) having any of the mutations of provided in Figure 2 or Figure 6 with a KD of between of between about 200 nM and about 0.01 nM, or between 100 nM and 0.1 nM, or between 100 nM and 1 nM.
- the antibody is the fully human S7E504 antibody or antibody fragment, or an antigen-binding protein derived therefrom, having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the SI protein of SARS-CoV-2 WA1/2020 (SEQ ID NO:4), as well as an SI protein having the mutations HV69-70 (deletion), Y144 deletion, N501Y, A570D, D614G, and P681H found in variant B.1.1.7, and an SI protein having the mutations K417N, E484K, N501Y, D614G of variant B.1.351 with a KD of between about 10 nM and about 1 nM or between about 5 nM and about 1 nM.
- an antigen-binding protein provided herein which can be in various embodiments be an antibody, such as a fully human antibody, and may be, as nonlimiting examples, an IgG, a Fab fragment, or a single chain antibody, or can be an antigen-binding protein derived from any thereof, specifically binds the receptor binding domain (RBD) of a coronavirus S protein (e.g., SEQ ID NO:5 or an RBD of a spike protein of a coronavirus comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:5) with a K d of less than 100 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, or less than 0.01 nM.
- RBD receptor binding domain
- the antigen-binding protein can bind the RBD of the S protein of a coronavirus (e.g., the RBD the SARS-CoV-2 S protein) with a K d of between of between about 200 nM and about 0.01 nM, or between 100 nM and 0.1 nM, or between 100 nM and 1 nM.
- a coronavirus e.g., the RBD the SARS-CoV-2 S protein
- the antigen binding protein is the fully human S7E5041 antibody having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the RBD of the S protein of SARS-CoV-2 with a KD of between about 100 nM and about 1 nM or between about 60 nM and about 2 nM.
- the antigen binding protein may be the fully human S7E5041 antibody disclosed herein having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally having L234A and L235A mutations in the Fc region, where the antibody binds the RBD of the S protein of SARS-CoV-2 with a K D of between about 100 nM and about 1 nM, or between about 60 nM and about 5 nM, or between 50 nM and 40 nM as measured by SPR.
- the antigen-binding proteins described herein block binding between the S protein of a coronavirus (such as HCoV-NL63, SARS-CoV, or SARS-CoV-2) and the ACE2 protein, for example, block binding of the ectodomain of the human ACE2 protein (hACE2) by the S protein of a coronavirus.
- a coronavirus such as HCoV-NL63, SARS-CoV, or SARS-CoV-2
- hACE2 human ACE2 protein
- the antigen-binding proteins described herein block binding between the S protein of SARS-CoV-2 and the human ACE2 protein with an IC50 of between about 0.01 pg/ml and about 100 pg/ml, between about 0.05 pg/ml and about 50 pg/ml, between about 0.1 pg/ml and about 10 pg/ml, or between about 0.1 pg/ml and about 5 pg/ml.
- the antigen binding proteins described herein block binding between the S 1 domain or subunit of SARS-CoV-2 and the human ACE2 protein with an IC50 of between about 0.01 pg/ml and about 100 pg/ml, between about 0.05 pg/ml and about 50 pg/ml, between about 0.1 pg/ml and about 10 pg/ml, or between about 0.1 pg/ml and about 5 pg/ml.
- an antigen binding protein as disclosed herein can block the binding of the SI subunit of SARS-CoV-2 and the human ACE2 protein with an IC50 of less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 1 nM.
- an antigen-binding protein as disclosed herein that includes an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:6 and an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:7 can in some embodiments block the binding of the SI subunit of SARS-CoV-2 (e.g., SEQ ID NO:4) to the ACE2 polypeptide (or the ACE2 ectodomain, e.g., SEQ ID NO:23 or SEQ ID NO:24) with an IC50 of less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, or less than 1 nM, for example, with an IC50 of between about 100 nM, less than 50 nM, less than 10 nM, less than
- the antigen binding protein is the fully human S7E5041 antibody having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the SI subunit of the S protein of SARS-CoV-2 and can block binding of the SARS-CoV-2 SI subunit to the ACE2 protein or the ectodomain thereof with an IC50 of between about 10 nM and about 1 nM, for example between about 5 nM and about 1 nM.
- an antigen-binding protein as disclosed herein which can be or comprise, as nonlimiting examples, an IgGl, IgG2, IgG3, or IgG4, a Fab fragment or a single chain antibody, or can be an antigen binding protein derived from or comprising any of these, is a neutralizing antigen binding protein that is able to inhibit binding to a target cell by a coronavirus such as HCoV-NL63, SARS-CoV, or SARS-CoV-2.
- the antigen-binding protein when included in a mixture that includes coronavirus and target cells expressing the ACE2 receptor, can reduce binding of the coronavirus to cells expressing the ACE2 receptor with an IC50 of between about 0.001 pg/ml and about 200 pg/ml, or between about 0.01 pg/ml and about 100 pg/ml, or between about 0.01 pg/ml and about 50 pg/ml, or between about 0.01 pg/ml and about 10 pg/ml, or between about 0.01 pg/ml and about 5 pg/ml, or between about 0.01 pg/ml and about 1 pg/ml, or between about 0.1 pg/ml and about 100 pg/ml, or between about 0.1 pg/ml and about 50 pg/ml.
- an IC50 of between about 0.001 pg/ml and about 200 pg/ml, or between about 0.
- an antigen-binding protein can reduce binding of the coronavirus to a cell expressing the ACE2 receptor with an IC50 of between about 1 pg/ml and about 50 pg/ml, or between about 1 pg/ml and about 10 pg/ml, or between about 1 pg/ml and about 5 pg/ml.
- an antigen-binding protein can reduce binding of the coronavirus to a cell expressing the ACE2 receptor with an IC50 of between about 0.1 pg/ml and about 10 pg/ml, or between about 0.1 pg/ml and about 5 pg/ml, or between about 0.1 pg/ml and about 1 pg/ml.
- the anti-S antigen binding proteins disclosed herein are neutralizing antigen-binding proteins, e.g., neutralizing antibodies, that can block infection of target cells, for example, can inhibit a cytopathic effect (CPE) resulting from infection by a coronavirus such as SARS- CoV or SARS-CoV-2 of susceptible cells with an IC50 of between about 0.1 ng/ml and about 500 ng/ml, or between about 1 ng/ml and about 200 ng/ml, or between about 2 ng/ml and about 100 ng/ml, or between about 5 ng/ml and about 50 ng/ml.
- CPE cytopathic effect
- the antigen binding protein is the fully human S7E5041 antibody having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the SI subunit of the S protein of SARS-CoV-2 and can inhibit CPE by SARS-CoV-2 WA1/2020 with an IC50 of between about 10 ng/mL and about 50 ng/mL.
- anti-S antigen binding proteins that are neutralizing antigen-binding proteins, e.g., neutralizing antibodies, that can block infection of target cells, for example, can inhibit a cytopathic effect (CPE) resulting from infection by a variant SARS-CoV-2, such as the B.l.1.7 variant or the B 1.351 variant, of susceptible cells with an IC50 of between about 0.1 ng/ml and about 10 pg/ml, or between about 0.1 ng/ml and about 500 ng/ml, or between about 1 ng/ml and about 1 pg/ml, or between about 10 ng/ml and about 1 pg/ml, or between about 10 ng/ml and about 500 ng/ml, or between about 50 ng/ml and about 500 nM.
- CPE cytopathic effect
- the antigen binding protein is the fully human S7E5041 antibody having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7, and optionally comprising one or more mutations in the Fc region, that binds the S 1 subunit of the S protein of SARS-CoV-2 and can inhibit CPE by SARS-CoV-2 variant B 1.351 with an IC50 of between about 50 ng/ml and about 500 ng/ml.
- antigen binding proteins comprising a neutralizing antibody or antigen binding fragment thereof that blocks infection of susceptible cells by SARS-CoV-2 isolate WAl/2020 with an IC50 of 100 ng/mL or lower.
- antigen binding proteins comprising a neutralizing antibody that blocks infection of susceptible cells by SARS-CoV-2 variant B.1.351 with an IC50 of 200 ng/mL or lower.
- monoclonal fully humanized antibodies and antigen binding proteins derived therefrom having a variable heavy chain sequence of SEQ ID NO:6 and a variable light chain sequence of SEQ ID NO:7 that are able to block infection of target cells, e.g., inhibit CPE, by SARS-CoV-2 WA1/2020 with an IC50 of between about 10 ng/ml and about 100 ng/ml and are also able to block infection of target cells, e.g., inhibit CPE, by SARS- CoV-2 variant B E352 with an IC50 of between about 50 ng/ml and about 500 ng/ml.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein and a second antigen-binding protein comprising a CDRH1, and/or a CDRH2, and/or CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO.
- kits comprising: a first antigen binding protein according to one or more embodiments disclosed herein and a second antigen-binding protein comprising a CDRL1, and/or a CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and second antigen-binding protein comprising a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO.
- CDRL1, CDRL2, and/or CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein comprising a heavy chain variable region comprising a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen binding protein comprising a light chain variable region comprising a CDRL1, and/or a CDRL2, and/or CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein comprising a heavy chain variable region comprising: a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO.
- a light chain variable region comprising a CDRL1, and/or CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein comprising a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein comprising a light chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein wherein the second antigen-binding protein comprises: a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28; and a light chain variable region comprising having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein, wherein the second antigen-binding protein comprises antibody S1D7270 or antibody STI-2020.
- kits comprising: a first antigen-binding protein according to one or more embodiments disclosed herein; and a second antigen-binding protein according to one or more embodiments disclosed herein and throughout, wherein the kit comprises a single composition comprising the first antigen-binding protein and the second antigen-binding protein.
- compositions comprising: an antigen-binding protein disclosed herein and throughout; or a kit as disclosed herein and throughout; and a pharmaceutically-acceptable excipient.
- a first antigen-binding protein comprises an IgG antibody, a Fab, Fab’ or F(ab’)2 fragment, or a single chain antibody; and/or a kit comprises a second antigen-binding protein comprising an IgG antibody, a Fab, Fab’ or F(ab’)2 fragment, or a single chain antibody.
- compositions wherein a first antigen-binding protein comprises an IgG antibody; and/or a kit comprises a second antigen-binding protein comprising an IgG antibody.
- a first antigen-binding protein comprises an IgG antibody having an Fc region comprising F234A and F235A mutations; and/or a kit comprises a second antigen-binding protein comprising F234A and F235A mutations.
- compositions comprising an antigen-binding protein as disclosed herein that specifically binds the S protein of a coronavirus, such as the S protein of SARS-CoV-2, and a pharmaceutically acceptable carrier.
- the pharmaceutical can be formulated for intramuscular or subcutaneous injection, or for intravenous, oral, intranasal, or pulmonary delivery, as nonlimiting examples.
- pharmaceutical compositions may optionally be formulated as a liquid, solid, or gel, depending on the mode of delivery and/or storage and packaging considerations, and can optionally be formulated and packaged in single doses.
- nucleic acid molecules comprising a nucleic acid sequence encoding an antigen-binding protein disclosed herein and throughout and/or a second antigen binding protein of a kit as disclosed herein and throughout.
- nucleic acid molecules comprising a vector, wherein the vector comprises a promoter operably linked to the nucleic acid sequence encoding an antigen-binding protein disclosed herein and throughout and/or the second antigen binding protein of a kit disclosed herein and throughout.
- methods comprising administering an effective amount of an antigen-binding protein as disclosed herein and throughout; a kit as disclosed herein and throughout; a pharmaceutical composition as disclosed herein and throughout; a nucleic acid as disclosed herein and throughout; a vector as disclosed herein and throughout; and/or a host cell as disclosed herein and throughout, to a subject.
- administration may be, as nonlimiting examples, by intramuscular or subcutaneous injection, by intravenous delivery (by bolus injection or infusion), by oral delivery, by intranasal delivery, or by pulmonary delivery, for example by inhalation.
- kits as disclosed herein and throughout; a pharmaceutical composition as disclosed herein and throughout; a nucleic acid as disclosed herein and throughout; a vector as disclosed herein and throughout; and/or a host cell as disclosed herein and throughout, to a subject.
- administration may be, as nonlimiting examples, by intramuscular or subcutaneous injection, by intravenous delivery (by bolus injection or infusion), by oral delivery, by intranasal delivery, or by pulmonary delivery, for example by inhalation.
- methods of detecting a coronavims using an antigen-binding protein that specifically binds the S protein of a coronavims as disclosed herein comprise detecting the presence of a coronavims, or a protein of a coronavims, e.g., an S protein or SI subunit of a coronavims, in a sample, comprising: (a) contacting the sample with an antigen binding protein as disclosed herein under conditions suitable to form an antibody-antigen complex; and (b) detecting the presence of the antibody-antigen complex to detect the presence of a coronavims or protein thereof.
- such methods mayh be used to detect the presence of a coronavims in a sample from a subject and thereby diagnose a subject suspected of having a coronavims infection.
- such methods comprise use of a sample from a subject comprises phlegm, mucous, saliva, blood, pleural fluid, cheek scaping, tissue biopsy, or semen.
- such methods comprise use of an antigen-binding protein that specifically binds the S protein, which can be an antibody or antibody fragment that specifically binds the S protein, can be labeled for direct or indirect detection of an antigen- antibody complex, where the label can comprise a radionuclide, fluorophore, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, or ligand (e.g., biotin, a hapten).
- an antigen-binding protein that specifically binds the S protein which can be an antibody or antibody fragment that specifically binds the S protein, can be labeled for direct or indirect detection of an antigen- antibody complex, where the label can comprise a radionuclide, fluorophore, enzyme, enzyme substrate, enzyme cofactor, enzyme inhibitor, or ligand (e.g., biotin, a hapten).
- the presence of the antibody-antigen complex can be detected using any detection mode including detection of radioactivity, detection of fluorescence, detection of luminescence, or colorimetric, antigenic, or enzymatic detection, or detection of a magnetic or electrodense (e.g., gold) bead, and may optionally use binding moieties such as but not limited to biotin, streptavidin, or protein A.
- detection mode including detection of radioactivity, detection of fluorescence, detection of luminescence, or colorimetric, antigenic, or enzymatic detection, or detection of a magnetic or electrodense (e.g., gold) bead, and may optionally use binding moieties such as but not limited to biotin, streptavidin, or protein A.
- nucleic acid molecules encoding an antigen-binding protein as provided herein comprising a heavy chain variable domain sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:6 and a light chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:7, and/or having the heavy chain CDR sequences having the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10, and the light chain CDR sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.
- nucleic acid molecules that encode one or both of a heavy chain variable domain sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:6 and a light chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:7 or includes the heavy chain CDRs 1, 2, and 3 of SEQ ID NOs:8, 9, and 10, respectively and the light chain CDRs 1, 2, and 3 of SEQ ID NOs:ll, 12, and 13, respectively can be an expression vector that includes a promoter operably linked to the antigen-binding protein encoding sequence.
- such expression vectors may comprise a viral or plasmid vector.
- promoters comprise a eukaryotic promoter and can be, as nonlimiting examples, an EFla promoter, a CMV promoter, a JeT promoter, an RSV promoter, an SV40 promoter, a CAG promoter, a beta-actin promoter, an HTLV promoter, or an EFla/HTLV hybrid promoter.
- provided expression vectors may be, for example, a viral or plasmid vector, and in some examples can be a nanoplasmid vector having fewer than 500 bp of a bacterial plasmid and having fewer than 10 CpG sequences.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein disclosed, for example, in WO 2022/032139, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a CDRL1, and/or a CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a CDRH1, and/or a CDRH2, and/or CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO.
- SEQ ID NO: 32 31, and/or SEQ ID NO:32, respectively, and a CDRL1, and/or a CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a heavy chain variable region comprising a CDRH1, and/or a CDRH2, and/or CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a light chain variable region comprising a CDRL1, and/or a CDRL2, and/or CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a heavy chain variable region comprising a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO.
- SEQ ID NO: 32 31, and/or SEQ ID NO:32, respectively, and a light chain variable region comprising a CDRL1, and/or CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a light chain variable region comprising having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28 and a light chain variable region comprising having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising antibody S1D7270 as disclosed in WO 2022/032139, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
- compositions comprising: a first antigen binding protein as disclosed herein and throughout; and a second antigen binding protein comprising antibody STI-2020 as disclosed in WO 2022/032139, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
- nucleic acid molecules that encode an antigen-binding protein as provided herein and throughout.
- pharmaceutical formulations comprising such nucleic acid molecule(s) and one or more excipients or carriers.
- pharmaceutical compositions comprising one or more compounds that enhance delivery of such nucleic acid molecules into cells, such as for example a cationic lipid or amphiphilic block copolymers, for example, one or more poloxamers or poloxamines.
- a coronavims infection by administering an effective amount of a pharmaceutical composition as provided herein and throughout that includes at least one nucleic acid molecule encoding an antigen-binding protein as disclosed herein and throughout.
- a pharmaceutical composition as provided herein and throughout that includes at least one nucleic acid molecule encoding an antigen-binding protein as disclosed herein and throughout.
- such administration may be, for example, by injection, such as intradermal or intramuscular injection.
- such administration may be, for example via single or multiple doses, including multiple doses over weeks or months.
- the amount of DNA e.g., plasmid or plasmids encoding a neutralizing antibody
- the amount of DNA e.g., plasmid or plasmids encoding a neutralizing antibody
- transgenic cells engineered to express an antibody comprising a heavy chain variable domain sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:6 and a light chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO:7.
- the cells can be prokaryotic or eukaryotic.
- the transgenic cells are mammalian cells, such as cells of a mammalian cell line.
- Figure 1 provides SPR sensorgrams of anti-S 1 fully human monoclonal antibody S7E5041LALA binding to the SARS-CoV2 SI proteins having amino acid sequences corresponding to the SI protein of WA1/2020 (“SI”), as well as SI proteins incorporating the K417N, E484K, N501Y, D614G mutations found in the SI protein of variant B.1.351 (“B.1.351”) and incorporating the mutations HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H of the S protein of variant B.1.1.17 (“B.1.1.17”), and a table providing the binding parameters, including binding affinities (KDS).
- KDS binding affinities
- Figure 2 is a table providing the EC50s for binding of the S7E5041LALA antibody to cells expressing SI variants engineered to include certain mutations of SARS-CoV-2 variants.
- the SI protein of variant “S.A B.1.351” in row three includes, in addition to the mutations listed in the table, the D614G mutation.
- Figure 3A-H provides the binding curves for S7E5041LALA antibody to cells expressing SI variants engineered to include certain mutations found in SARS-CoV-2 variants.
- Figure 4A provides a graph of the results of a plaque reduction neutralization titer (PRNT) assay on Vero cells using the WA1/2020 SARS-CoV-2 and increasing concentrations of S7E5041LALA antibody
- Figure 4B provides a graph of the results of a PRNT assay on Vero cells performed with the “South Africa” variant SARS-CoV-2 B.1.351 and increasing dilution of S7E5041LALA antibody.
- PRNT plaque reduction neutralization titer
- Figure 5 is a schematic for preparing pseudotyped VSV for testing the ability of the S7E5041 antibody to neutralized SARS-CoV-2 S protein-mediated infection of cells expressing ACE2.
- Figure 6 is a table of various engineered S protein mutants incorporating amino acid changes found in SARS-CoV-2 variants and the EC50s for neutralization of infection by VSVs pseudotyped with these S proteins by the S7E5041LALA antibody.
- Figure 7A is a graph in which the average % weight change over the course of a study in which hamsters infected with the SARS-CoV-2 B.1.351 variant were treated with either IgG (control) or S7E5041LALA antibody.
- the upper curve represents the average values for the S7E5041LALA -treated group and the lower curve represents the average values for the IgG-treated control group.
- Figure 7B provides a graph in which the titers of virus present in the lungs of animals sacrificed at day five are represented individually.
- Figure 8A provides a flow diagram depicting an exemplary process for discovery and characterization of antigen binding-proteins provided herein and throughout. Figurte and data associated therewith.
- the depicted phage display library was panned for SARS- CoV-2 Spike SI subunit-binding scFv fragments. Following confirmation of binding activity and blocking of S1:ACE2 interactions by candidate scFvs, the most potent of these candidates were converted to IgGl antibodies bearing the LALA Fc modification.
- Candidate nAbs were characterized for binding of Spike S 1 subunit and neutralization of related clinical SARS-CoV-2 isolates.
- FIG. 8B provides affinity measurements of STI-2020 and STI-5041 for Spike SI domain from the following isolates and VOCs: USA/WA-1/2020(WA-1) isolate, D614G 2020001 isolate,
- FIG. 8C provides STI-2020 and STI-5041 Spike protein binding data using Spike protein derived from the WA-1 and 2020001 (D614G) SARS-CoV-2 isolates, which were independently expressed on the surface of HEK 293 cells. Serially-diluted STI-2020 or STI-5041 were assayed for Spike protein binding by flow cytometry. To quantify antibody binding, mean fluorescent intensity was measured for each dilution tested and the EC50 value was calculated for each nAb.
- FIG. 8D STI-2020 and STI-5041 were evaluated in neutralizing test for potency against SARS- CoV-2 US A/WA- 1/2020, 2020001 (D614G), B.1.1.7 VOC (Alpha), and B.1.351 VOC (Beta).
- Figure 9A-H provides pharmacokinetic and bioavailability of neutralizing antibodies.
- Figure 9A Neutralization of SARS-CoV-2 Spike-pseudotyped VSV by STI-2020 and STI-5041. VSVAG-luciferase was pseudotyped with the indicated spike variant, incubated with STI-2020 or STI-5041 at a range of 0.0005-10 pg/mL for 30 minutes, then added to 293-ACE2 target cells. Absolute IC50 was calculated from luciferase values and are indicated. Experiments were performed at least three independent times and data presented as the mean ⁇ SD.
- Figure 9B Epitope binning performed as described in Example 7.
- the sensorgram shows S7E5041 can bind to SARS-CoV-2 SI and STI-2020 complex (blue line) and indicates that S7E5041 and STI-2020 bind to distinct epitopes.
- Figure 9C-H Biodistribution: Concentration of STI-2020 (C, D) or 5041 (E, F) in serum and lung lavage or lysates of spleens, lungs, small intestines, and large intestines collected from female CD-I mice administered STI-2020 IV at doses of 0.5 mg/kg, 0.05 mg/kg, or 0.005 mg/kg or IN at doses of 2.5 mg/kg, 0.5 mg/kg, 0.05 mg/kg, and 0.005 mg/kg or STI-5041 administered IV at doses of 2 mg/kg, 0.2 mg/kg, and 0.02 mg/kg or IN at doses of 10 mg/kg, 2 mg/kg, 0.2 mg/kg, and 0.02 mg/kg at 24 hours post-administration as compared to samples collected from
- Figure 10A-D provides efficacy of intravenous (IV) delivery of neutralizing antibodies in the Syrian golden Hamster Model of COVID-19.
- Female hamsters were inoculated with SARS-CoV-2 US A/WA- 1/2020 (Figure 10A-B) or SARS-CoV-2 Beta variant ( Figure 10C-D) on day 0.
- Isotype control IgG 500 pg
- STI-2020 100 pg, 300 pg, or 500 pg
- For 10C-D Isotype control IgG (1,000 pg) or STI-5041 (500 pg, or 1,000 pg).
- FIG. 10A Average % weight change ⁇ SEM was plotted for each group. Days in which there was a significant difference in average % weight change compared to Isotype control IgG 500 pg-treated animals are denoted by * (p-value ⁇ 0.05).
- Figure 10B Lung tissues collected from five animals per group and virus titers were determined on day 5. A broken line indicates the detection limit of the assay ( ⁇ 1.5 TCIDso/g).
- Figure IOC Average % weight change ⁇ SEM was plotted for each group.
- FIG. 10D Lung tissues collected from five animals per group administered Isotype control IgG (1,000 pg) or STI-5041 (500 pg or 1,000 pg) and virus titers were determined on day 5.
- Figure 11A-C provides efficacy of intranasal (IN) delivery of neutralizing antibodies in the Syrian golden Hamster Model of COVID-19.
- Figure 11A-B Female hamsters were inoculated with SARS-CoV-2 US A/WA- 1/2020, and then administrated with 500 pg or 400 pg Isotype control antibody or 500 pg or 400 pg STI-2020 intranasally at 12 hours post-infection.
- Figure 11A Average % weight change ⁇ SEM was plotted for each group. Days in which there was a significant difference in average % weight change for STI- 5020 at 500 pg compared to Isotype control IgG 500 pg-treated animals are denoted by * (p- value ⁇ 0.05).
- Figure 11B Upper panels show representative figures of nasal turbinates and nasal septum at 5 d.p.i.
- Average ⁇ SEM of OE thickness on the nasal septum in the lower graph for STI-5020 at 400 pg compared to Isotype control IgG 400 pg-treated animals are denoted with * (p-value ⁇ 0.05).
- Figure 11C Hamsters were inoculated with SARS-CoV-2 Beta variant, and then administrated with 500 pg Isotype control antibody or 100 pg, 300 pg, or 500 pg STI-5041 intranasally at 12 hours post-infection. Average % weight change ⁇ SEM was plotted for each group.
- Figure 12 provides Intravenous isotype control antibody weight change data of uninfected hamsters. Female hamsters were administrated 2000 pg Isotype control antibody. Average % weight change ⁇ SEM was plotted.
- a or B “A” (alone), and “B” (alone).
- the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
- the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
- “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art.
- “about” or “approximately” can mean a range of up to 10% (i.e., ⁇ 10%) or more depending on the limitations of the measurement system.
- about 5 mg can include any number between 4.5 mg and 5.5 mg.
- the terms can mean up to an order of magnitude or up to 5-fold of a value.
- the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition.
- polypeptide and “protein” and other related terms used herein are used interchangeably and refer to a polymer of amino acids that is not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids. Polypeptides include recombinant and chemically-synthesized polypeptides. Polypeptides include precursor molecules and mature (e.g., processed) molecules. Precursor molecules include those that have not yet been subjected to cleavage, for example cleavage of a secretory signal peptide or by enzymatic or non-enzymatic cleavage at certain amino acid residue(s). Polypeptides include mature molecules that have undergone cleavage.
- proteins encompass native proteins, recombinant proteins, and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins.
- polypeptides that bind the S protein of a coronavirus and that are produced using recombinant procedures are described herein.
- nucleic acid refers to polymers of nucleotides that are not limited to any particular length.
- Nucleic acids include recombinant and chemically- synthesized forms.
- Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA, expression constructs, DNA fragments, etc.), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof, as well as peptide nucleic acids, locked nucleic acids, and other synthetic nucleic acid analogs and hybrids thereof.
- a nucleic acid molecule can be single-stranded or double-stranded.
- nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment or scFv, derivative, mutein, or variant thereof.
- nucleic acids comprise one type of polynucleotides or a mixture of two or more different types of polynucleotides. Nucleic acids encoding anti-S protein antibodies or antigen-binding portions thereof are described herein.
- the term “recover” or “recovery” or “recovering”, and other related terms refer to obtaining a protein (e.g., an antibody or an antigen binding portion thereof), from host cell culture medium or from host cell lysate or from the host cell membrane.
- the protein is expressed by the host cell as a recombinant protein fused to a secretion signal peptide sequence (e.g., leader peptide sequence) which mediates secretion of the expressed protein.
- the secreted protein can be recovered from the host cell medium.
- the protein is expressed by the host cell as a recombinant protein that lacks a secretion signal peptide sequence which can be recovered from the host cell lysate.
- the protein is expressed by the host cell as a membrane-bound protein which can be recovered using a detergent to release the expressed protein from the host cell membrane.
- the protein can be subjected to procedures that remove cellular debris from the recovered protein.
- the recovered protein can be subjected to chromatography, gel electrophoresis and/or dialysis.
- the chromatography comprises any one or any combination or two or more procedures including affinity chromatography, hydroxyapatite chromatography, ion-exchange chromatography, reverse phase chromatography and/or chromatography on silica.
- affinity chromatography comprises protein A or protein G (cell wall components from Staphylococcus aureus).
- isolated refers to a protein (e.g., an antibody or an antigen binding portion thereof) or polynucleotide that is substantially free of other cellular material.
- isolated also refers in some embodiments to protein or polynucleotides that are substantially free of other molecules of the same species, for example other proteins or polynucleotides having different amino acid or nucleotide sequences, respectively.
- the purity or homogeneity of the desired molecule can be assayed using techniques well known in the art, including low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrometry.
- any of the anti-S antibodies or antigen binding protein thereof disclosed herein are isolated.
- Antibodies can be obtained from sources such as serum or plasma that contain immunoglobulins having varied antigenic specificity. If such antibodies are subjected to affinity purification, they can be enriched for a particular antigenic specificity. Such enriched preparations of antibodies usually are made of less than about 10% antibody having specific binding activity for the particular antigen. Subjecting these preparations to several rounds of affinity purification can increase the proportion of antibody having specific binding activity for the antigen. Antibodies prepared in this manner are often referred to as "monospecific.” Monospecific antibody preparations can be made up of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody having specific binding activity for the particular antigen. Antibodies can be produced using recombinant nucleic acid technology as described below.
- leader sequence or “leader peptide” or “[peptide] signal sequence” or “signal peptide” or “secretion signal peptide” refers to a peptide sequence that is located at the N-terminus of a polypeptide.
- a leader sequence directs a polypeptide chain to a cellular secretory pathway and can direct integration and anchoring of the polypeptide into the lipid bilayer of the cellular membrane.
- a leader sequence is about 10-50 amino acids in length and is cleaved from the polypeptide upon secretion of the mature polypeptide or insertion of the mature polypeptide into the membrane.
- proteins provided herein such as membrane proteins and antibodies having signal peptides that are identified by their precursor sequences that include a signal peptide sequence are also intended to encompass the mature forms of the polypeptides lacking the signal peptide, and proteins provided herein such as membrane proteins and antibodies having signal peptides that are identified by their mature polypeptide sequences that lack a signal peptide sequence are also intended to encompass forms of the polypeptides that include a signal peptide, whether native to the protein or derived from another secreted or membrane-inserted protein.
- a leader sequence includes signal sequences comprising CD8a, CD28 or CD16 leader sequences.
- the signal sequence comprises a mammalian sequence, including for example mouse or human Ig gamma secretion signal peptide.
- a leader sequence comprises a mouse Ig gamma leader peptide sequence MEWS W VFLFFLS VTT G VHS (SEQ ID NO: 17).
- an "antigen-binding protein” and related terms used herein refer to a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen-binding protein to the antigen.
- antigen-binding proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs.
- an “antigen-binding protein derived from [a referenced] antibody” is an antigen-binding protein that includes the variable light chain sequence and variable heavy chain sequence of the referenced antibody.
- the antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
- Such scaffolds include, but are not limited to, antibody- derived scaffolds comprising mutations introduced to, for example, stabilize the three- dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Komdorfer et ah, 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129; Roque et ah, 2004, Biotechnol. Prog. 20:639-654.
- peptide antibody mimetics can be used, as well as scaffolds based on antibody mimetics utilizing fibronection components as a scaffold.
- PAMs peptide antibody mimetics
- Antigen binding proteins that bind the spike protein of SARS-CoV-2 are described herein.
- An antigen binding protein can have, in some examples, the structure of an immunoglobulin.
- an "immunoglobulin” refers to a tetrameric molecule composed of two identical 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.
- the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains.
- Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
- the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for ah purposes).
- the heavy and/or light chains may or may not include a leader sequence for secretion.
- an antigen binding protein can be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but still binds a target antigen or binds two or more target antigens.
- a synthetic antigen binding protein can comprise antibody fragments, 1-6 or more polypeptide chains, asymmetrical assemblies of polypeptides, or other synthetic molecules.
- variable regions of immunoglobulin chains exhibit the same general structure of three hypervariable regions, also called complementarity determining regions or CDRs, joined by relatively conserved framework regions (FR). From N-terminus to C-terminus, both light and heavy chains comprise the segments FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
- One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein.
- An antigen binding protein may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently.
- the CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.
- an "antibody” and “antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof (or an antigen binding fragment thereof) that binds specifically to an antigen.
- Antigen binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
- Antigen binding portions include, inter alia, Fab, Fab', F(ab') 2 , Fv, single domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, nanobodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
- Antibodies include recombinantly produced antibodies and antigen binding portions.
- Antibodies include non-human, chimeric, humanized and fully human antibodies.
- Antibodies include monospecific, multispecific (e.g., bispecific, trispecific and higher order specificities).
- Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers.
- Antibodies include F(ab’) 2 fragments,
- Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies (anti-id), minibodies.
- Antibodies include monoclonal and polyclonal antibody populations.
- the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible spontaneous mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
- Monoclonal antibodies include monoclonal antibodies produced using hybridoma methods that provide a cell line producing a population of identical antibody molecules, and also include chimeric, hybrid, and recombinant antibodies produced by cloning methods such that a cell transfected with the construct or constructs that include the antibody-encoding sequences and the progeny of the transfected cell produce a population of antibody molecules directed against a single antigenic site.
- variable regions of an antibody may be cloned into an antibody framework that includes constant regions of any species, including human constant regions, where expression of the construct in a cell can produce a single antibody molecule or antigen-binding protein that is referred to herein as monoclonal.
- the modifier "monoclonal” thus indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
- the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
- the “monoclonal antibodies” may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), for example.
- an “antigen binding domain,” “antigen binding region,” or “antigen binding site” and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains.
- telomere binding refers to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., an antibody specifically binds to a particular antigen relative to other available antigens).
- an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant (K d ) of 10 "5 M or less, or 10 "6 M or less, or 10 "7 M or less, or 10 "8 M or less, or 10 "9 M or less, or 10 "10 M or less, or 10 "11 or less, or 10 "12 or less.
- K d dissociation constant
- Binding affinity of an antigen-binding protein for a target antigen can be reported as a dissociation constant (K d ) which can be measured using a surface plasmon resonance (SPR) assay.
- SPR surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using a BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).
- An "epitope" and related terms as used herein refers to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or an antigen binding portion thereof).
- An epitope can comprise portions of two or more antigens that are bound by an antigen binding protein.
- An epitope can comprise non-contiguous portions of an antigen or of two or more antigens (e.g., amino acid residues that are not contiguous in an antigen’s primary sequence but that, in the context of the antigen’s tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding protein).
- the variable regions, particularly the CDRs, of an antibody interact with the epitope.
- the term “antagonist” and “antagonistic” refers to a blocking antibody that binds its cognate target antigen and inhibits or reduces the biological activity of the bound antigen.
- the term “agonist” or “agonistic” refers to an antibody that binds its cognate target antigen in a manner that mimics the binding of the physiological ligand which causes antibody-mediated downstream signaling.
- an "antibody fragment”, “antibody portion”, “antigen-binding fragment of an antibody”, or “antigen-binding portion of an antibody” and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide.
- Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
- Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.
- Fab fragment
- a Fab is capable of binding an antigen.
- An F(ab') 2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
- a F(Ab’) 2 has antigen binding capability.
- An Fd fragment comprises V H and C H I regions.
- An Fv fragment comprises V L and V H regions.
- An Fv can bind an antigen.
- a dAb fragment has a V H domain, a V L domain, or an antigen binding fragment of a V H or VF domain (U.S. Patents 6,846,634 and 6,696,245; U.S. published Application Nos. 2002/02512, 2004/0202995, 2004/0038291, 2004/0009507, 2003/0039958; and Ward et al., Nature 341:544-546, 1989).
- a single-chain antibody is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain.
- the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
- Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Hohiger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites.
- Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites.
- tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
- Diabody, tribody and tetrabody constructs can be prepared using antigen binding portions from any of the anti-Spike protein antibodies described herein.
- a “humanized antibody” refers to an antibody originating from a non-human species that has one or more variable and constant regions that has been sequence modified to conform to corresponding human immunoglobulin amino acid sequences.
- the constant regions of a humanized antibody may be human constant region sequences, where the amino acid sequence of a variable domains may be from an antibody sequence of another species, such as a mouse (in which the antibody may have been generated).
- a humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject.
- certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody.
- the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species.
- one or more amino acid residues in one or more CDR sequences of a non human antibody is changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immuno specific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.
- an antibody can be a “fully human” antibody in which all of the constant and variable domains (optionally excepting from the CDRs) are derived from human immunoglobulin sequences.
- a fully human antibody as disclosed herein may have one or more mutations (which may be, for example amino acid substitutions, deletions, or insertions) in the constant regions, such as for example the Fc constant regions of the heavy chain, with respect to a wild type human antibody sequence.
- a fully human antibody can have one or more mutation in the constant regions of either the light or heavy chain of the antibody, where the sequence of either or both of the light chain constant region or heavy chain constant regions (CHI, CH2, and CH3) of the fully human antibody are greater than 95%, greater than 96%, greater than 97%, and preferably greater than 98% or at least 99% identical to the sequence of the non-mutant human constant regions.
- CHI, CH2, and CH3 light chain constant region or heavy chain constant regions
- Humanized and fully human antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes, e.g., the "Xenomouse II" that, when challenged with an antigen, generates high affinity fully human antibodies Mendez et al. ((1997) Nature Genetics 15: 146-156). This was achieved by germ-line integration of megabase human heavy chain and light chain loci into mice with deletion of the endogenous J H region. The antibodies produced in these mice closely resemble that seen in humans in all respects, including gene rearrangement, assembly, and repertoire.
- phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from immunized or nonimmunized donors.
- V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
- the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
- the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats; see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993).
- V-gene segments can be used for phage display, e.g., the spleens of immunized mice (Clackson et al., Nature 352, 624-628 (1991)) or blood cells of nonimmunized human donors can be used to generate antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222, 581-597 (1991) or Griffith et al., EMBO J. 12, 725-734 (1993).
- chimeric antibody refers to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies.
- one or more of the CDRs are derived from a human antibody.
- all of the CDRs are derived from a human antibody.
- the CDRs from more than one human antibody are mixed and matched in a chimeric antibody.
- a chimeric antibody may comprise a CDR1 from the light chain of a first human antibody, a CDR2 and a CDR3 from the light chain of a second human antibody, and the CDRs from the heavy chain from a third antibody.
- the CDRs originate from different species such as human and mouse, or human and rabbit, or human and goat.
- the framework regions of a chimeric antibody may be derived from one of the same antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody.
- a portion of the heavy and/or light chain is identical with, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with, homologous to, or derived from an antibody (-ies) from another species or belonging to another antibody class or subclass.
- fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind a target antigen).
- variant polypeptides and variants of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residues inserted into, deleted from and/or substituted into the amino acid sequence relative to a reference polypeptide sequence.
- Polypeptide variants include fusion proteins.
- a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence.
- Polynucleotide variants include fusion polynucleotides.
- derivatives of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
- another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
- antibody includes, in addition to antibodies comprising full-length heavy chains and full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.
- a hinge region refers to an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the overall construct and movement of one or both of the domains relative to one another.
- a hinge region comprises from about 10 to about 100 amino acids, e.g., from about 15 to about 75 amino acids, from about 20 to about 50 amino acids, or from about 30 to about 60 amino acids.
- the hinge region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.
- the hinge region can be derived from is a hinge region of a naturally-occurring protein, such as a CD8 hinge region or a fragment thereof, a CD8a hinge region, or a fragment thereof, a hinge region of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibodies), or a hinge region that joins the constant domains CHI and CH2 of an antibody.
- a hinge region of a naturally-occurring protein such as a CD8 hinge region or a fragment thereof, a CD8a hinge region, or a fragment thereof, a hinge region of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibodies), or a hinge region that joins the constant domains CHI and CH2 of an antibody.
- the hinge region can be derived from an antibody and may or may not comprise one or more constant regions of the antibody, or the hinge region comprises the hinge region of an antibody and the CH3 constant region of the antibody, or the hinge region comprises the hinge region of an antibody and the CH2 and CH3 constant regions of the antibody, or the hinge region is a non- naturally occurring peptide, or the hinge region is disposed between the C-terminus of the scFv and the N-terminus of the transmembrane domain.
- the hinge region comprises any one or any combination of two or more regions comprising an upper, core or lower hinge sequences from an IgGl, IgG2, IgG3 or IgG4 immunoglobulin molecule.
- the hinge region comprises an IgGl upper hinge sequence EPKSCDKTHT. In one embodiment, the hinge region comprises an IgGl core hinge sequence CPXC, wherein X is P, R or S. In one embodiment, the hinge region comprises a lower hinge/CH2 sequence PAPELLGGP (SEQ ID NO: 18). In one embodiment, the hinge is joined to an Fc region (CH2) having the amino acid sequence SVFLFPPKPKDT (SEQ ID NO: 19). In one embodiment, the hinge region includes the amino acid sequence of an upper, core and lower hinge and comprises EPKSCDKTHTCPPCPAP EFFGGP (SEQ ID NO:20). In one embodiment, the hinge region comprises one, two, three or more cysteines that can form at least one, two, three or more interchain disulfide bonds.
- Fc or “Fc region” as used herein refers to the portion of an antibody heavy chain constant region beginning in or after the hinge region and ending at the C- terminus of the heavy chain.
- the Fc region comprises at least a portion of the CH2 and CH3 regions and may, or may not, include a portion of the hinge region.
- An Fc domain may bind Fc cell surface receptors and some proteins of the immune complement system.
- An Fc region may bind a complement component Clq.
- An Fc domain may exhibit effector function, including any one or any combination of two or more activities including complement- dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization and/or cell binding.
- CDC complement- dependent cytotoxicity
- ADCC antibody-dependent cell-mediated cytotoxicity
- ADP antibody-dependent phagocytosis
- An Fc domain may bind an Fc receptor, including FcyRI (e.g., CD64), FcyRII (e.g, CD32) and/or FcyRIII (e.g., CD16a).
- FcyRI e.g., CD64
- FcyRII e.g., CD32
- FcyRIII e.g., CD16a
- the Fc region may include a mutation that increases or decreases any one or any combination of these functions.
- the Fc domain comprises Fc region comprises one or more mutations selected from N297A, N297Q,
- the Fc domain comprises a FAFA mutation (e.g., equivalent to F234A, F235A according to Rabat numbering) which reduces effector function.
- the Fc domain comprises a FAFA-PG mutation (e.g., equivalent to F234A, F235A, P329G according to Rabat numbering) which reduces effector function.
- the Fc domain mediates serum half-life of the protein complex, and a mutation in the Fc domain can increase or decrease the serum half-life of the protein complex.
- the Fc domain affects thermal stability of the protein complex, and mutation in the Fc domain can increase or decrease the thermal stability of the protein complex.
- the Fc region comprises one or more mutations selected from M252Y, T256D, T307Q, T307W, M252Y, S254T, T256E, M428F, and N434S (e.g., according to Rabat numbering).
- the Fc region comprises the mutations M252Y, S254T, and T256E (YTE) (e.g., according to Rabat numbering).
- labeled refers to joinder antibodies and their antigen binding portions thereof that are unlabeled or joined to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
- detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
- a variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens). Any of the anti-PD-1 antibodies described herein can be unlabeled or can be joined to
- labeled refers to joinder thereof to a detectable label or moiety for detection.
- exemplary detectable labels or moieties include radioactive, colorimetric, antigenic, enzymatic labels/moieties, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
- a variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens). Any of the anti-spike protein antibodies described herein or antigen binding portions thereof that described herein can be unlabeled or can be joined to a detectable label or detectable moiety.
- a “neutralizing antibody” and related terms refers to an antibody that is capable of specifically binding to a target antigen (e.g., a coronavirus spike protein) and substantially inhibiting or eliminating the biological activity of the target antigen.
- a neutralizing antibody binds to a coronavirus and inhibits infection of susceptible cells by the coronavirus.
- An antibody that blocks binding of the coronavirus to a target cell is a neutralizing antibody as binding is required for infection of the target cell.
- a “neutralizing antibody”, an “antibody with neutralizing activity”, or “inhibitory antibody” is an antibody that neutralizes 100 times the tissue culture infectious dose required to infect 50% of cells (100 x TCID50) of a virus, for example, a SARS coronavirus, such as for example, SARS-CoV-1 or SARS-CoV-2.
- a “neutralizing antibody” is an antibody that neutralizes 200 times the tissue culture infectious dose required to infect 50% of cells (200 x TCID50) of a virus, for example, a SARS Coronavirus, such as SARS-CoV-1 or SARS-CoV-2.
- Neutralizing antibodies such as those disclosed herein are effective at antibody concentrations of less than 20 pg/ml, less than 15 pg/ml, less than 12.5 pg/ml, less than 10 pg/ml, less than 5 pg/ml, less than 3.5 pg/ml, less than 2 pg/ml or less than 1 mg/ml. In some preferred embodiments, neutralizing antibodies are effective at antibody concentrations of ⁇ 0.8 pg/ml. For the S7E5041 antibody, a concentration of 50 pg/ml corresponds to 333 nM.
- neutralizing antibodies are effective at antibody concentrations of less than 0.5 pg/ml and in some further preferred embodiments, neutralizing antibodies are effective at antibody concentrations of less than 0.2 pg/ml or less than 0.1 pg/ml.
- TCID50 or “median tissue culture infective dose” refers to the amount of vims necessary to infect 50% of cells in tissue culture.
- the lOOx and 200x refer to 100 and 200 times the TCID50 concentration of virus.
- the “percent identity” or “percent homology” and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences.
- the percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences.
- the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences.
- a comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm.
- the "percent identity” or “percent homology” of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters.
- Expressions such as “comprises a sequence with at least X% identity to Y” with respect to a test sequence mean that, when aligned to sequence Y as described above, the test sequence comprises residues identical to at least X% of the residues of Y.
- the amino acid sequence of a test antibody may be similar but not necessarily identical to any of the amino acid sequences of the polypeptides that make up any of the anti-spike protein antibodies, or antigen binding protein thereof, described herein.
- the similarities between the test antibody and the polypeptides can be at least 95%, or at or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, to any of the polypeptides that make up any of the anti-spike protein antibodies, or antigen binding protein thereof, described herein.
- similar polypeptides can contain amino acid substitutions within a heavy and/or light chain.
- the amino acid substitutions comprise one or more conservative amino acid substitutions.
- a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
- R group side chain
- a conservative amino acid substitution will not substantially change the functional properties of a protein.
- the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference in its entirety.
- Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
- a "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene).
- Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell).
- Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector.
- Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct.
- Expression vectors can include one or more origin of replication sequences. Vectors can be single-stranded or double-stranded nucleic acid molecules.
- Vectors can be linear or circular nucleic acid molecules.
- One type of vector is a "plasmid,” which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and/or translating the transgene.
- a viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modified to disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome.
- viral vectors examples include retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors.
- Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors).
- Other vectors e.g., non- episomal mammalian vectors
- An "expression vector” is a type of vector that can contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers. Expression vectors can include ribosomal binding sites and/or polyadenylation sites. Expression vectors can include one or more origin of replication sequences. Regulatory sequences direct transcription, or transcription and translation, of a transgene linked to or inserted into the expression vector which is transduced into a host cell. The regulatory sequence(s) can control the level, timing and/or location of expression of the transgene.
- the regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and/or the nucleic acid). Regulatory sequences can be part of a vector. Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606.
- a transgene is “operably linked” to a regulatory sequence (e.g., a promoter) when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.
- a regulatory sequence e.g., a promoter
- transfected or transformed or transformed or transduced or other related terms used herein refer to a process by which exogenous nucleic acid (e.g., transgene) is transferred or introduced into a host cell, such as an antibody production host cell.
- a "transfected” or “transformed” or “transduced” host cell is one which has been introduced with exogenous nucleic acid (transgene).
- the host cell includes the primary subject cell and its progeny. Exogenous nucleic acids encoding at least a portion of any of the anti-spike protein antibodies described herein can be introduced into a host cell.
- Expression vectors comprising at least a portion of any of the anti- spike protein antibodies described herein can be introduced into a host cell, and the host cell can express polypeptides comprising at least a portion of the anti- spike protein antibody.
- a host cell can be a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.
- the phrase “transgenic host cell” or “recombinant host cell” can be used to denote a host cell that has been introduced (e.g., transduced, transformed or transfected) with a nucleic acid either to be expressed or not to be expressed.
- a host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- host cell or “or a population of host cells” or related terms as used herein may refer to a cell (or a population thereof or a plurality of host cells) to be used for production of the antibody or fragment thereof, is a cell or cells into which foreign (exogenous or transgene) nucleic acids have been introduced, for example, to direct production of the anti- spike protein antibody by the production host cell.
- the foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and/or polypeptide encoded by the foreign nucleic acid (transgene).
- a host cell (or a population thereof) can be a cultured cell, can be extracted from a subject, or can be the cell of an organism, including a human subject.
- the host cell (or a population of host cells) includes the primary subject cell and its progeny without any regard for the number of generations or passages.
- the host cell (or a population thereof) includes immortalized cell lines. Progeny cells may or may not harbor identical genetic material compared to the parent cell.
- a production host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and/or manipulated in any way to express an antibody, as disclosed herein.
- the host cell (or population thereof) can be transfected or transduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, or an antigen binding portion thereof, as described herein.
- Production host cells and populations thereof can harbor an expression vector that is stably integrated into the host’s genome or can harbor an extrachromosomal expression vector.
- host cells and populations thereof can harbor an extrachromosomal vector that is present after several cell divisions or is present transiently and is lost after several cell divisions.
- the disclosure may use the term “host cell” or “host cells” to refer to a cell or cells that are infected with a vims (such as a coronavirus), cells capable of being infected by a vims (e.g., lung cells of a subject), or cells used in assays or experiments testing their ability to be infected by a vims.
- a vims such as a coronavirus
- Other terms for virally-infected cells, cells capable of being infected by a vims, or cells used in assays that include viral infection procedures may include, as nonlimiting examples, “target cells”, “susceptible cells”, “test cells”, “vims propagating cells”, “infected cells”, and the like.
- subject refers to human and non-human animals, including vertebrates, mammals and non-mammals.
- the subject can be human, non-human primates, simian, ape, murine (e.g., mice), bovine, porcine, equine, canine, feline, caprine, lupine, ranine, or piscine.
- administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
- exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
- parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
- the formulation is administered via a non-parenteral route, e.g., orally.
- non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
- Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
- Any of the anti- spike protein antibodies described herein (or antigen binding protein thereof) can be administered to a subject using art-known methods and delivery routes.
- an antigen binding protein e.g., any of the anti-spike protein antibodies described herein or antigen binding protein thereof
- therapeutically effective amounts of antibodies provided herein, when used alone or in combination will vary depending upon the relative activity of the antibodies and combinations (e.g. , in inhibiting cell growth) and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
- a therapeutically effective amount will depend on certain aspects of the subject to be treated and the disorder to be treated and may be ascertained by one skilled in the art using known techniques.
- the polypeptide is administered to a subject at about 0.01 g/kg - 50 mg/kg per day, about 0.01 mg/kg - 30 mg/kg per day, or about 0.1 mg/kg - 20 mg/kg per day.
- the polypeptide may be administered daily (e.g., once, twice, three times, or four times daily) or less frequently (e.g., weekly, every two weeks, every three weeks, monthly, or quarterly).
- adjustments for age as well as the body weight, general health, sex, diet, time of administration, drug interaction, and the severity of the disease may be necessary.
- the present disclosure provides methods for treating a subject testing positive for a coronavirus infection, such as an infection with SARS-CoV or SARS-CoV-2.
- a coronavirus infection such as an infection with SARS-CoV or SARS-CoV-2.
- the present disclosure also provides methods for treating a subject suspected of being infected or at risk of being infected with a coronavirus, such as SARS-CoV or SARS-CoV-2.
- the present disclosure provides nucleic acid molecules, such as vectors, encoding one or more polypeptides of an antigen-binding protein as disclosed herein that specifically binds the S protein of a coronavirus.
- the one or more nucleic acid molecules, such as one or more vectors encode an antigen-binding protein as disclosed herein that specifically binds the S protein of a coronavirus.
- the present disclosure provides a composition that includes a first nucleic acid molecule encoding a first polypeptide comprising a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO:9, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 10, and a second nucleic acid molecule encoding a second polypeptide comprising the light chain variable region having a light chain CDR1 having the amino acid sequence of SEQ ID NO: 11, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 13.
- the first and second nucleic acid molecules can encode a heavy chain and light chain of an antibody, for example.
- the disclosure provides a single nucleic acid molecule encoding a first polypeptide that comprises a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:8, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO:9, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 10, and a second polypeptide comprising the light chain variable region having a light chain CDR1 having the amino acid sequence of SEQ ID NO: 11, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 13.
- the disclosure provides a nucleic acid molecule that encodes a polypeptide having a heavy chain CDR1 having the amino acid sequence of SEQ ID NO:8, a heavy chain CDR2 region having the amino acid sequence of SEQ ID NO:9, and a heavy chain CDR3 region having the amino acid sequence of SEQ ID NO: 10, a light chain CDR1 having the amino acid sequence of SEQ ID NO: 11, a light chain CDR2 region having the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 region having the amino acid sequence of SEQ ID NO: 13.
- An antigen binding protein encoded by one or more nucleic acid molecules as provided herein can be, as nonlimiting examples, an IgG or a single chain antibody.
- the present disclosure provides a first nucleic acid molecule encoding a first polypeptide comprising a heavy chain variable region having a heavy chain variable region having at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:6, and a second nucleic acid molecule encoding a second polypeptide comprising the light chain variable region having at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO:7.
- the first and second nucleic acid molecules can encode a heavy chain and light chain of an antibody, for example.
- the disclosure provides a single nucleic acid molecule encoding a first polypeptide that comprises a heavy chain variable region having a heavy chain CDR1 having an amino acid having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:6, and a second polypeptide comprising a light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:7.
- the disclosure provides a nucleic acid molecule that encodes a polypeptide having a heavy chain amino acid sequence having at least 95% identity to SEQ ID NO:6 and a light chain amino acid sequence having at least 95% identity to SEQ ID NO:7.
- An antigen-binding protein encoded by one or more nucleic acid molecules as provided herein can be, as nonlimiting examples, an IgG or a single chain antibody.
- a nucleic acid molecule encoding an antigen-binding protein that specifically binds the S protein of a coronavirus as disclosed herein can be an expression vector that includes a promoter operably linked to the protein-encoding sequence.
- a promoter operably linked to the polypeptide-encoding sequence(s) can be a eukaryotic or prokaryotic promoter but is preferably a eukaryotic promoter that is active in a mammalian cell.
- the expression vector(s) can direct transcription and/or translation of the transgene in the host cell and can include ribosomal binding sites and/or polyadenylation sites.
- the polypeptide(s) that include heavy and light chain sequences as disclosed above can be displayed on the surface of the transgenic host cell or secreted into the cell culture medium.
- the host cell, or population of host cells harbor one or more expression vectors that can direct transient introduction of the transgene into the host cells or stable insertion of the transgene into the host cells’ genome, where the transgene comprises nucleic acids encoding any of the first and/or second polypeptides described herein.
- nucleic acid molecule encodes two polypeptides (e.g., a first polypeptide having homology to SEQ ID NO:6 and a second polypeptide having homology to SEQ ID NO:7)
- the two polypeptide encoding sequences can be regulated by the same promoter and can be linked by an IRES or 2A sequence (Shao et al. (2009) Cell Research 19:296-306) or the two polypeptide-encoding sequences can be operably linked to different promoters.
- a production host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.
- a prokaryote for example, E. coli
- a eukaryote for example, a single-celled eukaryote (e.g., a yeast or other fungus)
- a plant cell e.g., a tobacco or tomato plant cell
- a mammalian cell e.g., a human cell, a monkey cell, a hamster cell, a rat
- a production host cell can be transfected with an expression vector operably linked to a nucleic acid encoding a desired antigen-binding protein thereby generating a transfected/transformed host cell which is cultured under conditions suitable for expression of the antigen-binding protein by the transfected/transformed host cell, and optionally recovering the antibody from the transfected/transformed host cells (e.g., recovery from host cell lysate) or recovery from the culture medium.
- production host cells comprise non-human cells including CHO, BHK, NSO, SP2/0, and YB2/0.
- host cells comprise human cells including HEK293, HT-1080, Huh-7 and PER.C6.
- host cells examples include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum- free media (see Rasmussen et ah, 1998, Cytotechnology 28:31) or CHO strain DX-B 11, which is deficient in DHFR (see Urlaub et ah, 1980, Proc. Natl. Acad. Sci.
- COS-7 line of monkey kidney cells ATCC CRL 1651
- L cells C127 cells
- 3T3 cells ATCC CCL 163
- CHO Chinese hamster ovary
- HeLa cells can be lymphoid cells such as Y0, NSO or Sp20.
- a host cell is a mammalian host cell, but is not a human host cell.
- Polypeptides of the present disclosure can be produced using any methods known in the art.
- the polypeptides are produced by recombinant nucleic acid methods by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector which is introduced into a host cell and expressed by the host cell under conditions promoting expression.
- a nucleic acid sequence e.g., DNA
- the recombinant DNA can also encode any type of protein tag sequence that may be useful for purifying the protein.
- protein tags include but are not limited to a histidine (his) tag, a FLAG tag, a myc tag, an HA tag, or a GST tag.
- His histidine
- FLAG FLAG
- myc myc
- HA HA
- GST GST
- the expression vector construct can be introduced into a host cell, e.g., a production host cell, using a method appropriate for the host cell.
- a host cell e.g., a production host cell
- methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; non-viral transfection; microprojectile bombardment; lipofection; and infection (e.g., where the vector is a viral vector).
- Suitable bacteria include gram negative or gram positive organisms, for example, E. coli or Bacillus spp. Yeast, for example from the Saccharomyces species, such as S. cerevisiae, may also be used for production of polypeptides.
- Saccharomyces species such as S. cerevisiae
- Various mammalian or insect cell culture systems can also be employed to express recombinant proteins.
- Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, ( Bio/Technology , 6:47, 1988).
- suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines.
- Purified polypeptides are prepared by culturing suitable host/vector systems to express the recombinant proteins.
- E. coli host cells are suitable for expressing small polypeptides. The protein can then be purified from culture media or cell extracts.
- Antibodies and antigen binding proteins disclosed herein can also be produced using cell-translation systems.
- the nucleic acids encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the particular cell-free system being utilized (eukaryotic such as a mammalian or yeast cell-free translation system or prokaryotic such as a bacterial cell-free translation system).
- Nucleic acids encoding any of the various polypeptides disclosed herein may be synthesized chemically or using gene synthesis methods (available for example through commercial entities such as Blue Heron, DNA 2.0, GeneWiz, etc.). Codon usage may be selected so as to improve expression in a cell. Such codon usage will depend on the production host cell type. Specialized codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et ah, Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002 (1):96-105; Connell N D. Ciirr. Opin. Biotechnol. 2001 12(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.
- Antibodies and antigen binding proteins described herein can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications to the protein can also be produced by chemical synthesis.
- Antibodies and antigen binding proteins described herein can be purified by isolation/purification methods for proteins generally known in the field of protein chemistry.
- Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations of these.
- polypeptides may be exchanged into different buffers and/or concentrated by any of a variety of methods known to the art, including, but not limited to, filtration and dialysis.
- the purified antibodies and antigen binding proteins described herein can be at least 65% pure, at least 75% pure, at least 85% pure, at least 95% pure, or at least 98% pure. Regardless of the exact numerical value of the purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any of the anti-spike protein antibodies, or antigen binding protein thereof, described herein can be expressed by transgenic host cells and then purified to about 65-98% purity or high level of purity using any art-known method.
- the antibodies and antigen binding proteins herein can further comprise post-translational modifications.
- post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation or addition of a polypeptide side chain or of a hydrophobic group.
- the modified polypeptides may contain non-amino acid elements, such as lipids, poly- or mono-saccharide, and phosphates.
- a form of glycosylation can be sialylation, which conjugates one or more sialic acid moieties to the polypeptide.
- Sialic acid moieties improve solubility and serum half- life while also reducing the possible immunogenicity of the protein. See Rajuetal. Biochemistry 2001 31; 40:8868-76.
- the antibodies and antigen binding proteins described herein can be modified to increase their solubility and/or serum half-life which comprises linking the antibodies and antigen binding proteins to non-pro teinaceous polymers.
- polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes can be conjugated to antigen-binding proteins, for example in the manner as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.
- polyethylene glycol or “PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented by the formula: X — 0(CH 2 CH 2 0) n — CH 2 CH 2 OH (1), where n is 20 to 2300 and X is H or a terminal modification, e.g., a C 1-4 alkyl.
- the PEG terminates on one end with hydroxy or methoxy, i.e., X is H or CH 3 (“methoxy PEG”).
- a PEG can contain further chemical groups which are necessary for binding reactions; which results from the chemical synthesis of the molecule; or which is a spacer for optimal distance of parts of the molecule.
- a PEG can consist of one or more PEG side-chains which are linked together.
- PEGs with more than one PEG chain are called multiarmed or branched PEGs.
- Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol.
- Branched PEG molecules are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462.
- One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et ah, Bioconjugate Chem. 6 (1995) 62-69).
- kits comprising any of the anti-spike protein antibodies, antigen-binding proteins, kits, including “kits” comprising a first antigen-binding protein and a second antigen-binding protein described herein and throughout, and a pharmaceutically acceptable excipient.
- An excipient encompasses carriers and stabilizers.
- the pharmaceutical compositions comprise an anti-spike protein antibody or antigen binding protein as disclosed herein, comprising a heavy chain variable region with an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6 (the heavy chain variable region of antibody S7E5041) and an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:7 (the light chain variable region of antibody S7E5041).
- a pharmaceutical composition can comprise an anti-spike protein antibody or antigen binding protein as disclosed herein that comprises a heavy chain variable region comprising a heavy chain CDR1 sequence having the amino acid sequence of SEQ ID NO:8, a heavy chain CDR2 sequence having the amino acid sequence of SEQ ID NO:9, and a heavy chain CDR3 sequence having the amino acid sequence of SEQ ID NO: 10, and further comprises a light chain variable region comprising a light chain CDR1 sequence having the amino acid sequence of SEQ ID NO: 11, a light chain CDR2 sequence having the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 sequence having the amino acid sequence of SEQ ID NO: 13.
- the pharmaceutical compositions can be produced to be sterile and stable under the conditions of manufacture and storage.
- the antigen-binding proteins provided herein can be in powder form, for example for reconstitution in the appropriate pharmaceutically acceptable excipient before or at the time of delivery.
- the antigen-binding proteins can be in solution with an appropriate pharmaceutically acceptable excipient or a pharmaceutically acceptable excipient can be added and/or mixed before or at the time of delivery, for example to provide a unit dosage injectable or inhalable form.
- the pharmaceutically acceptable excipient used in the present invention is suitable to high drug concentration, can maintain proper fluidity and, in some embodiments, can delay absorption.
- Examples of pharmaceutically acceptable excipients includes for example inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Additional examples include buffering agents, stabilizing agents, preservatives, non ionic detergents, anti-oxidants and isotonifiers.
- inert diluents or fillers e.g., sucrose and sorbitol
- lubricating agents e.g., glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).
- anti-adhesives e.g., magnesium stearate, zinc stearate, stearic
- Therapeutic compositions and methods for preparing them are well known in the art and are found, for example, in “Remington: The Science and Practice of Pharmacy” (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.).
- Therapeutic compositions can be formulated for parenteral administration may, and can for example, contain excipients, sterile water, saline, poly alky lene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
- Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the antibody (or antigen binding protein thereof) described herein.
- Nanoparticulate formulations e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes
- Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
- the concentration of the antibody (or antigen binding protein thereof) in the formulation varies depending upon a number of factors, including the dosage of the drug to be administered, and the route of administration.
- Any of the anti-spike protein antibodies disclosed herein (or antigen binding portions thereof) may be optionally administered as a pharmaceutically acceptable salt, such as non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry.
- acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like.
- Metal complexes include zinc, iron, and the like.
- the antibody (or antigen binding portions thereof) is formulated in the presence of sodium acetate to increase thermal stability.
- any of the anti-spike protein antibodies disclosed herein may be formulated for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients.
- Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.
- Kits comprising first and second antigen-binding proteins and uses therefore [00163]
- kits comprising a first antigen-binding protein as described herein and throughout, and a second a second antigen-binding protein as described herein and throughout.
- such “kits” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may therefore be used or administered to subjects separately.
- such a first antigen-binding protein and such a second antigen-binding protein may be independently obtained or prepared simultaneously or at different points in time, and be different persons or entities, and still constitute a “kit” as used herein.
- a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a CDRH1, and/or a CDRH2, and/or CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a CDRL1, and/or a CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively, and a CDRL1, CDRL2, and/or CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a heavy chain variable region comprising a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a light chain variable region comprising a CDRL1, and/or a CDRL2, and/or CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a heavy chain variable region comprising: a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a second antigen binding protein comprising a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises a second antigen binding protein comprising a light chain variable region comprising having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises: a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28; and a light chain variable region comprising having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the second antigen-binding protein of such a “kit” comprises antibody S1D7270 or antibody STI-2020 (see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes).
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are not in contact or otherwise mixed together, and may nonetheless be used or administered separately or, alternatively, simultaneously, to subjects.
- such a “kit” may comprise such a first antigen-binding protein and such a second antigen-binding protein, wherein the first antigen-binding protein and the second antigen-binding protein are placed in contact or otherwise mixed together, and may be used or administered simultaneously, to subjects.
- the first antigen-binding protein and the second antigenbinding protein of such “kits” are provided and/or are administered to subjects in a single composition.
- the first antigen-binding protein and the second antigenbinding protein of such “kits” are provided and/or are administered to subjects as separate compositions.
- such “kits” can further include one or more sterile pharmaceutically acceptable solutions for resuspension or dilution of the the first antigenbinding protein and/or the second antigen-binding protein, and may include one or more additional pharmaceutical formulations, which may be, as nonlimiting examples, any of an additional antibody, an analgesic, an antibiotic, an anti-inflammatory drug, a bronchodilator, or an antiviral drug.
- the “kit” can be used for treating a subject having a coronavims infection, or for providing prophylaxis against infection with a coronavims.
- the components of the “kit” of can be provided in suitable containers and labeled for diagnosis, prophylaxis and/or treatment of coronavims infection.
- the above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution.
- the containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
- the “kit” may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, HBSS, Tyrode’s solution, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts.
- a pharmaceutically acceptable buffer such as phosphate-buffered saline, HBSS, Tyrode’s solution, Ringer's solution, or dextrose solution.
- a pharmaceutically acceptable buffer such as phosphate-buffered saline, HBSS, Tyrode’s solution, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts.
- the first antigen-binding protein and/or the second antigen-binding protein can be provided as a dry powder, which may be formulated with one or more suitable excipients, or as a liquid formulation.
- the “kit” can further include: solutions for resuspension or dilution of the the first antigen-binding protein and/or the second antigenbinding protein and a means for dispensing the pharmaceutical composition comprising the antibody into a nebulizer or metered dose inhaler.
- the “kit” may in some embodiments include a metered dose inhaler.
- the “kit” can be used for treating a subject having a coronavirus-associated infection or disease.
- kits can be instructions customarily included in commercial packages of therapeutic, prophylactic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic, prophylactic or diagnostic products.
- kit comprising an anti-S protein as disclosed herein.
- the kit comprises an antigen binding protein that specifically binds the S protein of a coronavirus as disclosed herein, such as an anti-S protein comprising a heavy chain variable region having at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to SEQ ID NO:6 and a light chain variable region having at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to SEQ ID NO:7.
- an antigen binding protein that specifically binds the S protein of a coronavirus as disclosed herein, such as an anti-S protein comprising a heavy chain variable region having at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to SEQ ID NO:6 and a light chain variable region having at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least
- the kit comprises an antigen binding protein that specifically binds the S protein of a coronavirus as disclosed herein, such as an anti-S protein comprising heavy chain CDRs having the amino acid sequences of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10, and further comprising light chain CDRs having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.
- the antibody can be provided in solution or as a solid, for example, a powder for reconstitution.
- compositions that include nucleic acid molecules that may be administered to a subject, such as a human subject for treatment or prevention of a coronavirus infection.
- a nucleic acid molecule that encodes a neutralizing antigen binding protein as provided herein can be an RNA molecule or a DNA molecule and can include one or more non-naturally occurring linkages (e.g., backbone linkages) or nucleobases.
- a nucleic acid molecule provided in a pharmaceutical composition can be, for example, a DNA molecule encoding a neutralizing antigen-binding protein as described hererinabove.
- a pharmaceutical composition can include one or more nucleic acid molecules, for example, can include a nucleic acid molecule that encodes a heavy chain of an antibody and a second nucleic molecule that encodes a light chain of an antibody, or a pharmaceutical composition can include a single nucleic acid construct that includes two open reading frames or genes, each operably linked to its own promoter, for example, encoding a light chain of an antibody and a heavy chain of an antibody.
- the composition is formulated for intramuscular injection, and a first gene encoding a light chain of an antibody and a second gene encoding a heavy chain of the antibody are each independently linked to a promoter active in muscle cells.
- the heavy and light chain genes can be on the same or different nucleic acid molecules.
- the nucleic acid molecule(s) in the pharmaceutical composition can be a plasmid, for example, a nanoplasmid having fewer than 500 base pairs of sequence of a bacterial plasmid, ten or fewer, five or fewer, or three or fewer CpG sequences, and/or can lack an antibiotic resistance marker such as any disclosed in US 9,550,998; US 10,047,365; or US 10,844,388, all of which are incorporated herein by reference in their entireties.
- a pharmaceutical composition that comprises one or more nucleic acid molecules as provided herein can include compounds that enhance delivery of nucleic acid molecules into cells, such as for example a cationic lipid or amphiphilic block copolymers, for example, linear and/or X-shaped copolymers, and can include one or more poloxamers or poloxamines, or of any of an ethylene oxide/propylene oxide copolymer, synperonics®, pluronics®, kolliphor®, poloxamer 181, poloxamer 188, or poloxamer 407, poloxamines, tetronics®, T/908, or T/1301, for example.
- compounds that enhance delivery of nucleic acid molecules into cells such as for example a cationic lipid or amphiphilic block copolymers, for example, linear and/or X-shaped copolymers, and can include one or more poloxamers or poloxamines, or of any of an ethylene oxide/propylene oxide copolymer, syn
- the pharmaceutical composition can further include any of alginate, or a PEG polymer or copolymer, e.g., DSPE-PEG, as nonlimiting examples, and can be formulated for injection and can include a buffer such as PBS, TBS, HBSS, Ringer’s, or Tyrode’s.
- the pharmaceutical composition that comprises one or more nucleic acid molecules can be formulated for injection and can be provided in a vial or other container as a liquid solution or solid (e.g., a lyophilate).
- the present disclosure provides methods for treating a subject having a coronavirus infection or suspected of having a coronavirus infection, the method comprising: administering to the subject an effective amount of a therapeutic composition comprising an anti-Sl antigen-binding protein as described herein, e.g., an antibody as described herein.
- the (suspected or actual) coronavirus infection is a SARS-CoV-2 infection.
- the subject can be a human subject or an animal.
- the subject may be a subject who has tested positive for the coronavirus, a subject who has had close and/or prolonged contact with another individual or animal that has tested positive for coronavirus, and/or can be a subject exhibiting symptoms associated with coronavirus infection.
- the antigen-binding protein or antibody is preferably a fully human neutralizing antigen-binding protein or antibody and may be a fully human neutralizing antibody having one or more mutations in the Fc region that result in reduced Fc effector function.
- the subject in infected with or suspected of being infected with HCoV-NL63, SARS-CoV, or SARS-CoV- 2 for example, the subject may be a human subject infected with or suspected of being infected with SARS-CoV-2.
- the present disclosure also provides methods for preventing a coronavirus infection in a subject, the method comprising: administering to a subject at risk of becoming infected with a coronavirus an effective amount of a therapeutic composition comprising an anti-S 1 antigen-binding protein as described herein, which may be an antibody or antibody fragment as described herein.
- the subject can be a human subject or an animal.
- the subject may be a health care worker, a first responder, a transportation worker, a delivery person, a worker in a meat-packing plant, or a warehouse.
- the subject may be an incarcerated subject in a jail or prison.
- the subject may be a person in an assisted living facility.
- the subject may live in an area with a high rate of increase of people testing positive for the coronavirus.
- the antigen-binding protein or antibody is preferably a fully human neutralizing antigen-binding protein or antibody as disclosed herein and may be a fully human neutralizing antibody having one or more mutations in the Fc region that result in reduced Fc effector function.
- the subject is at risk of being infected with HCoV-NL63, SARS-CoV, or SARS-CoV-2, for example, the subject may be a human subject at risk of becoming infected with SARS-CoV-2.
- administration of the antigen binding protein that specifically binds the S protein of a coronavirus can be by oral delivery.
- Oral dosage forms can be formulated for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules, soft gelatin capsules, syrups or elixirs, pills, dragees, liquids, gels, or slurries.
- formulations can include pharmaceutically excipients including, but not limited to, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as com starch or alginic acid; binding agents such as starch, gelatin or acacia; lubricating agents such as calcium stearate, glyceryl behenate, hydrogenated vegetable oils, magnesium stearate, mineral oil, polyethylene glycol, sodium stearyl, fumarate, stearic acid, talc, zinc stearate; preservatives such as n-propyl-p- hydroxybenzoate; coloring, flavoring or sweetening agents such as sucrose, saccharine, glycerol, propylene glycol or sorbitol; vegetable oils such as arachis oil, olive oil, sesame oil or coconut oil; mineral oils such as liquid paraffin; wetting agents such as benzalkonium chloride, docu
- administration can be by injection or intravenous or intra-arterial delivery, and may be, for example, by epidermal, intradermal, subcutaneous, intramuscular, intraperitoneal, intrapleural, intra-abdominal, or intracavitary delivery.
- Formulations for parenteral administration can be inter alia in the form of aqueous or non-aqueous isotonic sterile non-toxic injection or infusion solutions or suspensions.
- Preferred parenteral administration routes include intravenous, intra-arterial, intraperitoneal, epidural, and intramuscular injection or infusion.
- Intravenous delivery can be by infusion or by bolus (“push”) injection.
- the solutions or suspensions may comprise agents that are non-toxic to recipients at the dosages and concentrations employed such as 1,3-butanediol, Ringer's solution, Hank's solution, isotonic sodium chloride solution, oils such as synthetic mono- or diglycerides or fatty acids such as oleic acid, local anesthetic agents, preservatives, buffers, viscosity or solubility increasing agents, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like, oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like, and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
- an antibody (or antibody fragment) that specifically binds an epitope of a coronavirus S 1 subunit can be incorporated into a pharmaceutical composition suitable for pulmonary administration to a subject.
- an antibody as provided herein antibody that specifically binds an epitope of a coronavirus S 1 subunit can be formulated into a liquid pharmaceutical composition that includes a pharmaceutical excipient, where pharmaceutical composition is suitable for inhalation by a subject.
- the neutralizing antibody can comprise a heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6 and can comprise a light chain variable region having 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:7.
- the neutralizing antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO:6 or a sequence having at least 99% identity to SEQ ID NO:6.
- the antigen-binding proteins have a heavy chain CDR1 sequence of SEQ ID NO:8, a heavy chain CDR2 sequence of SEQ ID NO:9, a heavy chain CDR3 sequence of SEQ ID NO:10, a light chain CDR1 sequence of SEQ ID NO:ll, a light chain CDR2 sequence of SEQ ID NO: 12, and a light chain CDR3 sequence of SEQ ID NO: 13.
- the neutralizing antibody comprises a light chain variable region comprising the sequence of SEQ ID NO:7 or a sequence having at least 99% identity to SEQ ID NO:7.
- the neutralizing antibody can be an immunoglobulin molecule than optionally includes one or more mutations in the Fc region, for example one or both of a LALA mutation and a YTE mutation as described hereinabove.
- a liquid composition that comprises an anti-neutralizing antibody that specifically binds an epitope of a coronavirus S protein as disclosed herein formulated for pulmonary administration comprises a neutralizing antibody formulated into a solution or suspension, e.g., an isotonic saline solution, which is optionally buffered, at an appropriate concentration for pulmonary administration as an aerosol, mist, or vapor.
- a solution or suspension that includes the neutralizing antibody is isotonic with respect to pulmonary fluids and of about the same pH, for example, has a pH of from about pH 4.0 to about pH 8.5 or from pH 5.5 to pH 7.8, or, for example, from about 7.0 to about 8.2.
- Suitable buffering agents that can be present in a liquid pharmaceutical composition for pulmonary delivery include, but are not limited to, citrate buffer, phosphate buffer, and succinate buffer.
- imidazole, histidine, or another compound that maintains pH in the range of about pH 4.0 to about 8.5 can be used.
- Ringer’s solution, isotonic sodium chloride, and phosphate buffered saline may be used.
- One of skill in the art can determine an appropriate saline content and pH for an aqueous solution for pulmonary administration.
- Additional compounds that may be present in a liquid formulation for pulmonary delivery include, without limitation, sugars, sugar alcohols, alcohols (e.g., benzyl alcohol), polyols, amino acids, salts, polymers, surfactants, and preservatives (e.g., ethyl or n-propyl p- hydroxybenzoate).
- Other possible ingredients include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone, and gum tragacanth, and a wetting agent such as lecithin.
- the compositions can include any of a variety of compounds to aid in solubility, stability, or delivery, where the added compounds do not negatively affect the coronavirus S -protein binding activity of the neutralizing antibody.
- a liquid pharmaceutical composition for pulmonary delivery of an SI -binding neutralizing antibody as provided herein may include an excipient or stabilizer including but not limited to a sugar, alcohol, sugar alcohol, or an amino acid.
- Preferred sugars include sucrose, trehalose, raffinose, stachyose, sorbitol, glucose, lactose, dextrose, or any combination thereof.
- a sugar can optionally be present in the range of about 0% to about 9.0% (w/v), preferably about 0.5% to about 5.0%, for example about 1.0%.
- An amino acid for example, can be optionally be present in the range of about 0% to about 1.0% (w/v), preferably about 0.3% to about 0.7%, for example about 0.5%.
- a buffering agent such as phosphate, citrate, succinate, histidine, imidazole, or Tris can also optionally be present in the liquid neutralizing antibody formulation.
- EDTA may be present as a stabilizer.
- Any of various surfactants may also be present, such as for example, polyoxyethylene sorbitol esters such as polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20); polyoxypropylene-polyoxyethylene esters such as Poloxamer 188; polyoxyethylene alcohols such as Brij 35; a mixture of polysorbate surfactants with phospholipids (such as phosphatidylcholine and derivatives), dimyristolglycerol and other members of the phospholipid glycerol series; lysophosphatidylcholine and derivatives thereof; mixtures of polysorbates with lysolecithin or cholesterol; bile salts and their derivatives such as sodium cholate, sodium deoxycholate, sodium glycodeoxycholate, sodium
- Suitable surfactants include L-alpha- phosphatidylcholine dipalmitoyl ("DPPC"), diphosphatidyl glycerol (DPPG), 1,2- Dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), l,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DSPC), l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1- palmitoyl-2-oleoylphosphatidylcholine (POPC), fatty alcohols, polyoxyethylene-9-lauryl ether, surface active fatty acids, sorbitan trioleate (Span 85), glycocholate, surfactin, poloxomers, sorbitan fatty acid esters, tyloxapol, phospholipids, and alkylated sugars.
- DPPC L-alpha- phosphatidylcholine dipalmitoyl
- Such pharmaceutical compositions may be administered for example, as a propellant-free inhalable solution comprising a soluble S protein-binding neutralizing antibody and may be administered to the subject via a nebulizer.
- suitable preparations include, but are not limited to, mist, vapor, or spray preparations so long as the particles comprising the protein composition are delivered in a size range consistent with that described for the delivery device.
- compositions are preferably sterile and stable under the conditions of manufacture and storage.
- the formulation can be formulated as a solution, microemulsion, dispersion, or suspension.
- Sterile inhalable solutions can be prepared by incorporating the active compound (i.e., a soluble S 1-binding neutralizing antibody as provided herein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. Fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants.
- the concentration of S protein-binding neutralizing antibody in a liquid formulation for pulmonary delivery can range for example, from about 1 pg per ml to about 500 mg per ml, and may be in the range of, for example, from about 10 pg per ml to about 200 mg per ml, or from about 20 pg per ml to about 100 mg per ml, although these ranges are not limiting.
- a pharmaceutical composition as provided herein is formulated for nasal delivery.
- “Nasal delivery” is used herein to refer to deposition of the pharmaceutical composition within one or preferably both nares (nostrils or nasal passages) of a subject; “nasal delivery” and “intranasal delivery” are used interchangeably herein.
- Nasal delivery may be topical administration, i.e., deposition or application of a liquid, gel, paste, powder, or particles within the nasal passages, for example using a dropper, squeeze bottle, or applicator.
- nasal delivery may be by inhalation, including by means of a dry powder inhaler, metered-dose inhaler, or nebulizer that generates aerosols for delivery of particles or droplets to the lung.
- a dry powder inhaler metered-dose inhaler
- nebulizer that generates aerosols for delivery of particles or droplets to the lung.
- Contemplated herein are methods of topical nasal delivery that do not require an inhalation device such as a dry powder inhaler, metered dose inhaler, or nebulizer, although the formulations and methods provided herein are not limited to such methods.
- the subject may use a dropper or squeeze bottle to deposit one, two, or more drops of a pharmaceutical composition for nasal delivery in one or both nostrils.
- the user (or another person) may administer the nasal drops when the subject is lying down, or has the head tilted back to avoid having the composition drain from the nostrils.
- the subject may remain supine (or with head tilted back) for several seconds to two minutes, for example.
- a coronavirus-neutralizing antigen-binding protein as provided herein can be formulated with any suitable excipient(s) for nasal delivery.
- suitable excipient(s) for nasal delivery Reference may be made to standard handbooks, such as for example Remington's Introduction to the Pharmaceutical Sciences, 2nd Ed., Lippincott Williams and Wilkins, USA (2011) or Remington, the Science and Practice of Pharmacy, 23rd Edition, Academic Press (2020).
- a neutralizing antibody for nasal administration is formulated as a composition or pharmaceutical composition comprising at least a therapeutically effective amount of a neutralizing antibody, such as a neutralizing antibody disclosed herein, and at least one pharmaceutically acceptable nasal carrier, and optionally one or more additional pharmaceutically acceptable additives and/or agents.
- a “nasal carrier” as set forth in the present invention is a carrier that is suitable for application through the nasal route, i.e. deposition within the nostril or application to the nasal mucosa.
- the nasal carrier may be a solid, semi-liquid, or liquid filler, diluent, or encapsulating material, for example.
- the nasal composition can be provided in a variety of forms, including fluid or semi-liquid or viscous solutions, gels, creams, pastes, powders, microspheres, and films for direct application to the nasal mucosa including application as liquid drops into the nasal passage.
- the nasal carrier should be "pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not eliciting an unacceptable deleterious effect in the subject.
- Preferred nasal carriers are those which maintain the solubility of the neutralizing antibody (when applied as a liquid or in a gel or paste) and those that improve the contact of the pharmaceutical composition with the nasal mucus or nasal mucosa.
- a carrier provided in the composition may also facilitate the diffusion of the antibody from the composition to the nasal mucosa and/or may prolong the nasal residence time of the composition allowing dissemination to the lungs via respiration, for example.
- Suitable nasal carriers are known to those skilled in the art of pharmacology.
- a carrier used in a composition for intranasal delivery can be a liquid or solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof.
- Other preferred liquid carriers are aqueous saline, e.g. physiological saline, or an aqueous buffer, e.g. a phosphate/citric acid buffer.
- compositions which can be soluble, in semi-solid or gelatinous form, sparingly soluble, or in solid form can include, without limitation, additional salts, one or more sugars, or polymers, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
- additional salts one or more sugars, or polymers, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
- Further carriers include polyacrylates, sodium carboxy methyl cellulose, starches and their derivatives, alginic acid and salts, hyaluronic acid and salts, pectic acid and salts, gelatin and its derivatives, gums, polylactic acid and its copolymers, polyvinyl acetate, celluloses and their derivatives, coated celluloses, crosslinked dextrans, polylactic acid and its copolymers, and polyvinyl acetate.
- Non-limiting examples of other solid nasal carriers are described in US 5,578,5674, WO 04/093917 and WO 05/120551, incorporated herein by reference.
- the neutralizing antibody may be released from the composition by diffusion or by disintegration of the nasal carrier.
- the neutralizing antibody is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, e.g., isobutyl 2-cyanoacrylate (see, e.g., Michael et al., J. Pharmacy Pharmacol. 1991; 43: 1-5).
- microspheres e.g., Michael et al., J. Pharmacy Pharmacol. 1991; 43: 1-5
- These particular microspheres not only demonstrate mucoadhesion properties, but also protect against enzymatic degradation. They may further allow manipulation of the rate of release of the neutralizing antibody or antibodies to provide sustained delivery and biological activity over a protracted time (Morimoto et al., Eur. J. Pharm. Sci. 2001 May; 13(2): 179-85).
- the pharmaceutical composition can include a bioadhesive nasal carrier, e.g., a compound that adheres to the nasal mucosa by chemical or physical binding such as Van der Waals interaction, ionic interaction, hydrogen bonding or by polymer chain entanglement.
- the adhesion may be to the epithelial (cellular) surface or to the mucus overlying the surface (a mucoadhesive).
- bioadhesive or mucoadhesive materials include, without limitation, carhopol, cellulose and cellulose derivatives (e.g., hydroxypropyl methylcellulose, hydroxypropylcellulose) or cellulose-containing compounds, coated cellulose (e.g., microcrystalline cellulose coated with glycerol monooleate) starch, dextran, and chitosan (See, for example, Ilium, Bioadhesive formulations for nasal peptide delivery. In: E Mathiowitz, D E Chickering III, C Lehr, eds. Bioadhesive Drug Delivery Systems. New York: Marcel Dekker, 1999: 507-541; EP 0490806; and WO 96/03142, all incorporated herein by reference).
- carhopol cellulose and cellulose derivatives (e.g., hydroxypropyl methylcellulose, hydroxypropylcellulose) or cellulose-containing compounds, coated cellulose (e.g., microcrystalline cellulose coated with gly
- formulations comprising neutralizing antibody may also contain a hydrophilic low molecular weight compound as a nasal carrier, base, or excipient.
- a hydrophilic low molecular weight compound as a nasal carrier, base, or excipient.
- Such hydrophilic low molecular weight compounds provide a passage medium through which a water-soluble active agent, such as the neutralizing antibody of the invention, may diffuse.
- hydrophilic low molecular weight compound examples include polyol compounds, such as oligo-, di- and monosaccarides such as sucrose, mannitol, lactose, L-arabinose, D-erytrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, and polyethylene glycol.
- polyol compounds such as oligo-, di- and monosaccarides such as sucrose, mannitol, lactose, L-arabinose, D-erytrose, D-ribose, D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose, glycerin, and polyethylene glycol.
- hydrophilic low molecular weight compounds useful as such carriers or bases include N- methylpyrrolidone, and alcohols (e.
- a carrier in a nasal formulation may further contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, stabilizers, tonicity adjustors, wetting agents or other biocompatible materials.
- pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, stabilizers, tonicity adjustors, wetting agents or other biocompatible materials.
- Examples of pharmaceutically acceptable preservatives include benzalkonium chloride, an alkyl p-hydroxybenzoate (paraben) such as methyl p- hydroxybenzoate and propyl p-hydroxybenzoate, or sodium methylmercurithiosalicylate (Thiomersal). Further non-limiting examples of pharmaceutically acceptable preservatives are described in U.S. Pat. No. 5,759,565, US 20100129354, and WO 04/093917.
- antioxidants examples include alkali metal sulfites, alkali metal bisulfites, alkali metal pyrosulfites, sodium thiosulfate, thiodipropionic acid, cysteine in free or salt form (such as cysteine hydrochloride), ascorbic acid, citraconic acid, propyl or ethyl gallate, nordihydroguaiaretic acid, butylated hydroxyanisole or -toluene, and tocol.
- Further non-limiting examples of pharmaceutically acceptable antioxidants are provided in US 20100129354, WO 04/093917, and WO 05/120551.
- the desired viscosity for the compositions of the invention will depend on the particular form for administration, e.g. whether administration is to be by nasal drops or nasal spray.
- an appropriate viscosity may be from about 2 to about 40 x 10 -3 Pa s, and for nasal sprays the viscosity may be less than 2 x 10 -3 Pa s, e.g. from 1 to 2 x 10 -3 Pa s.
- Such values are exemplary only, and acceptable viscosities for a therapeutic or prophylactic neutralizing antibody composition can be determined empirically.
- Examples of pharmaceutically acceptable compounds for enhancing viscosity include, for example, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, (which can also serve as mucoadhesives) sucrose, PVA, PVP, polyacrylic acid, or natural polymers. Further non-limiting examples of viscosity builders are described in US 5,578,567.
- Examples of pharmaceutically acceptable stabilizers include albumin, e.g. human serum albumin, aprotinin or d-aminocaproic acid.
- the activity or physical stability of proteins can also be enhanced by various additives to aqueous solutions of the neutralizing antibody or antibodies.
- additives such as polyols (including sugars), amino acids, and various salts may be used.
- Examples of pharmaceutically acceptable tonicity adjustors include nasally acceptable sugars, e.g. glucose, mannitol, sorbitol, ribose, mannose, arabinose, xylose or another aldose or glucosamine.
- Further non-limiting examples of tonicity adjustors are provided in US201GQ129354.
- Such additives may be used in suitable amounts as known in the art and as can be determined by the skilled person based on the disclosure and art, including art cited herein.
- Enzyme inhibitors may also be optionally added to the composition for nasal delivery to reduce the activity of any hydrolytic enzymes in the nasal mucosa that can potentially degrade the neutralizing antibody or other components of the pharmaceutical composition.
- Enzyme inhibitors that can reduce degradative activities for use within the invention can be selected from a wide range of non-protein inhibitors that vary in their degree of potency and toxicity (see, e.g., L. Stryer, Biochemistry, WI-1: Freeman and Company,
- Non-limiting examples include amastatin and bestatin (O'Hagan et ah, Pharm. Res. 1990, 7: 772-776).
- Various classes of enzyme inhibitors that may be considered are extensively described and exemplified in WO 05/120551.
- Another means to inhibit degradation is pegylation with PEG molecules, preferably low molecular weight PEG molecules (e.g. 2 kDa; Lee et ah, Calcif Tissue Int. 2003, 73: 545-549).
- the tonicity of the formulation is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the nasal mucosa at the site of administration.
- the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7.
- Liquid compositions of the invention further preferably have a mildly acid pH, e.g. from about pH 3 to about pH 6.5, from about pH 3.5 to about pH 6.5, or preferably from about pH 4.5 to about pH 6.5 to minimize nasal irritation (Betel et al. Adv. Drug Delivery Rev. 1998;
- the required degree of acidity may conveniently be achieved, e.g by the addition of a buffering agent, e.g. a mixture of citric acid and disodium hydrogen phosphate, or an acid such as HC1 or another appropriate mineral or an organic acid, e.g. phosphoric acid.
- Solid compositions may also comprise a buffering agent when they are prepared by lyophilization of a liquid composition buffered to a pH value as indicated above.
- a buffering agent e.g. a mixture of citric acid and disodium hydrogen phosphate
- an acid such as HC1 or another appropriate mineral or an organic acid, e.g. phosphoric acid.
- Solid compositions may also comprise a buffering agent when they are prepared by lyophilization of a liquid composition buffered to a pH value as indicated above.
- Non limiting examples of pharmaceutically acceptable buffering agents are provided in US20100129354, WO 04/093917, and WO 05/120551.
- a composition for topical nasal delivery that includes a neutralizing antibody as provided herein, such as the STI-2020 antibody, comprises: 20mM Histidine, 240 mM Sucrose, .,2 - 0.3% Hydroxypropyl methyl cellulose (HPMC), and 0.05% Polysorbate 80, at a pH of about 5.8.
- the concentration of antibody can be, for example, from about 1 mg/mL to about 200 mg/mL, from about 2 mg/mL to about 100 mg/mL, or from about 5 mg/mL to about 80 mg/mL, or from about 10 mg/mL to about 50 mg/mL.
- a neutralizing antibody such as a neutralizing antibody provided herein may be formulated for intranasal delivery in the form of a powder (such as a freeze- dried or micronised powder) or mist; for example with a particle size within the ranges indicated herein.
- Compounds may also be included in a pharmaceutical composition for intranasal delivery to reduce or prevent aggregation of the neutralizing antigen-binding protein.
- Aggregation inhibitory agents include, for example, polymers such as polyethylene glycol, dextran, diethylaminoethyl dextran, and carboxymethyl cellulose, which significantly increase the stability and reduce the solid-phase aggregation of peptides and proteins.
- additives in particular sugars and other polyols, also impart significant physical stability to dry, e.g., lyophilized proteins.
- These additives can also be used within the invention to protect the proteins against aggregation not only during lyophilization but also during storage in the dry state.
- sucrose and Ficoll 70 a polymer with sucrose units
- These additives may also enhance the stability of solid proteins embedded within polymer matrices.
- additional additives for example sucrose, stabilize proteins against solid-state aggregation in humid atmospheres at elevated temperatures, as may occur in certain sustained-release formulations of the invention.
- polypeptide microparticles can be prepared by simply lyophilizing or spray drying a solution containing various stabilizing additives described above. Sustained release of unaggregated peptides and proteins can thereby be obtained over an extended period of time.
- suitable methods and anti-aggregation agents are available for incorporation within the compositions of the invention such as disclosed in WO 05/120551, Breslow et al. (/. Am. Chem. Soc. 1996; 118: 11678-11681), Breslow et al. ( PNAS USA 1997; 94: 11156-11158), Breslow et al. ( Tetrahedron Lett.
- the particle size of the components including the carriers, of the invention may be from 5 to 500m, preferably from 10 to 250 m, more preferably from 20 to 200 m.
- the average particle size may be in the range of 50 to 100m.
- the present disclosure provides methods for treating a subject having a coronavirus infection, the method comprising: administering to the subject an effective amount of a therapeutic composition comprising a neutralizing antibody as provided herein by inhalation.
- the present disclosure also provides methods of preventing infection with a coronavirus such as SARS-CoV or SARS-Cov-2.
- the method includes administering to a subject at risk of becoming infected with a coronavirus such as SARS-CoV or SARS-Cov-2 an effective amount of a neutralizing antibody as disclosed herein, for example in a pharmaceutical formulation as disclosed herein, to the subject.
- Administration is by bronchial or pulmonary delivery, such as by inhalation.
- the composition is administered by pulmonary delivery, for example by oral inhalation.
- Pulmonary delivery can use any delivery device that can deliver a liquid (e.g., droplets) to the lungs, e.g., can deliver aerosols comprising a therapeutic composition such as a liquid pharmaceutical composition comprising a neutralizing antibody as provided herein to the lungs.
- the subject can be a human subject and can be a patient testing positive for a coronavirus such as hCov-NL63, SARS-CoV, or SARS-CoV-2. In some embodiments the subject is a subject testing positive for SARS-CoV-2 or exhibiting symptoms of infection with SARS-CoV-2.
- the neutralizing antibody(ies) can be administered to the subject in combination with at least one anti-viral agent and/or at least one viral entry inhibitor.
- One skilled in the art can routinely select an appropriate anti-viral agent or viral entry inhibitor to be administered with a neutralizing antibody.
- the anti viral agent and/or the viral entry inhibitor can be administered prior to, during, or after, administration of the neutralizing antibody.
- Administration of a pharmaceutical formulation that include a neutralizing S 1- binding antibody by pulmonary delivery via inhalation can use any device that provides respiratable droplets or particles that are able to reach the lungs by inhalation, preferably by oral inhalation.
- pulmonary delivery can be by means of a delivery device such as but not limited to a nebulizer or a metered dose inhaler.
- the formulations of the invention may include a "therapeutically effective amount" of a neutralizing SI -binding antibody as provided herein.
- a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
- a therapeutically effective amount of the neutralizing antibody may vary according to factors such as the viral load, disease state, age, sex, and weight of the individual, and the ability of the neutralizing SI -binding antibody to elicit a desired response in the individual.
- a therapeutically effective amount is also one in which any toxic or detrimental effects of the neutralizing SI -binding antibody are outweighed by the therapeutically beneficial effects.
- Toxicity and therapeutic efficacy of the active ingredients described herein can be determined using standard pharmaceutical procedures including in vitro, in cell cultures, and experimental animals.
- the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
- the dosage may vary depending upon the dosage form employed and the route of administration utilized.
- the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).
- Dosage amount and interval may be adjusted individually, for example, to provide serum and cell levels of the active ingredient which are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). Dosages necessary to achieve the MEC will depend on individual characteristics including the severity of the viral infection and related pathologies, the condition of the patient, and the judgment of the physician. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
- the delivery device can deliver, in a single dose or in multiple doses, a pharmaceutically effective amount of the composition to the subject's lungs by pulmonary inhalation.
- Devices suitable for pulmonary delivery of a dry powder form of a protein composition as a nonaqueous suspension are commercially available. Examples of such devices include the Ventolin metered-dose inhaler (Glaxo Inc., Research Triangle Park, N.C.) and the Intal Inhaler (Fisons, Corp., Bedford, Mass.). See also the aerosol delivery devices described in U.S. Pat. Nos. 5,522,378, 5,775,320, 5,934,272 and 5,960,792, herein incorporated by reference.
- An aerosol propellant used in an aerosol delivery device may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochloro-fluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoro-methane, dichlorotetrafluoromethane, dichlorodifluoro-methane, dichlorotetrafluoroethanol, and 1,1,1,2-tetra-fluoroethane, or combinations thereof.
- a chlorofluorocarbon such as a chlorofluorocarbon, a hydrochloro-fluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoro-methane, dichlorotetrafluoromethane, dichlorodifluoro-methane, dichlorotetrafluoroethanol, and 1,1,1,2-t
- a dry powder inhaler or other appropriate delivery device is preferably used.
- the dry powder form of the formulation is preferably prepared as a dry powder aerosol by dispersion in a flowing air or other physiologically acceptable gas stream.
- the delivery device can be any of dispenser is of a type selected from the group consisting of a reservoir dry powder inhaler (RDPI), a multi-dose dry powder inhaler (MDPI), and a metered dose inhaler (MDI).
- Fiquid aerosol delivery by nebulizer is another form of pulmonary drug delivery that can be employed.
- Nebulizers as they are generally more effective for delivery to the deep lung and may be preferred for delivering protein therapeutics in active form.
- Nebulizers create liquid aerosols, which are forced from a small orifice at high velocity by the release of compressed air, resulting in low pressure at the exit region due to the Bernoulli effect. See, e.g., U.S. Pat. No. 5,511,726. The low pressure is used to draw the fluid to be aerosolized out of a second tube. This fluid breaks into small droplets as it accelerates in the air stream.
- nebulizers for liquid formulation aerosolization include, for example, air jet nebulizers, liquid jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers.
- Nonlimiting examples of nebulizers include the AkitaTM (Activaero GmbH) (see U.S. Pat. No. 7,766,012, EP1258264 and the portable AeronebTM Go, Pro, and Lab nebulizers (AeroGen).
- the nebulizer can use a pharmaceutical composition that includes any pharmaceutically acceptable carrier, including a saline solution.
- a dry powder formulation can also be delivered by a nebulizer.
- Nebulizers can be customized for delivery of the particular protein, e.g. a neutralizing anti-S protein antibody, to reduce any denaturation, aggregation, and loss of activity during nebulization.
- Ultrasonic nebulizers use flat or concave piezoelectric disks submerged below a liquid reservoir to resonate the surface of the liquid reservoir, forming a liquid cone which sheds aerosol particles from its surface (U.S. 2006/0249144 and U.S. Pat. No. 5,551,416). Since no airflow is required in the aerosolization process, high aerosol concentrations can be achieved. Smaller and more uniform liquid respirable dry particles can be obtained by passing the liquid to be aerosolized through micron-sized holes. See, e.g., U.S. Pat. Nos. 6,131,570; 5,724,957; and 6,098,620.
- Vibrating mesh nebulizers which are considered less likely to cause protein denaturation (See, Bodier-Montagutelli et al. (2016) Exp Op Drug Deliv 15:729-736), may be used to generate aerosols for delivery of a liquid composition the includes a neutralizing anti- S 1 antibody to the lungs of a subject. Vibrating mesh nebulizers force liquid through a vibrating membrane with apertures of specific sizes, resulting in droplets having diameters within a specified range, and may be customized for optimal delivery and stability of specific protein formulations.
- Nonlimiting examples of vibrating mesh nebulizers include the ALX- 0171 NanobodyTM nebulizer, the Vectura FOS-Flamingo®, the PARI eFlow®, the Philips I- neb AAD®, and the AeronebTM Pro (Rohm et al. (2017) Inti J Pharmaceutics 532:537-546; Bodier-Montagutelli et al. (2018)).
- a dosage regimen can include a single dose of a liquid formulation of the invention, of 0.001 to 500 mg neutralizing anti-S protein antibody, or about 10 pg to 200 mg neutralizing anti- S protein antibody, administered daily, every other day, or weekly, or a plurality of doses administered at least twice, 2-3 times, 2-4 times or 2-6 times daily; or a plurality of doses administered once every 36 hours, once every 36-48 hours, once every 36-72 hours, once every 2-3 days, once every 2-4 days, once every 2-5 days, or once every week; or a plurality of doses administered once every 36 hours, once every 36-48 hours, once every 36-72 hours, once every 2-3 days, once every 2-4 days, once every 2-5 days, or once every week.
- the method further comprises detecting reduced infection or reduced viral load of the coronavims in a subject diagnosed as having a coronavirus infection after pulmonary delivery of a neutralizing antibody as provided herein to the
- the disclosure provides methods for treating coronavims infection by delivering to the lungs of a subject having or suspected of having a coronavirus infection a composition that includes a polypeptide comprising a neutralizing anti-S protein antibody formulated for pulmonary administration.
- the composition can be a pharmaceutical composition that includes, in addition to a neutralizing anti-S protein antibody, at least one pharmaceutically acceptable excipient or carrier compound.
- the pharmaceutical formulation that includes a neutralizing anti-S 1 antibody can be a formulation for delivery by aerosol inhalation such as by a nebulizer and can be in liquid form.
- Compositions as provided herein can be packaged in single dose units, for example, in vials or dispensers such as nebulizers that generate aerosols for delivery of particles or droplets to the lung.
- compositions as described that includes at least one nucleic acid construct that encodes a neutralizing antibody that binds a coronavirus.
- the subject can be a human subject, for example, a human subject that has a coronavirus infection, is suspected of having a coronavirus infection, or is at risk of becoming infected with a coronavirus.
- the subject can also be a non-human animal.
- the administering can be by injection, for example, intramuscular injection. Single or multiple doses, including multiple doses over weeks or months, can be administered.
- the amount of DNA (e.g., plasmid or plasmids encoding a neutralizing antibody) to be delivered can be determined for example, at least in part by experiments on non-human animals.
- the present disclosure provides methods (e.g., in vitro ) for detecting the presence of a coronavirus, or a protein from a coronavims, in a sample, comprising: (a) contacting the sample (containing a target antigen) with any one or any combination of two or more of the neutralizing antibodies having increased in vivo serum half-life and/or reduced effector function described herein, under conditions suitable to form an antibody-antigen complex; and (b) detecting the presence of the antibody-antigen complex.
- this method can be used to detect the presence of a coronavims in a sample from a subject and thus can be used to diagnose a subject suspected of having a coronavirus infection.
- the sample from the subject comprises phlegm, saliva, blood, cheek scaping, tissue biopsy, hair or semen.
- the sample from the subject can be obtained from an acutely coronavirus infected subject or a convalescing subject.
- the subject can be human, non-human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.
- the sample can comprise cells expressing a coronavirus membrane protein, or a coronavirus.
- the neutralizing antibodies can be labeled so permit detection of an antigen-antibody complex, where the label comprises a radionuclide, fluorescer, enzyme, enzyme substrate, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).
- the presence of the antibody-antigen complex can be detected using any detection mode including radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
- the present disclosure further provides methods (e.g., in vitro ) for identifying a compound that modulates (increases or decreases) binding between a coronavirus S protein and an ACE2 target receptor (or cells expressing ACE2 e.g., Vero E6 cells), comprising: (a) contacting (i) a candidate compound with (ii) a coronavirus S protein and with (iii) any one or any combination of two or more of the neutralizing antibodies having increased in vivo serum half-life and/or reduced effector function described herein, under conditions suitable to form an antibody-S 1 complex; and (b) detecting the presence or absence of the antibody-S protein complex.
- the lack of formation of the complex may indicate that the candidate compound competes for the same or overlapping epitope on the S protein subunit as the neutralizing antibody.
- the concentration of the candidate compound and/or the neutralizing antibody can be increased to obtain a dose response curve.
- the neutralizing antibodies can be labeled so permit detection of an antigen-antibody complex, where the label comprises a radionuclide, fluorescer, enzyme, enzyme substrate, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).
- the presence of the antibody-antigen complex can be detected using any detection mode including radiation, fluorescence, colorimetric, absorption wavelength, electron density antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin, streptavidin or protein A.
- a detectable bead such as a magnetic or electrodense (e.g., gold) bead
- biotin streptavidin or protein A.
- Embodiment 1 An isolated antigen-binding protein that specifically binds the spike (S) protein of SARS-CoV-2, wherein the antigen-binding protein comprises one or more heavy chain complimentary determining regions (“CDRH”) having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1 as set forth in SEQ ID NO:6, and/or a CDRH2 as set forth in SEQ ID NO:6, and/or a CDRH3 as set forth in SEQ ID NO:6.
- CDRH heavy chain complimentary determining regions
- Embodiment 2 An isolated antigen-binding protein that specifically binds the spike (S) protein of SARS-CoV-2, wherein the antigen-binding protein comprises one or more light chain complimentary determining regions (“CDRL1”) having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1 as set forth in SEQ ID NO:7, and/or a CDRL2 as set forth in SEQ ID NO:7, and/or a CDRL3 as set forth in SEQ ID NO:7.
- CDRL1 light chain complimentary determining regions
- Embodiment 3 The isolated antigen-binding protein according to embodiment 1 or embodiment 2, wherein the antigen-binding protein comprises a heavy chain variable domain comprising a CDR having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1 as set forth in SEQ ID NO: 6, and/or a CDRH2 as set forth in SEQ ID NO: 6, and/or a CDRH3 as set forth in SEQ ID NO:6.
- Embodiment 4 The isolated antigen-binding protein according to any of embodiments 1-3, wherein the antigen-binding protein comprises a light chain variable domain comprising at least one CDR at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1 as set forth in SEQ ID NO:7, and/or a CDRL2 as set forth in SEQ ID NO:7, and/or a CDRL3 as set forth in SEQ ID NO:7.
- Embodiment 5 The isolated antigen-binding protein according to any of embodiments 1-4, wherein the antigen-binding protein comprises a CDRH1, a CDRH2, and a CDRH3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1, a CDRH2, and a CDRH3 as set forth in SEQ ID NO:6.
- Embodiment 6 The isolated antigen-binding protein according to any of embodiments 1-5, wherein the antigen-binding protein comprises a CDRL1, a CDRL2, and a CDRL3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1, a CDRL2, and a CDRL3 as set forth in SEQ ID NO:7.
- Embodiment 7 The isolated antigen-binding protein according to any of embodiments 1-6, wherein the isolated antigen-binding protein comprises:
- a CDRH1, a CDRH2, and a CDRH3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRH1, CDRH2, and/or CDRH3 set forth in SEQ ID NO:6; and
- a CDRL1, a CDRL2, and a CDRL3 that each independently has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of a CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NO:7.
- Embodiment 8 The isolated antigen-binding protein according to any of embodiments 1-7, wherein the isolated antigen-binding protein comprises a heavy chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:6.
- Embodiment 9 The isolated antigen-binding protein according to any of embodiments 1-8, wherein the isolated antigen-binding protein comprises a light chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:7.
- Embodiment 10 The isolated antigen-binding protein according to any of embodiments 1-9, wherein the isolated antigen-binding protein comprises a heavy chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:6 and a a light chain variable domain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of SEQ ID NO:7.
- Embodiment 11 The isolated antigen-binding protein according to any of embodiments 1-10, wherein the isolated antigen-binding protein comprises CDRH1 having the amino acid sequence of SEQ ID NO:8, a CDRH2 having the amino acid sequence of SEQ ID NO:9, and a CDRH3 having the amino acid sequence of SEQ ID NO: 10.
- Embodiment 12 The isolated antigen-binding protein according to any of embodiments 1-11, wherein the isolated antigen-binding protein comprises and a light chain variable region comprising a CDRL1 having the amino acid sequence of SEQ ID NO: 11, a CDRL2 having the amino acid sequence of SEQ ID NO: 12, and a CDRL3 having the amino acid sequence of SEQ ID NO: 13.
- Embodiment 13 The isolated antigen-binding protein according to any of embodiments 1-12, wherein the isolated antigen-binding protein comprises a CDRH1 having the amino acid sequence of SEQ ID NO:8, a CDRH2 having the amino acid sequence of SEQ ID NO:9, and a CDRH3 having the amino acid sequence of SEQ ID NO: 10 and a CDRL1 having the amino acid sequence of SEQ ID NO: 11, a CDRL2 having the amino acid sequence of SEQ ID NO: 12, and a CDRL3 having the amino acid sequence of SEQ ID NO:13.
- the isolated antigen-binding protein comprises a CDRH1 having the amino acid sequence of SEQ ID NO:8, a CDRH2 having the amino acid sequence of SEQ ID NO:9, and a CDRH3 having the amino acid sequence of SEQ ID NO: 10 and a CDRL1 having the amino acid sequence of SEQ ID NO: 11, a CDRL2 having the amino acid sequence of SEQ ID NO: 12, and a CDRL3 having the amino
- Embodiment 14 The isolated antigen-binding protein according to any of embodiments 1-13, wherein the heavy chain variable domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6 and the light chain variable domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:7.
- Embodiment 15 The isolated antigen-binding protein according to any of embodiments 1-14, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:6 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:7.
- Embodiment 16 The isolated antigen-binding protein according to of any of embodiments 1-15, wherein the antigen-binding protein binds the SI protein of SARS-CoV-2 WA1/2020 with a binding affinity of (K D ) of between about 1 nM and about 5 nM.
- Embodiment 17 The isolated antigen-binding protein according to of any of embodiments 1-16, wherein the antigen-binding protein binds an SI protein having the mutations HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, and P681H with respect to SEQ ID NO:4 with a binding affinity of (K D ) of between about 1 nM and about 5 nM.
- Embodiment 18 The isolated antigen-binding protein according to of any of embodiments 1-17, wherein the antigen-binding protein binds the SI protein having the mutations K417N, E484K, N501Y, and D614G with respect to SEQ ID NO:4 with a binding affinity of (K D ) of between about 1 nM and about 5 nM.
- Embodiment 19 The isolated antigen-binding protein according to of any of embodiments 1-18, wherein the antigen-binding protein binds the SI protein having having the mutations K417N, E484K, N501Y, and D614G with a binding affinity of (K D ) of between about 1 nM and about 5 nM.
- Embodiment 20 The isolated antigen-binding protein according to of any of embodiments 1-19, wherein the antigen-binding protein comprises an antibody or antibody fragment.
- Embodiment 21 The isolated antigen-binding protein according to of any of embodiments 1-20, wherein the antigen-binding protein comprises a fully human antibody, a heavy chain variable region and a light chain variable region of a fully human antibody, or an antibody fragment derived from a fully human antibody.
- Embodiment 22 The isolated antigen-binding protein according to of any of embodiments 1-21, wherein the antigen-binding protein comprises an IgG antibody, which is optionally an IgGl, IgG2, IgG3, or IgG4 antibody.
- Embodiment 23 The isolated antigen-binding protein according to of any of embodiments 1-22, wherein the antigen-binding protein comprisesan IgGl or IgG4 antibody.
- Embodiment 24 The isolated antigen-binding protein according to of any of embodiments 1-23, wherein the antigen-binding protein comprises a mutant Fc region.
- Embodiment 25 The isolated antigen-binding protein according to of any of embodiments 1-24, wherein the antigen-binding protein comprises an Fc region comprising one or more mutations selected from the group consisting of N297A, N297Q, N297D,
- Embodiment 26 The isolated antigen-binding protein according to of any of embodiments 1-25, wherein the antigen-binding protein comprises an Fc region comprising the mutations L234A and L235A (LALA), optionally wherein the Fc region comprises the sequence of SEQ ID NO: 14 or 16.
- LALA L234A and L235A
- Embodiment 27 The isolated antigen-binding protein according to of any of embodiments 1-26, wherein the antigen-binding protein comprises an Fc region comprising at least one mutation that increases antibody half-life.
- Embodiment 28 The isolated antigen-binding protein according to of any of embodiments 1-27, wherein the antigen-binding protein comprises an Fc region comprising one or more mutations selected from M252Y, T256D, T307Q, T307W, M252Y, S254T, T256E, M428L, and N434S.
- Embodiment 29 The isolated antigen-binding protein according to of any of embodiments 1-28, wherein the antigen-binding protein comprises a Fab fragment, a Fab’ fragment, or a F(ab’)2 fragment.
- Embodiment 30 The isolated antigen-binding protein according to of any of embodiments 1-29, wherein the antigen-binding protein comprises a single chain antibody, optionally wherein the single chain antibody comprises an ScFv.
- Embodiment 31 The isolated antigen-binding protein according to of any of embodiments 1-30, wherein the antigen-binding protein comprises a neutralizing antibody or antigen-binding fragment thereof that blocks infection of susceptible cells by SARS-CoV-2 isolate WA 1/2020.
- Embodiment 32 The isolated antigen-binding protein according to of any of embodiments 1-31, wherein the antigen-binding protein comprises a neutralizing antibody or antigen-binding fragment thereof that blocks infection of susceptible cells by SARS-CoV-2 isolate WA1/2020 with an IC50 of 100 ng/mL or lower.
- Embodiment 33 The isolated antigen-binding protein according to of any of embodiments 1-32, wherein the antigen-binding protein comprises a neutralizing antibody or antigen-binding fragment thereof that blocks infection of susceptible cells by SARS-CoV-2 variant B.1.351.
- Embodiment 34 The isolated antigen-binding protein according to of any of embodiments 1-33, wherein the antigen-binding protein comprises a neutralizing antibody that blocks infection of susceptible cells by SARS-CoV-2 variant B.1.351 with an IC50 of 200 ng/mL or lower.
- Embodiment 35 A kit comprising: a first antigen-binding protein according to of any of embodiments 1-34; and a second antigen-binding protein.
- Embodiment 36 The kit according to embodiment 35, wherein the second antigen-binding protein comprises a CDRH1, and/or a CDRH2, and/or CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively.
- Embodiment 37 The kit according to embodiment 35 or embodiment 36, wherein the second antigen-binding protein comprises a CDRL1, and/or a CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- Embodiment 38 The kit according to any of embodiments 35-37, wherein the second antigen-binding protein comprises a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively, and a CDRL1, CDRL2, and/or CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- Embodiment 39 The kit according to any of embodiments 35-38, wherein the second antigen-binding protein comprises a heavy chain variable region comprising a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO. 31, and/or SEQ ID NO:32, respectively.
- Embodiment 40 The kit according to any of embodiments 35-39, wherein the second antigen-binding protein comprises a light chain variable region comprising a CDRL1, and/or a CDRL2, and/or CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- Embodiment 41 The kit according to any of embodiments 35-40, wherein the second antigen-binding protein comprises a heavy chain variable region comprising: a CDRH1, and/or a CDRH2, and/or a CDRH3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 30, SEQ ID NO.
- a light chain variable region comprising a CDRL1, and/or CDRL2, and/or a CDRL3 independently having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 33, SEQ ID NO. 34, and/or SEQ ID NO:35, respectively.
- Embodiment 42 The kit according to any of embodiments 35-41, wherein the second antigen-binding protein comprises a second antigen binding protein comprising a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28.
- Embodiment 43 Embodiment 43.
- the second antigen-binding protein comprises a second antigen binding protein comprising a light chain variable region comprising having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- Embodiment 44 The kit according to any of embodiments 35-43, wherein the second antigen-binding protein comprises: a heavy chain variable region having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 28; and a light chain variable region comprising having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the amino acid sequence set forth as SEQ ID NO: 29.
- Embodiment 45 The kit according to any of embodiments 35-44, wherein the second antigen-binding protein comprises antibody S1D7270 or antibody STI-2020.
- Embodiment 46 The kit according to any of embodiments 35-45, wherein the kit comprises a single composition comprising the first antigen-binding protein and the second antigen-binding protein.
- Embodiment 47 A pharmaceutical composition, comprising: the antigen-binding protein of any of embodiments 1-34; or the kit of any of embodiments 35-46; and a pharmaceutically-acceptable excipient.
- Embodiment 48 The pharmaceutical composition of embodiment 47, wherein: the first antigen-binding protein comprises an IgG antibody, a Fab, Fab’ or F(ab’)2 fragment, or a single chain antibody; and/or the kit comprises a second antigen-binding protein comprising an IgG antibody, a Fab, Fab’ or F(ab’)2 fragment, or a single chain antibody.
- the first antigen-binding protein comprises an IgG antibody, a Fab, Fab’ or F(ab’)2 fragment, or a single chain antibody
- the kit comprises a second antigen-binding protein comprising an IgG antibody, a Fab, Fab’ or F(ab’)2 fragment, or a single chain antibody.
- Embodiment 49 The pharmaceutical composition of embodiment 47 or embodiment 48, wherein: the first antigen-binding protein comprises an IgG antibody; and/or the kit comprises a second antigen-binding protein comprising an an IgG antibody.
- Embodiment 50 The pharmaceutical composition of any of embodiments 47-49, wherein: the first antigen-binding protein comprises an IgG antibody having an Fc region comprising L234A and L235A mutations; and/or the kit comprises a second antigen-binding protein comprising L234A and L235A mutations.
- Embodiment 51 A nucleic acid molecule that comprises a nucleic acid sequence encoding the antigen-binding protein of any of embodiments 1-34 and/or the second antigen binding protein of the kit of any of embodiments 35-46.
- Embodiment 52 The nucleic acid molecule of embodiment 51, wherein the nucleic acid molecule comprises a vector, wherein the vector comprises a promoter operably linked to the nucleic acid sequence encoding the antigen-binding protein and/or the second antigen binding protein of the kit .
- Embodiment 53 A vector comprising a nucleic acid molecule of embodiment 51 or embodiment 52.
- Embodiment 54 A host cell comprising: a nucleic acid molecule according to embodiment 51 or embodiment 52;or a vector according to embodiment 53.
- Embodiment 55 A pharmaceutical composition comprising: a nucleic acid molecule according to embodiment 51 or embodiment 52; a vector according to embodiment 53; and/or a host cell according to embodiment 54.
- Embodiment 56 A method for preparing: an antigen-binding protein according to any of embodiments 1-34 or neutralizing antibody or a heavy or light chain thereof; a kit according to any of embodiments 35-46; or a pharmaceutical composition according to any of embodiments 47-50; the method comprising culturing a population of host cells of embodiment 54 under conditions suitable for expressing the antigen-binding protein or the heavy chain variable region and/or the light chain variable thereof or the kit.
- Embodiment 57 The method of embodiment 56, further comprising: recovering from the population of host cells the expressed antigen-binding protein or the heavy chain variable region and/or the light chain variable thereof, or the kit.
- Embodiment 58 A method for treating a subject having or suspected of having a coronavirus infection, the method comprising administering to the subject an effective amount of one or more of: the antigen-binding protein of any of embodiments 1-34; the kit of any of embodiments 35-46; the pharmaceutical composition of any one of embodiments 47- 50 and 55; the nucleic acid of embodiment 51 or embodiment 52; the vector of embodiment 53; or the host cell of embodiment 54.
- Embodiment 59 The method according to embodiment 58, wherein the administration is by pulmonary delivery by inhalation.
- Embodiment 60 The method according to embodiment 59, wherein the pulmonary delivery is by a nebulizer.
- Embodiment 61 The method according to embodiment 58 wherein the administration is by intranasal delivery.
- Embodiment 62 The method according to embodiment 58, wherein the administration is by intramuscular injection, intravenous injection, or intravenous infusion.
- S SARS-Cov-2 spike (S) protein (including leader sequence) with SI and S2 subunits
- SARS-Cov-2 spike protein without leader sequence that includes the SI subunit and the S2 subunit up to amino acid 1213 (amino acids 16-1213 of SEQ ID NO:l)
- SARS-Cov-2 spike SI subunit (including leader sequence)
- SARS-Cov-2 spike SI subunit (no leader sequence)
- SARS-Cov-2 spike protein RBD amino acids 319-537 of SEQ ID NO:l
- SARS-Cov-2 spike SI subunit no leader sequence
- polyhistidine tag
- Antibody S1D7270 also known as antibody STI-2020
- Heavy Chain Variable region see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- Antibody S1D7270 antibody also known as antibody STI-2020
- Light Chain Variable region see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- Antibody S1D7270 antibody also known as antibody STI-2020
- Heavy Chain CDRl see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- Antibody S1D7270 antibody also known as antibody STI-2020
- Heavy Chain CDR2 see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- Antibody S1D7270 antibody also known as antibody STI-2020
- Heavy Chain CDR3 see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- Antibody S1D7270 antibody also known as antibody STI-2020
- Light Chain CDR1 see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- Antibody S1D7270 antibody also known as antibody STI-2020
- Light Chain CDR2 see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- Antibody S1D7270 antibody also known as antibody STI-2020
- Light Chain CDR3 see, e.g., WO 2022/032139, hereby incorporated by reference in its entirety for all purposes
- a phage-display library was screened using the protein S 1 (spike) protein derived from the SARS-CoV-2/human/USA/WA-CDC-WAl/2020 (US A- WA 1/2020, GenBank: MN985325.1). ScFv fragments binding SI and having the capacity to block interactions between SI and a recombinant ACE2 ectodomain were sequenced and converted to an IgGl and an IgGl LALA format. In some cases such antibodies were subjected to affinity maturation or further engineered for improved binding or neutralization properties.
- S7E5041 One of the fully human IgGl antibodies, designated S7E5041, was engineered to include the LALA mutation in the Fc region (Magnani et al. (2017) Molecular Therapy 25:2323-2331; See, SEQ ID NO:14).
- the heavy chain variable region of antibody S7E5041 is provided as SEQ ID NO:6, and the light chain variable region of antibody S7E5041 is provided as SEQ ID NO:7.
- the heavy chain variable region CDRs are provided as SEQ ID NO:8 (Heavy chain CDR1), SEQ ID NO:9 (Heavy chain CDR2), and SEQ ID NO: 10 (Heavy chain CDR3), and the light chain variable region CDRs are provided as SEQ ID NO: 11 (Light chain CDR1), SEQ ID NO: 12 (Light chain CDR2), and SEQ ID NO: 13 (Light chain CDR3).
- Binding kinetics of the S7E5041 anti-spike protein antibody with SARS-CoV-2 SI proteins of the were measured using surface plasmon resonance (SPR). Binding of the S7E5041 antibody to the spike protein of SARS-CoV-2 isolate WA1/2020 (SEQ ID NO:4; Aero Biosystems) as well as to S 1 proteins that incorporated some of the mutations found in two variants: the HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H found in B.1.1.7, and the spike protein mutations: K417N, E484K, N501Y, D614G of variant B.1.351.
- the B.l.1.7 and B.1.351 variant-type S proteins included C-terminal his tags (Sino Biologicals). Kinetic interactions between the S7E5041LALA antibody and spike SI proteins were measured at 25°C using BIAcore T200 surface plasmon resonance (SPR) (GE Healthcare). Anti-human fragment crystallizable region (Fc region) antibody was immobilized on a CM5 sensor chip to approximately 8000 resonance units (RU) using standard N hydroxysuccinimide/N Ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride (NHS/EDC) coupling methodology.
- S7E5041-LALA Fc (1-2 pg/mL) was captured for 60 seconds at a flow rate of 10 pL/minute.
- the SARS-CoV-2 Spike SI proteins were prepared at six different dilutions (range 1.56 nM-50 nM) in a running buffer of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20 (HBS EP+). All measurements were conducted in HBS-EP+ buffer with a flow rate of 30 pl/minute. A 1:1 binding model and was used to fit the data.
- SPR sensorgrams of anti-Sl antibody S7E5041 binding to the SARS-CoV2 SI proteins of WA1/2020 (“SI”), the variant SI having B.1.351 mutations, and the variant SI having B.l.1.17 mutations are provided in Figure 1.
- the S7E5041 antibody was found to have a binding affinity (KD) of 1.91 nM for the WA1/2020 SI protein, a binding affinity (KD) of 2.93 nM for the B.1.351-type SI protein, and a binding affinity (KD) of 1.34 nM for the B.1.17-type SI protein.
- KD binding affinity
- Example 3 Binding Affinity of S7E5140 for Spike Protein Variants Expressed on Cells.
- HEK293 cells were transfected with nucleic acid constructs encoding the SARS-CoV-2 variant S proteins as shown in Figure 2.
- Transfected cells were aliquoted into the wells of tissue culture plates and dilutions of anti-S 1 antibody S7E5041LALA were added to the cells at a series of dilutions. After incubating the cells with antibody the cells were washed once with FACS buffer and then stained with Goat Anti- Human IgG (H+L) F(ab')2 fragment conjugated with allophycocyanin (APC) on ice for 20 minutes. The cells were washed with FACS buffer and resuspended in 50 pF FACS buffer for analysis in a flow cytometer. The results are shown in Figure 3A-3H. EC50 values are provided in the accompanying tables.
- Figure 2 summarizes the variants, their mutations, and the EC50s for S7E5041LALA antibody binding to cells expressing the SI protein of the variants.
- Virus supernatant was removed and replaced with fresh medium after 1 hour at 37 °C.
- Cytopathic effect i.e., the appearance of plaques or discontinuity in the cell monolayer due to cell lysis, was recorded on day 3 post-infection.
- the media was aspirated, and the cells were then fixed with formalin and stained with 0.25% crystal violet.
- Figure 4A shows the percent reduction in plaques made by SARS-CoV-2 isolate, WA1/2020, with increasing concentration of antibody.
- the IC50 of the S7E5041LALA antibody in the neutralization assay against the WA1/2020 SARS-CoV-2 WA1/2020 virus was found to be 41.18 ng/mF.
- Figure 4B shows the results of a separate experiment with the percent reduction in plaques made by SARS-CoV-2 variant B 1.35 IK shown as antibody dilution increases (i.e., decreasing concentration of antibody).
- the IC50 of the S7E5041LALA antibody in the neutralization assay against the B 1.35 IK vims was found to be 307.5 ng/mF.
- Example 5 S7E5041LALA neutralization assay using pseudotyped viruses displaying engineered mutant S proteins.
- S protein genes encoding thirty-five different S protein mutants were constructed and cloned into plasmid pCDNA3.1. Nearly all of the mutant S proteins included the D614G mutation found in both the B 1.1.17 and B 1.351 SARS-CoV-2 variants, and all of the encoded S proteins had a deletion of the C-terminal 21 amino acids.
- the general strategy for generating pseudotyped viruses having SARS-Cov-2 S proteins integrated into and displayed on the viral membrane is shown in Figure 5.
- the plasmids were transfected into BHK21 cells, and 24 hours later, the cells were transduced with VSV deleted for the G gene and encoding a firefly lucif erase gene (VSVAG(FLuc)). The cells were incubated with VSVAG(FLuc) for 1 hour and then the medium containing the VSVAG(FLuc) was removed. The cells were washed 2x with DPBS prior to the addition of fresh culture medium. After 40-48hrs, culture supernatants containing pseudotyped viruses displaying the mutant S protein expressed by the host cells were collected, aliquoted and frozen.
- VSVAG(FLuc) firefly lucif erase gene
- the pseudotyped viruses were normalized for fluorescence intensity on 293-ACE2 cells by luciferase assay.
- the VSVAG(FLuc)-S pseudotypes were incubated with the indicated concentrations of anti-spike antibody as well as 1 pg/mL of anti-VSV-G antibody which was added to neutralize any remaining parental VSV in the virus preparation for 30 minutes at room temperature.
- the VSVAG(FLuc)-S pseudotype plus antibody was then added to 293-ACE2 cells in 96- or 384-well format, and luciferase output was measured after 40-48 hrs.
- Relative IC50 was determined using nonlinear fit variable slope (4PL), and absolute IC50 was determined using nonlinear fit with a top constraint of 100 (complete inhibition) and a baseline constraint of 0 (no inhibition).
- Figure 6 shows the results of these neutralization assays performed with multiple versions of pseudotyped VSVAG(FLuc)-S having different S protein mutations and the S7E5041LALA antibody.
- Example 6 Treatment of SARS-CoV-2 B.1.351 Variant Infection in Hamsters with S7E5041LALA.
- Figure 7B shows that the titer of SARS-CoV-2 in the lungs of animals sacrificed on Day 5 post infection was far higher for animals treated with the control antibody versus the STI-5041LALA antibody, demonstrating a protective effect of this neutralizing antibody against the SARS-CoV-2 B.1.351 variant.
- Example 7 Unbiased Approach to Identify and Assess Efficacy of Human SARS-CoV-2 Neutralizing Antibodies.
- the Sorrento G-MABTM library utilizes a single chain variable fragment (scFv) antibody phage display library constructed from the antibody repertoire of over 600 healthy individuals, encoding the donor paratopes s as well as an expanded novel collection of antigen recognition regions created by random pairings of heavy and light chain-encoding genes from within the donor pool.
- Selection of neutralizing antibodies (nAbs) within phage display human antibody libraries also allows for functional, unbiased selection of Spike binding nAbs independent of the selective pressures underlying natural immunogenicity.
- the identified nAbs can be characterized and tested for virus neutralizing potency without reliance on identification of immunoglobulin coding sequences from survivor patient populations or immunization of transgenic animals.
- the G-MAB system provides a means of pre-banking a safe and potent collection of nAbs with the capacity to treat pandemic threat pathogens as they emerge over time.
- ADE antibody dependent enhancement
- the IgGl Fc regions were modified by introducing specific amino acid substitutions (L234A, L235A [LALA]).
- L234A, L235A [LALA] specific amino acid substitutions
- the LALA Fc modification reduces binding affinity to the Fey receptors while providing a similar blockade to interactions between SARS-COV-2 and the angiotensin-converting enzyme 2 (ACE2) receptor expressed on susceptible cells in the lung and other organs.
- ACE2 angiotensin-converting enzyme 2
- Mammalian expression vectors were constructed by cloning of the synthesized gene fragments encoding SARS-CoV-2 Spike variant proteins (see Table 1 below, indicating mutations introduced into wild type [WA-1 strain] spike protein sequence).
- HEK293 cells were transfected using FuGeneHD transfection reagent according to manufacturer’s protocol (Promega, Cat # E2311). 48 hours post-transfection, cells were harvested using enzyme free cell dissociation buffer (ThermoFisher, Cat #13151014.), washed once and resuspended in FACS buffer (DPBS + 2% FBS) at 2xl0 6 cells/mL.
- the cells were dispensed into wells of a 96-well V bottom plate (40 pL per well), and an equal volume of 2x final concentration of serially-diluted anti-S 1 antibody solution was added. After incubation on ice for 45 minutes, the cells were washed with 2 times of 150 pL FACS buffer. Detection of bound antibody was carried out by staining the cells with 50 pL of 1:500 diluted APC AffiniPure F(ab')2 Fragment (Goat Anti- Human IgG (H+L). Jackson ImmunoResearch, Cat# 109-136-4098) for 20 minutes on ice.
- the cells were washed once with 150 pL FACS buffer and analyzed on IntelliCyt iQue® Screener (Sartorius) flow cytometry. Mean fluorescent intensity values were obtained from the histograms. A sigmoidal four-parameter logistic equation was used for fitting the MFI vs. mAb concentration data set to extract EC50 values (GraphPad Prism 8.3.0 software).
- Vero E6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning, NY) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, MA), 1% penicillin-streptomycin, and L-glutamine.
- DMEM Dulbecco modified Eagle’s medium
- FBS fetal bovine serum
- Thermo Fisher Scientific, MA fetal bovine serum
- the P3 stock of the SARS-CoV-2 US A/WA- 1/2020, 202001, USA/CA-CDC5574/2020 and, MD-HP01542/2021 isolates were obtained from The World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch.
- the viruses were propagated in Vero E6 cells and cell culture supernatant of P4 stocks were stored at -80 °C under BSL3 conditions.
- All SARS-CoV-2 Spike constructs for pseudotype generation were expressed from plasmid pCDNA3.1 (Thermo Fisher, Cat #V79020).
- codon optimized SARS-CoV-2 Wuhan Spike carrying the D614G amino acid change (Sino Biological, Cat #VG40589-UT(D614G)) was modified to remove the last 21 amino acids at the C-terminus (SpikcA21) and was used as the parental clone.
- amino acid changes included D80A, D215G, D242-244, K417N, E484K, N501Y, D614G, and A701V.
- codon optimized genes encoding full-length spike proteins.
- BHK21 cells (ATCC, Cat #CCL-10) were maintained in DMEM/F12 media (Thermo Fisher, Cat #21041025) supplemented with 10% fetal bovine serum (Omega Scientific, Cat #FB-02) and 5% trypose phosphate broth (Thermo Fisher, Cat #18050039).
- BHK21/WI-2 cells (Kerafast, Cat #EH1011) were maintained in DMEM (Thermo Fisher, Cat #11965092) supplemented with 5% fetal bovine serum.
- 293-ACE2 cells were maintained in DMEM supplemented with 10% fetal bovine serum and 200pg/mL G418 (Invivogen, Cat #ant-gn-2).
- 1.2E6 BHK21 cells were nucleofected with 2pg of Spike plasmid using an Amaxa Nucleofector II with cell line kit L (Lonza, Cat #VCA-1005) and program A-031. Cells were plated to one well of a 6-well dish and incubated overnight at 37°C/5%C0 2 . The next day, cells were transduced with Pseudotyped AG-luciferase (G*AGTucif erase) rVSV (Kerafast, Cat #EH1025-PM) at MOI-4 for 1 hour at 37°C/5%C0 2 .
- G*AGTucif erase Pseudotyped AG-luciferase
- rVSV Kerafast, Cat #EH1025-PM
- Female CD-l-IGS (strain code #022) were obtained from Charles River at 6-8 weeks of age.
- 100 pF of antibody diluted in IX HBSS was administered retro-orbitally to anesthetized animals.
- antibody was diluted in IX HBSS and administered by inhalation into the nose of anesthetized animals in a total volume of 20 pF (10 pF per nostril) using a pipette tip.
- Organs, blood, and lung lavage samples were collected 24 hours post-antibody administration.
- the syringe was removed from the catheter, and the recovered lavage fluid was transferred into F5 mF tubes and kept on ice. Favage samples were centrifuged at 800g for 10 min at 4°C. Supernatants were collected, transferred to fresh F5 mF tubes, and stored at -80 °C. Total spleen, total large intestine, and 150 to 400 mg of lungs and small intestine were suspended in 300 pF of PBS in pre-filled 2.0 mF tubes containing zirconium beads (Spectrum, Cat # 155-40945). Tubes were processed in a BeadBug-6 homogenizer at a speed setting of 3,000g and a 30 second cycle time for four cycles with a 30-second break after each cycle.
- Tissue homogenates were centrifuged at 15,000 rpm for 15 minutes at 4°C. Homogenate supernatants were then transferred into F5 mF tubes and stored at -80 °C. STI- 2020 antibody levels in each sample were quantified using the antibody detection EFISA method. Statistical significance was determined a two-way unpaired t-test and a Welch’s correction was used when the sample size was different between groups. Experiments were conducted and reported in accordance with the ARRIVE guidelines. For intranasal injections, mice were briefly anesthetized using a low dose isoflurane inhalation.
- mice Female CD-l-IGS (strain code #022) were obtained from Charles River Laboratories at 6-8 weeks of age.
- STI-2020 and STI-5041 dissolved in intranasal formulation buffer were administered as previously described for the IN biodistribution study.
- Lungs and blood were collected from 3 mice at each of the following timepoints: 10 min, 1.5 h, 6 h, 24 h, 72 h, 96 h, 168 h, 240 h, and 336 h.
- Serum and lung tissue samples were collected as described for the biodistribution study.
- STI-2020 and STI-5041 antibody levels in each sample were quantified using the antibody detection ELISA method.
- Tubes were centrifuged and clarified homogenate was serially diluted 10-fold with DMEM+2% FBS. From material representing each serial dilution step, 100 pL was transferred to each of four wells of a 96-well plate previously seeded with Vero E6 cells. Plates were then incubated for 72-96 hours at 37 °C, 5% CO2. Cells were subsequently fixed with 10% formalin and stained with 0.25% crystal violet solution. TCID50 values were calculated by the method of Reed and Muench 19 . Virus titers in lungs were compared with the isotype control mAb-treated group using one-way ANOVA following Dunnett’s multiple comparisons test.
- nasal turbinates from hamster model
- Histopathology analyses of nasal turbinates were conducted as previously reported. In brief, nasal turbinates were collected at the time of euthanasia and fixed with 10% formalin for 7 days before removal from the ABSL-3 facility. Nasal turbinates samples were decalcified with EDTA (10% w/v) and embedded in paraffin prior to sectioning. Sections were 5 pm thick, and subsequently were stained with Hematoxylin and Eosin. The OE thickness of the nasal septum was measured using light microscopy. OE thickness of STI- 2020 treated hamsters were compared to isotype-control treated hamsters using an unpaired t- test.
- a nAb candidate termed STI-1499 displayed potent SARS-CoV-2 superior neutralizing activity, wherein affinity maturation was undertaken and identified the most potent clones STI-2020 and STI-5041, which were further profiled for binding affinity using surface plasmon resonance (SPR).
- SPR surface plasmon resonance
- STI-2020 bound to the SI region of the Spike protein of the USA/WA-1/2020 (WA-1) isolate with an affinity of 2.84 nM and 42.0 nM to the Alpha variant, and binding to the Beta variant was not observed (Figure 8B).
- the affinity of STI- 5041 was determined to be 1.91 nM to the WA-1 isolate, 1.34 nM to the Alpha variant, and 2.93 nM to the Beta variant, demonstrating potent affinity to clinically meaningful variant Spike proteins.
- STI-2020 and STI-5041 were tested for the binding of full-length Spike protein derived from naturally emerging viruses expressed on a cell surface. These binding studies demonstrated STI-2020 bound the WA-1 isolate and Alpha variant with similar affinity, whereas binding affinity to the Beta variant was undetectable in the assay.
- STI-5041 bound the WA-1 isolate with an EC50 of 0.012 pg/mL and exhibited similar affinity for both the Alpha and Beta variants, 0.021 and 0.024 pg/mL, respectively ( Figure 8C).
- the neutralizing activity of STI-2020 and STI-5041 was tested against WA-1, an early variant isolate encoding the Asp (D) to Gly (G) mutation at amino acid residue 614 of the Spike protein (2020001), Alpha and Beta variants.
- STI-2020 neutralized all isolates tested except the Beta variant, whereas STI-5041 retained neutralizing activity across the isolates tested (Figure 8D).
- Pseudotyped vims was used to determine the IC50 of the nAbs STI-2020 and STI- 5041.
- the IC50 for Alpha and Beta-based pseudotypes was within 10-fold of that measured in assays with the 20200001 D614G pseudotype for each nAb, with the exception of the STI- 2020 IC50 against the Beta variant, which was estimated to be >10 pg/mL (Figure 9A).
- Epitope binning with coordinated binding against the WA-1 RBD demonstrated distinct epitope binding of STI-2020 versus STI-5041, which may in part explain the lack of neutralizing activity seen for STI-2020 against the Beta variant ( Figure 9B).
- STI-2020 and STI-5041 were evaluated following delivery by either the intravenous or intranasal route. These studies illustrate the potential effects of delivery route on the timing of antibody exposure in the lung tissue and blood of treated mice. Following IV treatment at a dose level of 0.5 mg/kg, STI-2020 was detected in the serum, spleen, lungs, small intestine, and large intestine. Detected levels in the serum following the 0.5 mg/kg dose averaged 4.5 pg/mL, while STI-2020 was present at average concentrations less than 0.01 pg/mL in lung lavage material at each of the IV doses tested (Figure 9C, upper left panel).
- STI-2020 Following intranasal (IN) administration of STI-2020, the concentration of antibody in the serum at 24 hours reached an average value of 0.21 pg/mL in the 2.5 mg/kg dose group and 0.08 pg/mL in the 0.5 mg/kg dose group.
- STI-2020 administered IN resulted in a 30-fold lower concentration of antibody in serum.
- STI-2020 concentrations in lung lavage samples following IN dosing reached average concentrations of 2.7 pg/mL in the 2.5 mg/kg dose group and 1.1 pg/mL in the 0.5 mg/kg group.
- STI-5041 At the 0.2 mg/kg IV dose of STI-5041, 1.4 pg/mL was measured in the serum, whereas all other concentrations of STI-5041 (0.2 mg/kg or 0.02 mg/kg) were below 1 pg/mL in fluid or 1 ng/mg in tissue of organs measured ( Figure 9E). STI-5041 was then administered IN at 10 mg/kg, 2 mg/kg, 0.2 mg/kg and 0.02 mg/kg. At the IN doses of 10, 2, and 0.2 mg/kg, STI- 5041 was detected in the serum, the lung lavage and lung tissue.
- the Cmax value of STI-2020 in the lungs was measured at 1.5 hours post-administration at a value of 43 ng/mL.
- an apparent terminal half-life (T1 / 2) of 32.21 h was measured when analyzed between 0.15 and 240 h.
- the R 2 value equaled 0.932, however when the data were analyzed between 0.15 and 168 h the R 2 value increased to 0.987 but the T1/2 dropped to 25.07 h for the lung samples.
- Kinetics of STI-2020 exposure in the lungs following intranasal administration was accompanied by a slower kinetic of detectable antibody in the serum of treated mice (Figure 9G).
- Antibody was first detected in the serum at 6 hours post-administration and the Cmax of 871 ng/mL was reached at the 240 h timepoint (Tmax). Serum antibody concentrations were within 90% of the recorded Cmax by the 24 h timepoint. Antibody levels remained relatively constant in serum over the period spanning 24 - 240 h, which is in keeping with the calculated STI-2020 serum half-life observed following IV administration of 240 h in mice (data not shown). The total systemic exposure (AUClast) was significantly higher in the lungs than in the serum of treated mice (AUClast were 1,861,645.8 and 248,675.5 h*ng/mL respectively).
- STI-5041 was administered IN at 20 mg/kg and the PK parameters were assessed
- SARS-CoV-2 pathogenesis in the Syrian golden hamster model of infection provides a means of assessing nAb activity in a preclinical model of respiratory disease.
- Animals inoculated with 1x105 TCID50 of SARS-CoV-2 WA-1 strain intranasally were treated with STI-2020 (100, 300 or 500 pg) or STI-5041 (500 or 1000 pg) ( Figure 10) administered intravenously (IV) at 1-hour post- infection.
- mice treated with 500 pg of STI-2020 maintained an average body weight that as a percentage of day 0 weight was significantly different on days 3 and then throughout the study than the average weight measured among IsoCtl-treated animals.
- an average of 1.6x103 TCID50/g of virus was detected in lung at day 5 post-infection ( Figure 10B).
- Treatment with 500 pg STI-2020 resulted in reduction of virus titers below the level of detection in lungs of all animals tested, a STI-2020 -treatment- related lung titer reduction of 80-fold at minimum.
- STI-5041 was administered at either 500 pg or 1,000 pg IV at 1-hour postinfection.
- STI-5041 protected animals from the pathogenic effects of the Beta variant.
- a significant difference in weight was observed at 4 d.p.i. for animals treated with 1000 pg IV STI-5041 versus IsoCtl-treated animals (1000 pg), averaging -3.249% ⁇ 1.738 versus - 7.534% ⁇ 0.882, respectively (Figure IOC).
- Figure IOC On 5 d.p.i.
- STI-2020- treated animals at 500 pg maintained their average weight (average 2.0 % weight loss respectively) over the first four days of infection before steadily gaining weight, while IsoCtl- treated animals steadily lost weight across this timespan through day 5. Beginning on day 5 of infection, animals treated with 500 pg STI-2020 IN had a significant weight increase in comparison to IsoCtl-treated animals, with significant increases in weight also observed at 6, 7, 8, 12 and 15 d.p.i. ( Figure 11A).
- STI-5041 5x104 TCID50 of the SARS-CoV-2 Beta variant was administered to female hamsters intranasally, 12 hours post- infection STI -5041 was administered IN at 500, 300 or 100 pg, and IsoCtl was administered at 500 pg. In IsoCtl- treated animals, the greatest % weight change was observed on day 5 with a change of -7.7% ⁇ 2.5.
- Example 7 Provided in this Example 7, inter alia, is an exemplary use the G-MAB library for the rapid identification of SARS-CoV-2 neutralizing antibodies, demonstrating its potential for developing countermeasures against any potential pathogen. Screening for G-MAB nAbs requires neither animal immunization nor availability of B-cell donor survivors. We demonstrate this with the presented screen for nAbs with pan-specific activity against SARS- CoV-2 and VOCs using an unbiased approach. Once a candidate nAb has been identified, the LALA Fc modification can be incorporated to reduce the risk for ADE.
- STI-2020 and STI-5041 demonstrated potent neutralizing activity against SARS-CoV-2, as well as relevant VOCs Alpha and Beta.
- IV and IN delivery of STI-2020 and STI-5041 suggests that their use in treatment of COVID-19 might be well tolerated.
- STI-2020 provided neutralizing protection against virus isolates and virus pseudotypes bearing amino acid changes within Spike corresponding to 2020001 and Alpha but little effect on the Beta VOC.
- STI-5041 demonstrated efficacy against 2020001, Alpha and Beta, demonstrating the utility of a combination treatment as a potential future therapy.
- nAbs that provides a broad spectrum of VOC coverage should provide a potent means to treat early asymptomatic and mildly symptomatic COVID-19 patients, regardless of the variant causing infection.
- nAbs both intravenously and intranasally may have important therapeutic implications, as multiple modes of effective treatment delivery could ease the therapeutic relationship between provider and patient in the setting of respiratory infection.
- Phase 1 healthy subjects studies have been completed for either intravenous or intranasal administration of STI-2020, where the intranasal formulation of STI-2020 is termed STI-2099.
- the antibodies STI-2020 and STI- 2099 have been well tolerated at the concentrations tested, with no dose limiting toxicity, serious or severe adverse events (AEs), and no AEs resulting in early termination from participation.
Abstract
La présente divulgation concerne des protéines de liaison à l'antigène, telles que des anticorps entièrement humains, qui se lient spécifiquement à la protéine de spicule (S) du coronavirus SARS-CoV-2, y compris des variants du coronavirus SARS-CoV-2, ainsi que leurs utilisations. Dans divers modes de réalisation, les anticorps anti-protéine de spicule sont des anticorps neutralisants qui empêchent la liaison d'un coronavirus SARS-CoV-2, tel qu'un variant du coronavirus SARS-CoV-2, à une cellule cible. Sont également compris des anticorps anti-protéine S1, des fragments d'anticorps et des anticorps à chaîne unique, ainsi que des molécules d'acide nucléique et des vecteurs codant pour de telles protéines de liaison à l'antigène anti-S1. Sont en outre compris des cellules hôtes, des kits et des compositions pharmaceutiques comprenant de tels protéines de liaison à l'antigène, anticorps, fragments d'anticorps et anticorps à chaîne unique. Sont enfin comprises des méthodes de traitement ou de prévention d'une infection par un coronavirus, tel que le coronavirus SARS-CoV-2, y compris un variant du coronavirus SARS-CoV-2, par l'administration, à des sujets, de tels protéines de liaison à l'antigène, anticorps, fragments d'anticorps, anticorps à chaîne unique, molécules d'acide nucléique, vecteurs, cellules hôtes, ou kits et/ou compositions pharmaceutiques les comprenant.
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Citations (5)
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US20140234340A1 (en) * | 2010-11-30 | 2014-08-21 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule capable of binding to plurality of antigen molecules repeatedly |
US20180258175A1 (en) * | 2015-09-16 | 2018-09-13 | Ablexis, Llc | Anti-cd115 antibodies |
US10787501B1 (en) * | 2020-04-02 | 2020-09-29 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
WO2021226249A1 (fr) * | 2020-05-06 | 2021-11-11 | Sorrento Therapeutics, Inc. | Anticorps neutralisants se liant à la protéine s de sars-cov-2 |
WO2022032139A1 (fr) * | 2020-08-07 | 2022-02-10 | Sorrento Therapeutics, Inc. | Anticorps neutralisants se liant à la protéine s du sars-cov-2 |
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Publication number | Priority date | Publication date | Assignee | Title |
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US20140234340A1 (en) * | 2010-11-30 | 2014-08-21 | Chugai Seiyaku Kabushiki Kaisha | Antigen-binding molecule capable of binding to plurality of antigen molecules repeatedly |
US20180258175A1 (en) * | 2015-09-16 | 2018-09-13 | Ablexis, Llc | Anti-cd115 antibodies |
US10787501B1 (en) * | 2020-04-02 | 2020-09-29 | Regeneron Pharmaceuticals, Inc. | Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments |
WO2021226249A1 (fr) * | 2020-05-06 | 2021-11-11 | Sorrento Therapeutics, Inc. | Anticorps neutralisants se liant à la protéine s de sars-cov-2 |
WO2022032139A1 (fr) * | 2020-08-07 | 2022-02-10 | Sorrento Therapeutics, Inc. | Anticorps neutralisants se liant à la protéine s du sars-cov-2 |
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