Multivalent anti-spike protein binding molecules and uses thereof

Abstract

The present disclosure provides multivalent anti-spike protein binding molecules comprising a multimerization moiety linked to an anti-spike protein antigen-binding domain that specifically binds to the RBD region of SARS-CoV and / or SARS-CoV-2. The disclosure further relates to methods of producing the multivalent anti-spike protein binding molecules, pharmaceutical compositions comprising the multivalent anti-spike protein binding molecules, and methods of using the multivalent anti-spike protein binding molecules to treat conditions associated with SARS-CoV and SARS-CoV-2 infection, such as COVID-19.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint application of U.S. Provisional Application No. 63 / 487,408 (filed February 28, 2023), the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML Sequence Listing was created on February 27, 2024, is named RGN-028WO_SL.xml, and is 643,930 bytes in size. [Background technology]

[0003] 3.Background technology Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus of the Betacoronavirus genus, which also includes SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-OC43, and HCoV-HKU1 (Non-Patent Document 1). SARS-CoV-2 causes COVID-19, a potentially life-threatening disease that was first characterized in late 2019 and spread into a global pandemic in early 2020.

[0004] SARS-CoV-2 shares approximately 80% identity with SARS-CoV, and both viruses depend on interaction with angiotensin-converting enzyme 2 (ACE2) for cell entry, an enzyme expressed on the extracellular surface of many types of cells.

[0005] Several vaccines against SARS-CoV-2 are currently available to prevent the development of severe disease. However, vaccination rates vary across populations, and even in areas with high vaccination rates, breakthrough infections causing COVID-19 have been observed in individuals immunized against SARS-CoV-2. Because some individuals have been diagnosed with COVID-19 multiple times, previous SARS-CoV-2 infection does not appear to confer complete immunity against any future infection. Furthermore, in some individuals, SARS-CoV-2 infection causes prolonged illness associated with the persistence of one or more symptoms of COVID-19 for weeks to months after clearance of the infection. These observations highlight the serious population health threat posed by COVID-19 and the need to combat SARS-CoV-2 infection with effective treatments.

[0006] Small molecule treatments such as Paxlovid (a combination of the oral antiviral drugs nilmatrervir and ritonavir) can prevent hospitalization, but they are associated with a "Paxlovid rebound" effect, in which the virus re-emerges (NPL 2). On the other hand, biologic treatments such as monoclonal antibodies can directly slow viral growth, resulting in robust and long-lasting therapeutic effects. However, due to the rapid emergence of new SARS-CoV-2 variants, antibodies isolated from patients are typically strain-specific, rendering them ineffective against specific SARS-CoV-2 variants. Therefore, there remains a need to develop neutralizing therapies that are effective against SARS-CoV-2. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jackson et al.,2022,Nat Rev Mol Cell Biol.23(1):3-20 [Non-patent document 2] Callaway,Nature(News),11 August 2022 Summary of the Invention [Means for solving the problem]

[0008] 4. Overview The present disclosure relates to multivalent antigen-binding molecules (generically referred to herein as "multivalent anti-spike protein binding molecules") that bind to coronavirus spike proteins to inhibit interactions between coronaviruses and host cells. Multivalent anti-spike protein binding molecules of the present disclosure typically comprise multiple anti-spike protein antigen-binding domains (ABDs) operably linked by one or more multimerization moieties. Multivalent anti-spike protein binding molecules of the present disclosure are described in Section 6.2 and in numbered embodiments 1-96.

[0009] Multivalent anti-spike protein binding molecules of the present disclosure are typically tetravalent and comprise four spike protein ABDs, e.g., in the form of Fabs or scFvs. The spike protein ABDs of the multivalent anti-spike protein binding molecules of the present disclosure can be monospecific (e.g., all ABDs bind to the same region of the spike protein and, optionally, all have the same sequence) or multispecific (e.g., at least two of the ABDs bind to different regions or variants of the spike protein and have different sequences). Spike protein ABDs and spike protein ABD formats suitable for incorporation into multivalent anti-spike protein binding molecules of the present disclosure are described in Sections 6.3 and 6.4, and in numbered embodiments 3-15 and 48-61.

[0010] Multivalent anti-spike protein binding molecules of the present disclosure comprise one or more multimerization moieties, e.g., one or more multimerization moieties comprising or consisting of an Fc domain. Multimerization moieties suitable for incorporation into multivalent anti-spike protein binding molecules of the present disclosure are described in Section 6.5 and in numbered embodiments 28-32 and 36-39.

[0011] Two or more components of the multivalent anti-spike binding protein binding molecules of the present disclosure can be connected to each other by a linker, e.g., a peptide linker. By way of example and not limitation, a linker can be used to connect the spike protein ABD to the multimerization moiety. Linkers suitable for incorporation into the multivalent anti-spike binding protein binding molecules of the present disclosure are described in Section 6.6 and in numbered embodiments 44-47.

[0012] Multivalent anti-spike protein binding molecules of the present disclosure can include a linker that is a hinge region. Suitable hinge sequences for incorporation into multivalent anti-spike protein binding molecules of the present disclosure are described in Section 6.6.1 and in numbered embodiments 40-43.

[0013] The present disclosure further provides nucleic acids encoding the multivalent anti-spike protein binding molecules of the present disclosure, host cells engineered to express the multivalent anti-spike protein binding molecules of the present disclosure, and recombinant methods for producing the multivalent anti-spike protein binding molecules of the present disclosure. Such nucleic acids, host cells, and production methods are described in Section 6.7 and numbered embodiments 97-99.

[0014] The present disclosure further provides pharmaceutical compositions comprising the multivalent anti-spike protein binding molecules of the present disclosure, as well as therapeutic indications and methods of use. Pharmaceutical compositions are described in Section 6.8 and numbered embodiment 100. Methods of use of the multivalent anti-spike protein binding molecules are described in Section 6.9 and numbered embodiments 101-113.

[0015] Other features and advantages of embodiments of the multivalent anti-spike protein binding molecules of the present disclosure will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0016] [Figure 1A]Illustrated are exemplary multivalent anti-spike protein binding molecules of the present disclosure. Figure 1A depicts a monospecific tetravalent 2x2 N-Fab alternating format ("AF") antibody construct with identical Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1B depicts a bispecific tetravalent 2x2 N-Fab AF construct with different Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1C depicts a monospecific tetravalent 2x2 C-Fab AF construct with identical Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. Figure 1D depicts a bispecific tetravalent 2x2 C-Fab AF construct with different Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. [Figure 1B] Illustrated are exemplary multivalent anti-spike protein binding molecules of the present disclosure. Figure 1A depicts a monospecific tetravalent 2x2 N-Fab alternating format ("AF") antibody construct with identical Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1B depicts a bispecific tetravalent 2x2 N-Fab AF construct with different Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1C depicts a monospecific tetravalent 2x2 C-Fab AF construct with identical Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. Figure 1D depicts a bispecific tetravalent 2x2 C-Fab AF construct with different Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. [Figure 1C]Illustrated are exemplary multivalent anti-spike protein binding molecules of the present disclosure. Figure 1A depicts a monospecific tetravalent 2x2 N-Fab alternating format ("AF") antibody construct with identical Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1B depicts a bispecific tetravalent 2x2 N-Fab AF construct with different Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1C depicts a monospecific tetravalent 2x2 C-Fab AF construct with identical Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. Figure 1D depicts a bispecific tetravalent 2x2 C-Fab AF construct with different Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. [Figure 1D] Illustrated are exemplary multivalent anti-spike protein binding molecules of the present disclosure. Figure 1A depicts a monospecific tetravalent 2x2 N-Fab alternating format ("AF") antibody construct with identical Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1B depicts a bispecific tetravalent 2x2 N-Fab AF construct with different Fab arms, each comprising two consecutively linked Fab portions and attached to the N-terminus of the Fc portion. Figure 1C depicts a monospecific tetravalent 2x2 C-Fab AF construct with identical Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. Figure 1D depicts a bispecific tetravalent 2x2 C-Fab AF construct with different Fab arms, comprising Fab portions attached to the N-terminus of the Fc portion, which are also linked to a Fab portion on their C-terminus. [Figure 2A]Figure 2 shows the neutralization activity of different 2x2 N-Fab AF constructs against SARS-CoV-2 pseudovirus variants. Individual Fab arms from REGN10987, 14315, 10933, 14256, and 10985 were used and paired with each other or with each other. Figure 2A shows the pseudovirus neutralization IC50 values ​​of constructs grouped by Fab arm against the D614G variant, while Figure 2B shows the neutralization IC50 values ​​against the BA.2 variant. Black circles represent 2x2 N-Fab AF constructs with identical Fab arms and are therefore monospecific. White circles represent 2x2 N-Fab AF constructs with different Fab arms and are therefore bispecific. Black triangles for each Fab group represent the parental IgG control with that Fab. [Figure 2B] Figure 2 shows the neutralization activity of different 2x2 N-Fab AF constructs against SARS-CoV-2 pseudovirus variants. Individual Fab arms from REGN10987, 14315, 10933, 14256, and 10985 were used and paired with each other or with each other. Figure 2A shows the pseudovirus neutralization IC50 values ​​of constructs grouped by Fab arm against the D614G variant, while Figure 2B shows the neutralization IC50 values ​​against the BA.2 variant. Black circles represent 2x2 N-Fab AF constructs with identical Fab arms and are therefore monospecific. White circles represent 2x2 N-Fab AF constructs with different Fab arms and are therefore bispecific. Black triangles for each Fab group represent the parental IgG control with that Fab. [Figure 3A]Figure 3 shows the neutralizing activity of different 2x2 C-Fab AF constructs against SARS-CoV-2 pseudovirus variants. Figure 3A shows the pseudovirus neutralization IC50 values ​​of constructs against the D614G variant, grouped by Fab arm, while Figure 3B shows the neutralization IC50 values ​​of the same constructs against the BA.2 variant. Black circles represent 2x2 C-Fab AF constructs with identical Fab arms and are therefore monospecific. White circles represent 2x2 C-Fab AF constructs with different Fab arms and are therefore bispecific. Black triangles for each Fab group represent the parental IgG control with that Fab. [Figure 3B] Figure 3 shows the neutralizing activity of different 2x2 C-Fab AF constructs against SARS-CoV-2 pseudovirus variants. Figure 3A shows the pseudovirus neutralization IC50 values ​​of constructs against the D614G variant, grouped by Fab arm, while Figure 3B shows the neutralization IC50 values ​​of the same constructs against the BA.2 variant. Black circles represent 2x2 C-Fab AF constructs with identical Fab arms and are therefore monospecific. White circles represent 2x2 C-Fab AF constructs with different Fab arms and are therefore bispecific. Black triangles for each Fab group represent the parental IgG control with that Fab. [Figure 4A] Figure 4A shows the dose-dependent neutralization activity of REGN14287-based tetravalent AF molecules against the SARS-CoV-2 pseudovirus omicron variant BQ.1. Figure 4A shows the neutralization activity of COVAF-46-51 at a dose titration of 2x2 N-Fab with the bivalent parental IgG REGN14287 control and the REGN10933 / REGN10987 cocktail combination, in terms of percent neutralization against BQ.1. Figure 4B shows the neutralization activity of COVAF-52-57 at a dose titration of 2x2 C-Fab with the bivalent parental IgG REGN14287 control and the REGN10933 / REGN10987 cocktail combination, in terms of percent neutralization against BQ.1. [Figure 4B]Figure 4A shows the dose-dependent neutralization activity of REGN14287-based tetravalent AF molecules against the SARS-CoV-2 pseudovirus omicron variant BQ.1. Figure 4A shows the neutralization activity of COVAF-46-51 at a dose titration of 2x2 N-Fab with the bivalent parental IgG REGN14287 control and the REGN10933 / REGN10987 cocktail combination, in terms of percent neutralization against BQ.1. Figure 4B shows the neutralization activity of COVAF-52-57 at a dose titration of 2x2 C-Fab with the bivalent parental IgG REGN14287 control and the REGN10933 / REGN10987 cocktail combination, in terms of percent neutralization against BQ.1. DETAILED DESCRIPTION OF THE INVENTION

[0017] 6. Detailed Description 6.1.Definition As used herein, the following terms are intended to have the following meanings:

[0018] About, Approximately: The terms "about," "approximately," and the like are used throughout the specification before numerical values ​​to indicate that the numerical value is not necessarily exact (e.g., to account for fractions, variations in measurement precision and / or accuracy, timing, etc.). A disclosure of "about X" or "approximately X," where X is a numerical value, should be understood to also disclose "X." Thus, for example, disclosure of embodiments in which a sequence has "about X% sequence identity" to another sequence is also a disclosure of embodiments in which the sequence has "X% sequence identity" to the other sequence.

[0019] And / Or: Unless otherwise indicated, the conjunction "or" is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (A or B, where the selection of A is mutually exclusive of B) and the selection of conjoint features (A or B, where both A and B are selected). In several places in the text, the term "and / or" is used interchangeably and should not be construed to mean that "or" is used in reference to mutually exclusive alternatives.

[0020] Antibody: As used herein, the term "antibody" refers to a polypeptide (or set of polypeptides) of the immunoglobulin family that can non-covalently, reversibly, and specifically bind to an antigen. For example, a naturally occurring "antibody" of the IgG type is a tetramer comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-id) antibodies. Antibodies can be of any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, placental transport, Fc receptor binding, and complement fixation. By convention, the numbering of constant region domains increases further from the antigen-binding domain or amino-terminus of the antibody. The N-terminus is the variable region, the C-terminus is the constant region, and the CH3 and CL domains represent the carboxy-termini of the heavy and light chains of native antibodies, respectively. For convenience, and unless the context dictates otherwise, reference to an antibody also refers to antibody fragments and engineered antibodies containing non-naturally occurring antigen-binding domains and / or antigen-binding domains with non-native configurations.

[0021] Antigen-binding molecule or ABM: As used herein, the term "antigen-binding molecule" or "ABM" refers to a molecule (e.g., an assembly of multiple polypeptide chains) comprising two half antibodies. Typically, each half antibody comprises at least one antigen-binding domain. In some embodiments, the antigen is a coronavirus spike protein; therefore, ABMs of the present disclosure are generally referred to as "anti-spike protein-binding molecules." ABMs of the present disclosure can be monospecific or multispecific (e.g., bispecific). While the antigen-binding domains in a monospecific binding molecule all bind to the same epitope, a multispecific binding molecule has at least two antigen-binding sites that bind to different epitopes, which can be the same or different molecules (e.g., different spike protein variants).

[0022] Antigen-binding domain: As used herein, the term "antigen-binding domain" or "ABD" refers to a portion of an antibody or antibody fragment that has the ability to non-covalently, reversibly, and specifically bind to an antigen. Examples of antibody fragments that can contain an ABD include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of the VL domain, VH domain, CL domain, and CH1 domain, F(ab)2 fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region, Fd fragments consisting of the VH domain and CH1 domain, Fv fragments consisting of the VL domain and VH domain of a single antibody arm, dAb fragments consisting of the VH domain (Ward et al., 1989, Nature 341:544-546), and isolated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses both proteolytic fragments of antibodies (e.g., Fab fragments and F(ab)2 fragments) and engineered proteins containing one or more portions of antibodies (e.g., scFv). Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology 23:1126-1136).

[0023] Associated: The term "associated" in the context of a multivalent anti-spike protein-binding molecule refers to a functional relationship between two or more polypeptide chains. In particular, the term "associated" means that two or more polypeptides are associated with one another, e.g., noncovalently through molecular interactions or covalently through one or more disulfide or chemical crosslinks, to generate a functional multivalent anti-spike protein-binding molecule. Examples of associations that may be present in multivalent anti-spike protein-binding molecules of the present disclosure include (but are not limited to) associations between Fc domains to form an Fc region (e.g., as described in Section 6.5.1).

[0024] Bivalent: As used herein, the term "bivalent" refers to a binding molecule that contains two antigen-binding domains, whether in the same polypeptide chain or on different polypeptide chains.

[0025] Complementarity-determining region: The term "complementarity-determining region" or "CDR" as used herein refers to the sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. For example, each heavy chain variable region generally has three CDRs (CDR-H1, CDR-H2, and CDR-H3), and each light chain variable region generally has three CDRs (CDR-L1, CDR-L2, and CDR-L3). The precise amino acid sequence boundaries of a given CDR can be determined using the "Kabat" numbering scheme, Kabat et al., 1991, "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., 1997, JMB 273:927-948 ("Chothia" numbering scheme), and ImMunoGenTics (IMGT) numbering scheme (Lefranc, 1999, The Immunologist 7:132-136; Lefranc et al., 1999, The Immunologist 7:132-136). The CDR numbering scheme can be determined using any of several well-known schemes, including those described by Kabat et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme). For example, for the classical format, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3), and the CDR amino acid residues in the light chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3). The CDR amino acid residues in the VL are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3), and the amino acid residues in the VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3).Combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH, and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL. Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) ("Kabat" numbering). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0026] Constant domain: The term "constant domain" refers to a CH1, CH2, CH3, or CL domain of an immunoglobulin. The term "CH1 domain" refers to the heavy chain constant region that connects the variable domain to the hinge within the heavy chain constant domain. In some embodiments, the term "CH1 domain" refers to the region of an immunoglobulin molecule spanning amino acids 118-215 (EU numbering). The term "CH1 domain" encompasses wild-type CH1 domains and variants thereof (e.g., non-naturally occurring CH1 domains or modified CH1 domains). For example, the term "CH1 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH1 domains and variants thereof with 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, and / or additions. Exemplary CH1 domains include CH1 domains with mutations that modify antibody biological activity, such as ADCC, CDC, or half-life.

[0027] The term "CH2 domain" refers to the heavy chain constant region that connects the hinge to the CH3 domain within the heavy chain constant domain. In some embodiments, the term "CH2 domain" refers to the region of an immunoglobulin molecule spanning amino acids 238-340 (EU numbering). The term "CH2 domain" encompasses wild-type CH2 domains and variants thereof (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH2 domains and variants thereof with 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutation, e.g., substitution, deletion, and / or addition. Exemplary CH2 domains include CH2 domains with mutations that modify antibody biological activities, such as ADCC, CDC, purification, dimerization, and half-life.

[0028] The term "CH3 domain" refers to the heavy chain constant region C-terminal to the CH2 domain within the heavy chain constant domain. In some embodiments, the term "CH3 domain" refers to the region of an immunoglobulin molecule spanning amino acids 341-447 (EU numbering). The term "CH3 domain" encompasses wild-type CH3 domains and variants thereof (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH3 domains and variants thereof with 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, and / or additions. Exemplary CH3 domains include CH3 domains with mutations that modify antibody biological activities, such as ADCC, CDC, purification, dimerization, and half-life.

[0029] The term "CL domain" refers to the constant region of an immunoglobulin light chain. The term "CL domain" encompasses wild-type CL domains (e.g., kappa or lambda light chain constant regions) as well as variants thereof (e.g., non-naturally occurring CL domains or modified CL domains). For example, the term "CL domain" includes wild-type kappa and lambda constant domains as well as variants thereof having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5, and / or up to 5, 4, 3, 2, or 1 mutations, e.g., substitutions, deletions, and / or additions.

[0030] COVID-19: The term "COVID-19" stands for "Coronavirus disease 2019" and refers to the infectious disease caused by SARS-CoV-2 infection. Patients with COVID-19 can experience a wide range of symptoms, ranging from mild to severe. Symptoms may include, but are not limited to, fever, chills, cough, shortness of breath, difficulty breathing, fatigue, muscle aches, body aches, headache, loss of smell, loss of taste, sore throat, congestion, runny nose, nausea, and diarrhea.

[0031] EC50: The term "EC50" refers to the half maximal effective concentration of a molecule (e.g., a multivalent anti-spike protein-binding molecule) that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of a multivalent anti-spike protein-binding molecule at which 50% of its maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration of a multivalent anti-spike protein-binding molecule that confers half-maximal virus or pseudovirus neutralization in the assay described in Section 8.1.2.

[0032] Epitope: An epitope, or antigenic determinant, is the portion of an antigen that is recognized by an antibody or fragment thereof, e.g., an antigen-binding domain. Epitopes can be linear or conformational.

[0033] Fab: The term "Fab" refers to a pair of polypeptide chains, where the first polypeptide chain comprises an antibody variable heavy (VH) domain operably linked (typically N-terminally) to a first constant domain (referred to herein as C1), and the second polypeptide chain comprises an antibody N-terminal variable light (VL) domain operably linked (typically N-terminally) to a second constant domain (referred to herein as C2) that can pair with the first constant domain. In a native antibody, the VH is N-terminal to the first constant domain (CH1) of the heavy chain, and the VL is N-terminal to the constant domain (CL) of the light chain. The Fabs of the present disclosure can be oriented according to their natural orientation or can include domain substitutions or swaps that promote correct VH and VL pairing. For example, the CH1 and CL domain pair in a Fab can be replaced with a CH3 domain pair to promote modified correct Fab-chain pairing in a heterodimeric molecule. It is also possible to reverse the CH1 and CL, so that CH1 is attached to VL and CL is attached to VH, a configuration commonly known as a Crossmab. The term "Fab" encompasses single-chain Fab.

[0034] Fc domain and Fc region: The term "Fc domain" refers to the portion of a heavy chain that pairs with the corresponding portion of another heavy chain. In some embodiments, an Fc domain comprises a CH2 domain followed by a CH3 domain, with or without a hinge region N-terminal to the CH2 domain. The term "Fc region" refers to the region formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region may be the same or different from each other. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains may be modified to allow heterodimerization, for example, via knob-in-hole interactions.

[0035] Fv: The term "Fv" refers to the smallest antibody fragment derivable from an immunoglobulin that contains a complete target recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). In this configuration, the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. In many cases, six CDRs confer target binding specificity to the antibody. However, in some instances, even a single variable domain (or half of an Fv containing only three CDRs specific for a target) can have the ability to recognize and bind to a target. Reference to a VH-VL dimer herein is not intended to convey any particular configuration. When present on a single polypeptide chain (e.g., scFv), the VH and VL are at the N- or C-terminus.

[0036] Half antibody: The term "half antibody" refers to a molecule that contains at least an Fc domain and can associate with another molecule that contains an Fc domain, e.g., through disulfide bridges or molecular interactions (e.g., knob-in-hole interactions between Fc heterodimers). Half antibodies can be composed of one polypeptide chain or more than one polypeptide chain (e.g., heavy and light chains).

[0037] Hinge: As used herein, the term "hinge" is intended to include the region of consecutive amino acid residues connecting the C-terminus of the CH1 domain to the N-terminus of the CH2 domain of an immunoglobulin. Several amino acids at the N-terminus of the CH2 domain, encoded by the CH2 exon, are also considered part of the "lower hinge." Without being bound by any one theory, the amino acids in the hinge regions of IgG1, IgG2, and IgG4 have been characterized as including 12-15 consecutive amino acids encoded by different hinge exons and several N-terminal amino acids of the CH2 domain (encoded by the CH2 exon) (Brekke et al., 1995, Immunology Today 16(2):85-90). On the other hand, IgG3 contains a hinge region consisting of four segments: one upper segment resembling the hinge region of IgG1, and three segments with identical amino acid repeats unique to IgG3.

[0038] Host cell: As used herein, the term "host cell" refers to a cell into which a nucleic acid of the present disclosure has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer to the particular subject cell, and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the term as used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Exemplary eukaryotic host cells include yeast and mammalian cells, e.g., vertebrate cells such as mouse, rat, monkey, or human cell lines, e.g., HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0039] Immunoglobulin: The term "immunoglobulin" (Ig) refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains (one pair of light (L) chains and one pair of heavy (H) chains), all four of which may be interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Each heavy chain typically contains a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (CH or CH). The heavy chain constant region typically contains three domains: CH1, CH2, and CH3. The CH1 and CH2 domains are connected by a hinge. The Fc portion contains at least the CH2 and CH3 domains.

[0040] Typically, the numbering of amino acid residues in immunoglobulins is according to IMGT, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), or according to Kabat's EU numbering system (also known as "EU numbering" or "EU index"), e.g., as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

[0041] Linker: As used herein, the term "linker" refers to a connecting peptide between two moieties. For example, a linker can connect the spike protein ABD to the Fc domain.

[0042] Multivalent: As used herein, the term "multivalent" refers to an antigen-binding molecule that comprises two or more ABDs on one, two, or more polypeptide chains. Neutralizing, Blocking: A "neutralizing" or "blocking" spike protein ABD refers to an ABD whose binding to the spike protein inhibits the activity of the spike protein to any detectable extent, e.g., inhibiting the ability of the spike protein to bind to a receptor such as ACE2, to be cleaved by a protease such as TMPRSS2, or to mediate viral entry into or reproduction in a host cell.

[0043] Operably linked: The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of a fusion protein or other polypeptide, the term "operably linked" means that two or more amino acid segments are joined to produce a functional polypeptide. For example, in the context of the multivalent anti-spike protein binding molecules of the present disclosure, separate components (e.g., spike protein ABD and Fc domains) can be operably linked directly or via a peptide linker sequence. In the context of a nucleic acid encoding a fusion protein, such as a multivalent anti-spike protein binding molecule of the present disclosure, "operably linked" means that the two nucleic acids are connected such that the amino acid sequences encoded by the two nucleic acids remain in-frame.

[0044] Polypeptide, Peptide, and Protein: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0045] Recognize: As used herein, the term "recognize" refers to an antibody or antibody fragment (e.g., spike protein ABD) that finds and interacts with (e.g., binds to) the epitope.

[0046] Single-chain Fab or scFab: As used herein, the term "single-chain Fab" or "scFab" refers to a polypeptide chain comprising the VH, CH1, VL and CL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0047] Single-chain Fv or scFv: As used herein, the term "single-chain Fv" or "scFv" refers to an antibody comprising the VH and VL domains, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Plückthun in *The Pharmacology of Monoclonal Antibodies*, vol. 113, Rosenburg and Moore eds. (1994), Springer-Verlag, New York, pp. 269-315. The VH and VL can be arranged in N-terminal to C-terminal order, VH-VL or VL-VH, typically separated by a linker.

[0048] Subject: The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" and "subject" are used interchangeably herein.

[0049] Tetravalent: As used herein with respect to an antigen-binding molecule, the term "tetravalent" refers to an antigen-binding molecule that comprises four ABDs. In some embodiments, a tetravalent anti-spike protein-binding molecule refers to an anti-spike protein-binding molecule that comprises four spike protein ABDs. The four spike protein ABDs can be the same or different. In some embodiments, the tetravalent spike protein-binding molecule has a configuration shown in any one of Figures 1A-1D.

[0050] Treat, Treatment, Treating: As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or condition, and / or the alleviation of one or more symptoms (preferably one or more discernible symptoms) of a disease or condition, resulting from the administration of one or more multivalent anti-spike protein binding molecules of the disclosure.

[0051] In some embodiments, the disease or condition is caused by a coronavirus infection, e.g., SARS-CoV or SARS-CoV-2 (e.g., COVID-19). In some embodiments, the disease or condition is a SARS-CoV or SARS-CoV-2 infection, or any other disease associated with a similar infection. With reference to these diseases and conditions, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of disease progression, severity, and / or duration, or alleviation of one or more symptoms (preferably one or more discernible symptoms) of the disease, resulting from administration of one or more multivalent anti-spike protein binding molecules of the present disclosure. In certain embodiments, the terms "treat," "treatment," and "treating" refer to a reduction in at least one measurable (not necessarily discernible by the patient) physical parameter of COVID-19, such as blood oxygen saturation level. In other embodiments, the terms "treat," "treatment," and "treating" refer to an inhibition of COVID-19 progression, either physically (e.g., by stabilization of a discernible symptom), physiologically (e.g., by stabilization of a physical parameter), or both. In other embodiments, the terms "treat", "treatment" and "treating" refer to the reduction or elimination of an infection.

[0052] 6.2. Multivalent Anti-Spike Protein Binding Molecules Disclosed herein are multivalent antigen binding molecules (generically referred to herein as "multivalent anti-spike protein binding molecules") that bind to coronavirus spike proteins.

[0053] Anti-spike protein binding molecules of the present disclosure typically have a valency of more than 2. In some embodiments, an anti-spike protein binding molecule comprises at least four antigen binding domains (ABDs) that bind to a spike protein. In some embodiments, an anti-spike protein binding molecule is tetravalent.

[0054] Anti-spike protein binding molecules can be monospecific or multispecific. In some embodiments, the anti-spike protein binding molecule is monospecific, with all ABDs binding to the same epitope and, optionally, all having the same binding sequence.

[0055] In other embodiments, the anti-spike protein binding molecule is multispecific, with the ABD binding to two or more different epitopes. In some embodiments, the anti-spike protein binding molecule is bispecific, with antigen-binding domains that bind to two different epitopes. In some embodiments, the two different epitopes are spike protein epitopes, whether two different epitopes in the same spike protein, two different epitopes present in two spike protein variants, or a combination thereof.

[0056] In some embodiments, the tetravalent anti-spike protein binding molecule comprises four spike protein ABDs, e.g., in the form of Fabs or scFvs as described in Section 6.4.1 or Section 6.4.2, respectively.

[0057] The ABDs of the multivalent anti-spike protein binding molecules of the present disclosure are operably linked by one or more multimerization moieties, and there may be one or more polypeptide chains. Exemplary multimerization moieties are described in Section 6.5.

[0058] In some embodiments, one or more multimerizing moieties is a pair of Fc domains that associate to form an Fc dimer. The Fc dimer can be, for example, a homodimer as shown in Figure 1A or 1C, or a heterodimer as shown in Figure 1B or 1D. Each polypeptide chain comprising an Fc domain and an associated polypeptide chain (e.g., a light polypeptide chain) is referred to herein as a "half antibody."

[0059] Thus, the present disclosure provides multivalent anti-spike protein binding molecules comprising two half antibodies, hi some embodiments, each half antibody comprises at least two ABDs. In certain aspects, the present disclosure provides tetravalent multivalent spike protein binding molecules comprising two half antibodies, hi some embodiments, each half antibody comprises at least two ABDs.

[0060] In some embodiments, the present disclosure provides a multivalent spike protein binding molecule (sometimes referred to herein as a type 1 multivalent spike protein binding molecule or type 1 ABM) comprising two half antibodies: a) a first half antibody, i) a first ABD, typically comprising a first VH and a first VL; ii) an optional linker; iii) a second ABD, typically comprising a second VH and a second VL; iv) an optional linker (e.g., a hinge domain), and v) a first half antibody comprising a first Fc domain; and b) a second half antibody, i) a third ABD, typically comprising a third VH and a third VL; ii) an optional linker; iii) a fourth ABD, typically comprising a fourth VH and a fourth VL; iv) an optional linker (e.g., a hinge domain), and and v) a second half antibody comprising a second Fc domain.

[0061] In a further embodiment, the present disclosure provides a multivalent spike protein binding molecule comprising two half antibodies (sometimes referred to herein as a type 2 multivalent spike protein binding molecule or type 2 ABM), a) a first half antibody, i) a first ABD, typically comprising a first VH and a first VL; ii) an optional linker (e.g., a hinge domain); iii) a first Fc domain; iv) an optional linker, and v) a first half antibody comprising a second ABD, typically comprising a second VH and a second VL; b) a second half antibody, i) a third ABD, typically comprising a third VH and a third VL; ii) an optional linker (e.g., a hinge domain); iii) a second Fc domain; iv) an optional linker, and and v) a second half antibody comprising a fourth ABD, typically comprising a fourth VH and a fourth VL.

[0062] In both Type 1 and Type 2 ABMs, the first, second, third, and fourth ABDs can be the same or different. In some embodiments, the first, second, third, and fourth ABDs are the same. In other embodiments, the first and second ABDs are the same, and the third and fourth ABDs are the same (but different from the first and second ABDs). In further embodiments, the first and third ABDs are the same, and the second and fourth ABDs are the same (but different from the first and third ABDs).

[0063] In some embodiments, two of the ABDs, or all four of the ABDs, are scFvs, and thus the VH and VL of such ABDs are on the same polypeptide chain. Exemplary scFv structures are described in Section 6.4.2.

[0064] In some embodiments, two of the ABDs or all four of the ABDs are Fabs. Optionally, the Fab is not a single-chain Fab, and thus the VH and VL of such an ABD are on separate polypeptide chains. In some embodiments, the VH of the ABD is on the same polypeptide chain as the Fc domain of a half antibody comprising the ABD, and the VL is on a separate polypeptide chain. In some embodiments, the VL of the ABD is on the same polypeptide chain as the Fc domain of a half antibody comprising the ABD, and the VH is on a separate polypeptide chain. The Fab can be in a native immunoglobulin conformation, and the polypeptide chain comprising the VH can further comprise the CH1 domain of the Fab, or can be domain-swapped, such that the polypeptide chain comprising the VH can further comprise the CL domain of the Fab. Exemplary Fab structures (including domain-swapped structures) are described in Section 6.4.1.

[0065] Exemplary linkers for multivalent spike protein binding molecules of the present disclosure, including linkers for type 1 and type 2 multivalent spike protein binding molecules of the present disclosure, are described in Section 6.6.

[0066] In some embodiments, the multivalent spike protein-binding molecule is monospecific, and all ABDs bind to the same epitope. All ABDs in a monospecific multivalent spike protein-binding molecule may comprise the same CDR sequences or the same VH and VL sequences. In some embodiments, the ABDs of a monospecific multivalent spike protein-binding molecule are configured as shown in Figure 1A or 1C. Figure 1A shows a monospecific type 1 ABM, and Figure 1C shows a monospecific type 2 ABM. In Figure 1A, a Fab comprising VH3-VL3 represents the first ABD of the type 1 ABM, a Fab comprising VH1-VL1 represents the second ABD of the type 1 ABM, a Fab comprising VH4-VL4 represents the third ABD of the type 1 ABM, and a Fab comprising VH2-VL2 represents the fourth ABD of the type 1 ABM. In Figure 1B, a Fab comprising VH1-VL1 represents the first ABD of a type 2 ABM, a Fab comprising VH3-VL3 represents the second ABD of a type 2 ABM, a Fab comprising VH2-VL2 represents the third ABD of a type 2 ABM, and a Fab comprising VH4-VL4 represents the fourth ABD of a type 2 ABM. Monospecific type 1 and type 2 ABMs may comprise two identical half antibodies, such that association of the two half antibodies forms an Fc homodimer. Suitable Fc domains are described in Section 6.5.1. The Fc domain can include mutations that reduce effector function compared to a wild-type Fc domain, for example, as described in Section 6.5.1.1.

[0067] In some embodiments, the multivalent spike protein binding molecule is multispecific, with at least two ABDs binding different epitopes. At least two ABDs in the multispecific, multivalent spike protein binding molecule comprise different CDR sequences. In some embodiments, the multispecific, multivalent spike protein binding molecule is tetravalent, with a first pair of ABDs sharing the same CDR sequences or the same VH and VL sequences, and a second pair of ABDs sharing CDR sequences (different from the CDR sequences of the first pair of ABDs) or the same VH and VL sequences. In some embodiments, the ABDs of the multispecific, multivalent spike protein binding molecule are configured as shown in Figure 1B or Figure 1D. Figure 1B shows a multispecific type 1 ABM, and Figure 1D shows a multispecific type 2 ABM. In Figure 1B, a Fab comprising VH3-VL3 represents the first ABD of a type 1 ABM, a Fab comprising VH1-VL1 represents the second ABD of a type 1 ABM, a Fab comprising VH4-VL4 represents the third ABD of a type 1 ABM, and a Fab comprising VH2-VL2 represents the fourth ABD of a type 1 ABM. In Figure 1D, a Fab comprising VH1-VL1 represents the first ABD of a type 2 ABM, a Fab comprising VH3-VL3 represents the second ABD of a type 2 ABM, a Fab comprising VH2-VL2 represents the third ABD of a type 2 ABM, and a Fab comprising VH4-VL4 represents the fourth ABD of a type 2 ABM. Multispecific type 1 and type 2 ABMs may comprise two identical half antibodies or two different antibodies, and thus may form Fc homodimers or Fc heterodimers upon association of the two half antibodies. Suitable Fc domains are described in Section 6.5.1, and suitable approaches for heterodimerization are described in Section 6.5.1.2. The Fc domain may comprise mutations that reduce effector function compared to a wild-type Fc domain, e.g., as described in Section 6.5.1.1.

[0068] Exemplary spike protein ABD structures (e.g., CDR or VH / VL sequences) are disclosed in Section 6.3. 6.3. Spike Protein Antigen-Binding Domain The present disclosure relates to multivalent anti-spike protein binding molecules comprising multiple spike protein antigen binding domains (ABDs).

[0069] In some embodiments, a multivalent anti-spike protein binding molecule of the disclosure comprises two or more spike protein ABDs. In some embodiments, the multivalent anti-spike protein binding molecules of the present disclosure are monospecific, e.g., bind to the same epitope on the spike protein. In some of these embodiments, the spike protein ABDs are identical.

[0070] In other embodiments, the multivalent anti-spike protein binding molecules of the present disclosure are multispecific, e.g., bind to different epitopes. In some embodiments, the multispecific anti-spike protein binding molecules bind to different epitopes on the same spike protein. In other embodiments, the multispecific anti-spike protein binding molecules bind to different epitopes on different spike protein variants. The different epitopes can correspond to sequence variants of the same region in the spike protein or in entirely different regions.

[0071] In further embodiments, more than one or all of the ABDs in a multivalent anti-spike protein binding molecule of the present disclosure are capable of binding to the receptor binding domain (RBD) of the spike protein and / or blocking or neutralizing the spike protein, e.g., inhibiting the ability of the spike protein to bind to a receptor such as ACE2 that is cleaved by a protease such as TMPRSS2, or to mediate viral entry into or replication in a host cell.

[0072] Suitable spike protein ABD formats are described in Section 6.4. The spike protein ABD can be, for example, an antibody or an antigen-binding portion of an antibody, such as a Fab as described in Section 6.4.1 or an scFv as described in Section 6.4.2.

[0073] In some embodiments, the spike protein ABD competes for binding to spike protein with an exemplary antibody or an antibody having the sequence set forth in Table 1 below and / or comprises a binding portion of an exemplary antibody or an antibody having the antibody sequence set forth in Table 1. In some embodiments, the spike protein ABD competes for binding to spike protein with an antibody set forth in Table 1. In further embodiments, the spike protein ABD comprises CDRs having the CDR sequences of an antibody set forth in Table 1. In some embodiments, the spike protein ABD comprises all six CDR sequences of an antibody set forth in Table 1. In other embodiments, the spike protein ABD comprises at least a heavy chain CDR sequence (CDR-H1, CDR-H2, CDR-H3) of a universal light chain and a light chain CDR sequence. In a further aspect, the spike protein ABD comprises a VH comprising the amino acid sequence of the VH of an antibody shown in Table 1. In some embodiments, the spike protein ABD further comprises a VL comprising the amino acid sequence of the VL of an antibody shown in Table 1. In other embodiments, the spike protein ABD further comprises a universal light chain VL sequence.

[0074] [Table 1-1]

[0075] [Table 1-2]

[0076] [Table 1-3]

[0077] [Table 1-4]

[0078] [Table 1-5]

[0079] [Table 1-6]

[0080] In some embodiments, the spike protein ABD comprises the amino acid sequence or is encoded by the nucleotide sequence shown in Table 2 below. In particular aspects, the spike protein ABD comprises both the heavy chain CDRs and the light chain CDRs of an antibody shown in Table 2 below. In other embodiments, the spike protein ABD comprises at least the heavy chain CDR sequence and the light chain CDR sequence of a universal light chain. In further aspects, the spike protein ABD comprises a VH having the amino acid sequence of the VH of an antibody shown in Table 2 and a VL having the amino acid sequence of the VL of the same antibody as shown in Table 2. In other aspects, the spike protein ABD comprises a VH having the amino acid sequence of the VH of an antibody shown in Table 2 and a universal light chain VL sequence.

[0081] [Table 2-1]

[0082] [Table 2-2]

[0083] [Table 2-3]

[0084] [Table 2-4]

[0085] [Table 2-5]

[0086] Table 2-6

[0087] Table 2-7

[0088] Table 2-8

[0089] Table 2-9

[0090] Table 2-10

[0091] Table 2-11

[0092] In further embodiments, the spike protein ABD comprises the amino acid sequence set forth in Table 3 below. In particular aspects, the spike protein ABD comprises both the heavy chain CDRs and light chain CDRs of an antibody set forth in Table 3 below. In other embodiments, the spike protein ABD comprises at least the heavy chain CDR sequence and the light chain CDR sequence of a universal light chain. In further aspects, the spike protein ABD comprises a VH having the amino acid sequence of the VH of an antibody set forth in Table 3, and a VL having the amino acid sequence of the VL of the same antibody as set forth in Table 3. In other aspects, the spike protein ABD comprises a VH having the amino acid sequence of the VH of an antibody set forth in Table 3, and a universal light chain VL sequence. The initial sequence identifiers in Table 3 relate to the sequence listing of WO2021 / 045836A1, and the sequence identifiers are incorporated herein by reference, with the sequence identifiers provided in parentheses being those of the present disclosure.

[0093] [Table 3-1]

[0094] [Table 3-2]

[0095] [Table 3-3]

[0096] [Table 3-4]

[0097] In some embodiments, the spike protein ABD comprises the amino acid sequence shown in Table 4 below. In some embodiments, the spike protein ABD comprises both the heavy chain CDRs and the light chain CDRs of an antibody shown in Table 4 below. In particular embodiments, the spike protein ABD comprises a VH having the amino acid sequence of the VH of an antibody shown in Table 4, and a VL having the amino acid sequence of the VL of the same antibody as shown in Table 4. In other embodiments, the spike protein ABD comprises a VH having the amino acid sequence of the VH of an antibody shown in Table 4, and a universal light chain VL sequence. The initial sequence identifiers in Table 4 relate to the sequence listing of WO2023 / 287875A1, and the sequence identifiers are incorporated herein by reference, with the sequence identifiers provided in parentheses being those of the present disclosure.

[0098] [Table 4-1]

[0099] [Table 4-2]

[0100] [Table 4-3]

[0101] In some embodiments, the spike protein ABD of the multivalent anti-spike protein binding molecule comprises the heavy chain CDRs and light chain CDRs of antibody "mAb14287" shown in Table 4. Thus, in some embodiments, the spike protein ABD comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 579, 580, and 581, respectively, and a VL comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 398, 372, and 583, respectively. In some embodiments, the spike protein ABD comprises a VH 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 SEQ ID NO: 578, and a VL 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 SEQ ID NO: 582. In some embodiments, the spike protein ABD comprises a VH comprising the amino acid sequence of SEQ ID NO: 578 and a VL comprising the amino acid sequence of SEQ ID NO:582.

[0102] In some embodiments, the spike protein ABD of the multivalent anti-spike protein binding molecule comprises the heavy chain CDRs and light chain CDRs of antibody "mAb15160" shown in Table 4. Thus, in some embodiments, the spike protein ABD comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 507, 508, and 509, respectively, and a VL comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 511, 407, and 512, respectively. In some embodiments, the spike protein ABD comprises a VH 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 SEQ ID NO: 506, and a VL 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 SEQ ID NO: 510. In some embodiments, the spike protein ABD comprises a VH comprising the amino acid sequence of SEQ ID NO: 506, and a VL comprising the amino acid sequence of SEQ ID NO:510.

[0103] In some embodiments, the spike protein ABD of the multivalent anti-spike protein binding molecule comprises the heavy chain CDRs and light chain CDRs of antibody "mAb14315" shown in Table 4. Thus, in some embodiments, the spike protein ABD comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 450, 451, and 452, respectively, and a VL comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 454, 415, and 455, respectively. In some embodiments, the spike protein ABD comprises a VH 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 SEQ ID NO: 449, and a VL 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 SEQ ID NO: 453. In some embodiments, the spike protein ABD comprises a VH comprising the amino acid sequence of SEQ ID NO: 449 and a VL comprising the amino acid sequence of SEQ ID NO:453.

[0104] Exemplary formats for spike protein ABD are disclosed in Section 6.4 and include Fab (e.g., as described in Section 6.4.1) and scFv (e.g., as described in Section 6.4.2).

[0105] In some embodiments, the spike protein ABD is in the form of a Fab or scFv. In further embodiments, all spike protein ABDs in a multivalent spike protein binding molecule of the disclosure (e.g., a tetravalent spike protein binding molecule of the disclosure) are Fabs.

[0106] In some embodiments, the multivalent spike protein binding molecule is monospecific, and all ABDs bind to the same epitope. All ABDs in a monospecific, multivalent spike protein binding molecule may comprise the same CDR sequences or the same VH and VL sequences. In some embodiments, the ABDs of the multispecific, multivalent spike protein binding molecule are configured as shown in Figure 1A or 1C.

[0107] In some embodiments, the multivalent spike protein binding molecule is multispecific, with at least two ABDs binding different epitopes. At least two ABDs in the multispecific, multivalent spike protein binding molecule comprise different CDR sequences. In some embodiments, the multispecific, multivalent spike protein binding molecule is tetravalent, with a first pair of ABDs sharing the same CDR sequences or the same VH and VL sequences, and a second pair of ABDs sharing CDR sequences (different from the CDR sequences of the first pair of ABDs) or the same VH and VL sequences. In some embodiments, the ABDs of the multispecific, multivalent spike protein binding molecule are configured as shown in Figure 1B or Figure 1D.

[0108] [Table 5-1]

[0109] [Table 5-2]

[0110] 6.4. Spike Protein Antigen-Binding Domain Format In certain aspects, the multivalent anti-spike protein binding molecules of the present disclosure comprise an ABD of an anti-spike protein antibody that retains specific binding to an antigenic determinant. In one embodiment, the spike protein ABD is a naturally occurring (e.g., protease-cleaved) or engineered fragment of an immunoglobulin. Antibody fragments include, but are not limited to, VH (or VH fragment), VL (or VL fragment), Fab fragment, F(ab')2 fragment, scFv fragment, Fv fragment, minibody, diabody, triabody, and tetrabody.

[0111] 6.4.1.Fab Fab domains were traditionally generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can contain constant and variable region sequences from any suitable species and can therefore be murine, chimeric, human, or humanized.

[0112] A Fab domain typically comprises a CH1 domain attached to a VH domain, which pairs with a CL domain attached to a VL domain. In wild-type immunoglobulins, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding site. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0113] For anti-spike protein-binding antibodies of the present disclosure that are not homodimeric, particularly when the light chain of the anti-spike protein antibody is not a common or universal light chain, it is advantageous to use a Fab heterodimerization strategy to enable correct association of Fab domains belonging to the same antigen-binding domain and minimize aberrant pairing of Fab domains belonging to different antigen-binding domains. For example, the Fab heterodimerization strategy shown in Table 5 below can be used:

[0114] [Table 6]

[0115] Thus, in certain embodiments, correct association between the two polypeptides of a Fab is promoted by swapping the VL and VH domains of the Fab with one another, or by swapping the CH1 and CL domains with one another, as described, for example, in WO2009 / 080251.

[0116] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or by introducing one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain of the Fab. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces such that the Fab components preferentially pair with each other rather than with other Fab components.

[0117] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains, as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0118] In one embodiment, the modifications introduced in the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interface can be achieved based on steric and hydrophobic contacts, electrostatic / charge interactions, or a combination of various interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock and key fit, knob into hole, protrusion and cavity, donor and acceptor, etc., all of which suggest the nature of the structural and chemical match between the two interacting surfaces.

[0119] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds across the interface of the Fab component. In one embodiment, one or more of the introduced modifications introduce new salt bridges across the interface of the Fab component. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0120] In some embodiments, the Fab domain comprises a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0121] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which serve to exchange the hydrophobic and polar contact regions between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0122] In some embodiments, the Fab domain can contain modifications in some or all of the VH, CH1, VL, and CL domains to introduce an orthogonal Fab interface that promotes correct assembly of the Fab domain (Lewis et al., 2014, Nature Biotechnology 32:191-198). In one embodiment, a 39K, 62E modification is introduced in the VH domain, an H172A, F174G modification is introduced in the CH1 domain, a 1R, 38D, (36F) modification is introduced in the VL domain, and an L135Y, S176W modification is introduced in the CL domain. In another embodiment, a 39Y modification is introduced in the VH domain and a 38R modification is introduced in the VL domain.

[0123] Fab domains can also be modified to replace the native CH1:CL disulfide bond with an engineered disulfide bond, thereby increasing the efficiency of pairing of the Fab components. For example, an engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MABD 7:377-89).

[0124] Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that promote correct assembly. For example, Wu et al., 2015, MABD 7:364-76, describe replacing the CH1 domain with a T cell receptor constant domain and the CL domain with a T cell receptor b domain, pairing these domain replacements with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification in the VL domain and a 39K modification in the VH domain.

[0125] 6.4.2.scFv Single-chain Fv or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain, which can be expressed as a single-chain polypeptide and retain the specificity of the intact antibody from which they are derived. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding. Examples of linkers suitable for connecting the VH and VL chains of an scFv are the linkers identified in Section 6.6.

[0126] As used herein, unless otherwise specified, an scFv may have the VL variable region and the VH variable region in either order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0127] The scFv can comprise VH and VL sequences from any suitable species, such as murine, human, or humanized VH and VL sequences. To generate a nucleic acid encoding an scFv, DNA fragments encoding the VH and VL are operably linked to another fragment encoding a linker, for example, a fragment encoding any of the linkers described in Section 6.6 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:74)), such that the VH and VL sequences can be expressed as a contiguous single-chain protein with the VL and VH regions connected by a flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0128] 6.5. Multimerization moiety In some embodiments, the ABD of a multivalent anti-spike protein binding molecule of the disclosure comprises and / or is operably linked to one or more multimerization moieties, e.g., one or more multimerization moieties comprising or consisting of an Fc domain.

[0129] In certain embodiments, multivalent anti-spike protein binding molecules of the present disclosure comprise a single multimerization moiety (e.g., a single Fc domain), but more typically comprise two or more multimerization moieties (e.g., two or more Fc domains that can associate to form an Fc region). In some embodiments, the multivalent anti-spike protein binding molecule is a dimer, and the Fc region comprises two IgG-derived Fc domains, e.g., as described in Section 6.5.1.

[0130] Fc Domain The multivalent anti-spike protein binding molecules of the present disclosure can comprise an Fc domain, or a pair of Fc domains that associate to form an Fc region, from any suitable species operably linked to a spike protein ABD. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the spike protein ABD is fused to an IgG Fc domain.

[0131] The Fc domain that can be incorporated into the multivalent anti-spike protein binding molecule can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In another embodiment, the Fc domain is derived from IgG4.

[0132] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4), which dimerize to create the Fc region.

[0133] In the multivalent anti-spike protein binding molecules of the present disclosure, the Fc region, and / or the Fc domains therein, can comprise heavy chain constant domains from one or more different classes of antibodies, e.g., one, two, or three different classes.

[0134] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG1. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG2.

[0135] In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG3. In one embodiment, the Fc region comprises a CH2 domain and a CH3 domain derived from IgG4.

[0136] Heavy chain constant domains for use in generating Fc regions for the multivalent anti-spike protein binding molecules of the present disclosure may comprise variants of the naturally occurring constant domains described above. Such variants may comprise one or more amino acid mutations compared to the wild-type constant domain. In one example, the Fc region of the present disclosure comprises at least one constant domain whose sequence differs from that of the wild-type constant domain. It will be understood that the variant constant domain may be longer or shorter than the wild-type constant domain.

[0137] The Fc domain incorporated into the multivalent anti-spike protein binding molecules of the present disclosure may contain one or more modifications that alter the functional properties of the protein, for example, binding to an Fc receptor such as FcRn or a leukocyte receptor, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.5.1.1.

[0138] The Fc domain can also be altered to include modifications that improve the manufacturability of asymmetric multivalent anti-spike protein-binding molecules, for example, by enabling heterodimerization, the preferential pairing of non-identical over identical Fc domains. Heterodimerization allows for the generation of multivalent anti-spike protein-binding molecules in which different polypeptide components are connected to each other by Fc regions that contain Fc domains that differ in sequence. Examples of heterodimerization strategies are illustrated in Section 6.5.1.2.

[0139] It will be understood that any of the above modifications can be combined in any suitable way to achieve the desired functional properties and / or can be combined with other modifications to alter the properties of the multivalent anti-spike protein binding molecule.

[0140] 6.5.1.1. Fc Domains with Altered Effector Function In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0141] In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0142] In one embodiment, an Fc domain (e.g., an Fc domain of a multivalent anti-spike protein binding molecule polypeptide chain, or an Fc region (e.g., one or both Fc domains of a multivalent anti-spike protein binding construct that can associate to form an Fc region) comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain or Fc region comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to the Kabat EU index). In some embodiments, the Fc domain or Fc region comprises the amino acid substitutions L234A and L235A (numbering according to the Kabat EU index). In one such embodiment, the Fc domain or region is an IgD Fc domain or region, particularly a human IgD In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329. In a more particular embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to the Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and a further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numbering according to the Kabat EU index). In a more particular embodiment, the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). (Numbering according to the EU index). In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG").

[0143] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (Kabat EU index numbering).

[0144] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A, N297A mutations (EU numbering) to reduce effector function.

[0145] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. Exemplary IgG4 Fc domains with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table 6 below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below.

[0146] [Table 7-1]

[0147] [Table 7-2]

[0148] [Table 7-3]

[0149] [Table 7-4]

[0150] In certain embodiments, the IgG4 with reduced effector function comprises the bolded portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087, and is sometimes referred to herein as IgG4 or hIgG4.

[0151] For heterodimeric Fc regions, it is possible to incorporate combinations of the above-mentioned variant IgG4 Fc sequences, for example an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 30 of WO2014 / 121087 (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 37 of WO2014 / 121087 (or a bolded portion thereof), or an Fc region comprising an Fc domain comprising the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (or a bolded portion thereof) and an Fc domain comprising the amino acid sequence of SEQ ID NO: 38 of WO2014 / 121087 (or a bolded portion thereof).

[0152] 6.5.1.2. Fc Heterodimerization Variants Certain multivalent anti-spike protein binding molecules, unlike native immunoglobulins, involve dimerization between two Fc domains operably linked at non-identical N-terminal regions (e.g., one Fc domain connected to a Fab that binds a first spike protein epitope, and the other Fc domain connected to a different Fab that binds a second spike protein epitope). Inefficient heterodimerization of the two Fc domains to form an Fc region can be an obstacle to increasing the yield of the desired heterodimeric molecule, making purification challenging. Various approaches available in the art can be used to enhance dimerization of Fc domains that may be present in the ACE2 fusion proteins of the present disclosure, for example, as disclosed in EP 1870459 A1, U.S. Patent No. 5,582,996, U.S. Patent No. 5,731,168, U.S. Patent No. 5,910,573, U.S. Patent No. 5,932,448, U.S. Patent No. 6,833,441, U.S. Patent No. 7,183,076, U.S. Patent Application Publication No. 2006204493 A1, and PCT Publication No. WO2009 / 089004 A1.

[0153] The present disclosure provides multivalent anti-spike protein binding molecules comprising Fc heterodimers, i.e., Fc regions comprising heterologous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer comprises an antibody CH3 domain. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the preceding section.

[0154] Heterodimerization of two different heavy chains at their CH3 domains will result in the desired multivalent anti-spike protein-binding molecules, whereas homodimerization of identical heavy chains will reduce the yield of the desired multivalent anti-spike protein-binding molecules. Thus, in preferred embodiments, polypeptides that associate to form the multivalent anti-spike protein-binding molecules of the present disclosure will contain CH3 domains with modifications that favor heterodimeric association compared to unmodified Fc domains.

[0155] In certain embodiments, the modification that promotes Fc heterodimer formation is a so-called "knob-into-hole" or "knob-in-hole" modification, which includes a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168, U.S. Pat. No. 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, allowing the protrusion to be positioned within the cavity, to promote heterodimer formation and impede homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protrusions are created in the interface of the second polypeptide by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).

[0156] Thus, in some embodiments, amino acid residues in the CH3 domain of a first subunit of an Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion in the CH3 domain of the first subunit that can be positioned in a cavity in the CH3 domain of a second subunit, and amino acid residues in the CH3 domain of a second subunit of an Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity in the CH3 domain of the second subunit into which the protrusion in the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0157] In certain such embodiments, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In further embodiments, the first Fc domain additionally has a serine residue at position 354 replaced with a cysteine ​​residue (S354C) or a glutamic acid residue at position 356 replaced with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue), and the second Fc domain additionally has a tyrosine residue at position 349 replaced with a cysteine ​​residue (Y349C) (Kabat EU index numbering). In a specific embodiment, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (Kabat EU index numbering).

[0158] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote association of a first Fc domain and a second Fc domain of an Fc region.

[0159] Alternatively, or in addition to using an Fc domain modified to promote heterodimerization, the Fc domain can be modified to enable a purification strategy that allows for the selection of Fc heterodimers. In one such embodiment, one polypeptide contains a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that results in a heterodimeric protein. See, e.g., U.S. Patent No. 8,586,713. Thus, the IL12 receptor agonist comprises a first CH3 domain and a second Ig CH3 domain, wherein the first Ig CH3 domain and the second Ig CH3 domain differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the IL12 receptor agonist to Protein A compared to a corresponding IL12 receptor agonist lacking that amino acid difference. In one embodiment, the first CH3 domain binds Protein A and the second CH3 domain contains a mutation / modification that reduces or abolishes Protein A binding, for example, an H95R modification (according to IMGT exon numbering, H435R according to EU numbering). The second CH3 may further contain a Y96F modification (according to IMGT, Y436F according to EU). This class of modifications is referred to herein as "star" mutations.

[0160] In some embodiments, the Fc can contain one or more mutations to promote heterodimerization (eg, knob and hole mutations) and a star mutation to facilitate purification.

[0161] Linker In certain embodiments, the present disclosure provides multivalent anti-spike protein binding molecules in which two or more components are connected to each other by a peptide linker. By way of example and not limitation, a linker can be used to connect the spike protein ABD to a multimerization moiety.

[0162] Peptide linkers can range from 1 amino acid to 60 or more amino acids, and in certain embodiments, peptide linkers range from 3 to 50 amino acids in length, 4 to 30 amino acids in length, 5 to 25 amino acids in length, 10 to 25 amino acids in length, 10 to 60 amino acids in length, 12 to 20 amino acids in length, 20 to 50 amino acids in length, or 25 to 35 amino acids in length.

[0163] In certain embodiments, the peptide linker is at least 1 amino acid long, at least 2 amino acids long, at least 3 amino acids long, at least 4 amino acids long, at least 5 amino acids long, at least 6 amino acids long, or at least 7 amino acids long, and optionally up to 30 amino acids long, up to 40 amino acids long, up to 50 amino acids long, or up to 60 amino acids long.

[0164] In certain embodiments, the peptide linker is between 1 and 50 amino acids in length, e.g., between 1 and 50, 1 and 45, 1 and 40, 1 and 35, 1 and 30, 1 and 25, or 1 and 20 amino acids in length. In other certain embodiments, the peptide linker is between 2 and 50 amino acids in length, e.g., between 2 and 50, 2 and 45, 2 and 40, 2 and 35, 2 and 30, 2 and 25, or 2 and 20 amino acids in length. In other certain embodiments, the peptide linker is between 3 and 50 amino acids in length, e.g., between 3 and 50, 3 and 45, 3 and 40, 3 and 35, 3 and 30, 3 and 25, or 3 and 20 amino acids in length. In other certain embodiments, the peptide linker is between 4 and 50 amino acids in length, e.g., between 4 and 50, 4 and 45, 4 and 40, 4 and 35, 4 and 30, 4 and 25, or 4 and 20 amino acids in length. In some other specific embodiments, the peptide linker is between 5 and 50 amino acids in length, e.g., between 5 and 50, 5 and 45, 5 and 40, 5 and 35, 5 and 30, 5 and 25, or 5 and 20 amino acids in length. In other specific embodiments, the peptide linker is between 6 and 50 amino acids in length, e.g., between 6 and 50, 6 and 45, 6 and 40, 6 and 35, 6 and 30, 6 and 25, or 6 and 20 amino acids in length. In yet other specific embodiments, the peptide linker is between 7 and 50 amino acids in length, e.g., between 7 and 50, 7 and 45, 7 and 40, 7 and 35, 7 and 30, 7 and 25, or 7 and 20 amino acids in length.

[0165] In some embodiments, the linker is a G4S linker (SEQ ID NO: 80). In some embodiments, the linker comprises two consecutive G4S sequences (SEQ ID NO: 81), three consecutive G4S sequences (SEQ ID NO: 74), four consecutive G4S sequences (SEQ ID NO: 82), five consecutive G4S sequences (SEQ ID NO: 83), or six consecutive G4S sequences (SEQ ID NO: 84).

[0166] 6.6.1. Hinge arrangement In other embodiments, the multivalent anti-spike protein binding molecules of the present disclosure comprise a linker that is a hinge region. The hinge region can be a natural hinge region or a modified hinge region. Hinge regions are typically found at the N-terminus of the Fc region. The term "hinge region," unless otherwise indicated by context, refers to a natural or non-naturally occurring hinge sequence, which can be a monomeric hinge domain with respect to a single or monomeric polypeptide chain, or two associated hinge sequences on separate polypeptide chains with respect to a dimeric polypeptide (e.g., a homodimeric or heterodimeric multivalent anti-spike protein binding molecule formed by the association of two IgG Fc domains).

[0167] A native hinge region is typically the hinge region found between the Fab and Fc domains in naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges can include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat. Other modified hinge regions may include a complete hinge region derived from an antibody of a different class or subclass than that of the heavy chain Fc domain or Fc region. Alternatively, the modified hinge region may include a portion or repeat units of a native hinge, with each repeat unit derived from a native hinge region. In a further alternative, the native hinge region may be altered by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting appropriately positioned residues to cysteine ​​residues. The number of cysteine ​​residues in the hinge region may be increased or decreased by such means. Other modified hinge regions may be entirely synthetic and may be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0168] Several modified hinge regions have been previously described, for example, in U.S. Pat. No. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0169] In some embodiments, a multivalent anti-spike protein binding molecule of the present disclosure comprises an Fc region in which one or both Fc domains possess an intact hinge region at their N-terminus. In various embodiments, positions 233-236 in the hinge region may be G, G, G, empty, empty, G, G, empty, empty, empty, empty, or all empty, and the positions are numbered according to EU numbering.

[0170] In some embodiments, a multivalent anti-spike protein binding molecule of the disclosure comprises a modified hinge region that has reduced binding affinity for Fcγ receptors compared to a wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0171] In one embodiment, a multivalent anti-spike protein binding molecule of the present disclosure comprises an Fc region, each Fc domain having an intact hinge region at its N-terminus, wherein each Fc domain and hinge region is derived from IgG4, and each hinge region comprises the modified sequence CPPC (SEQ ID NO: 85). The core hinge region of human IgG4 comprises the sequence CPSC (SEQ ID NO: 86), compared to IgG1, which comprises the sequence CPPC (SEQ ID NO: 85). The serine residues present in the IgG4 sequence provide increased flexibility in this region, and therefore a proportion of the molecules form disulfide bonds within the same protein chain (intrachain disulfides) rather than cross-linking to other heavy chains within the IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Changing the serine residues to prolines to obtain the same core sequence as IgG1 allows complete formation of interchain disulfides within the IgG4 hinge region, thus reducing heterogeneity in the purified product. This altered isotype is called IgG4P.

[0172] 6.7. Nucleic Acids and Host Cells In another aspect, the present disclosure provides nucleic acids encoding the multivalent anti-spike protein-binding molecules of the present disclosure. In some embodiments, the multivalent anti-spike protein-binding molecules are encoded by a single nucleic acid. In other embodiments, the multivalent anti-spike protein-binding molecules can be encoded by multiple (e.g., two, three, four, or more) nucleic acids.

[0173] A single nucleic acid can encode a multivalent anti-spike protein-binding molecule comprising a single polypeptide chain, a multivalent anti-spike protein-binding molecule comprising two or more polypeptide chains, or a portion of a multivalent anti-spike protein-binding molecule comprising three or more polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of a multivalent anti-spike protein-binding molecule comprising three, four, or more polypeptide chains, or three polypeptide chains of a multivalent anti-spike protein-binding molecule comprising four or more polypeptide chains). To separately control expression, open reading frames encoding two or more polypeptide chains can be under the control of separate transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory element and separated by an internal ribosome entry site (IRES) sequence, allowing translation into separate polypeptides.

[0174] In some embodiments, multivalent anti-spike protein binding molecules comprising two or more polypeptide chains are encoded by two or more nucleic acids. The number of nucleic acids encoding the multivalent anti-spike protein binding molecule can be equal to or less than the number of polypeptide chains in the multivalent anti-spike protein binding molecule (e.g., when two or more polypeptide chains are encoded by a single nucleic acid).

[0175] The nucleic acids of the present disclosure can be DNA or RNA (eg, mRNA). In another aspect, the present disclosure provides host cells and vectors comprising the nucleic acids of the present disclosure. The nucleic acids may be present in a single vector or may be present in separate vectors that are present in the same host cell or in separate host cells, as described in more detail herein below.

[0176] Vectors The present disclosure provides vectors comprising a nucleotide sequence encoding a multivalent anti-spike protein-binding molecule described herein or a component thereof (e.g., one or two of the polypeptide chains of a multivalent anti-spike protein-binding molecule). Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0177] Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, eastern equine encephalitis virus, and flavivirus.

[0178] Additionally, cells that have stably integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers may provide, for example, prototropy to auxotrophic hosts, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be either directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0179] After the construct containing expression vector or DNA sequence is prepared for expression, the expression vector can be transfected or introduced into suitable host cells.To achieve this, various techniques can be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.The method and conditions for culturing the obtained transfected cells and recovering the expressed polypeptide are known to those skilled in the art, and can be modified or optimized according to the specific expression vector and mammalian host cell used based on this specification.

[0180] 6.7.2.Cells The present disclosure also provides a host cell comprising a nucleic acid of the present disclosure. In one embodiment, the host cell is genetically engineered to contain one or more nucleic acids described herein.

[0181] In one embodiment, the host cell is genetically engineered using an expression cassette. The term "expression cassette" refers to a nucleotide sequence capable of affecting the expression of a gene in a host compatible with such sequence. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression, such as an inducible promoter, may also be used.

[0182] The present disclosure also provides host cells comprising the vectors described herein. The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0183] Pharmaceutical Compositions The multivalent anti-spike protein-binding molecules of the present disclosure may be in the form of a composition comprising the multivalent anti-spike protein-binding molecule and one or more carriers, excipients, and / or diluents. The composition may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the multivalent anti-spike protein-binding molecule and, in the case of therapeutic uses, the mode of administration.

[0184] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition can be in any suitable form (depending on the desired method of administration to a patient). Pharmaceutical compositions can be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or topical. The most suitable route for administration in any given case will depend on the particular antibody, the subject, the nature and severity of the disease, and the physical condition of the subject. Typically, pharmaceutical compositions will be administered intravenously or subcutaneously.

[0185] Pharmaceutical compositions can be conveniently presented in unit dosage forms containing a predetermined amount of the multivalent anti-spike protein-binding molecule of the present disclosure per dose. The amount of multivalent anti-spike protein-binding molecule contained in a unit dose will depend not only on the disease being treated but also on other factors well known in the art. Such unit dosages may be in the form of a lyophilized dry powder containing an amount of multivalent anti-spike protein-binding molecule suitable for a single administration, or in liquid form. The dry powder unit dosage form may be packaged in a kit together with a syringe, a suitable amount of diluent, and / or other components useful for administration. The liquid unit dosage may conveniently be supplied in the form of a syringe pre-filled with an amount of multivalent anti-spike protein-binding molecule suitable for a single administration.

[0186] Pharmaceutical compositions may also be supplied in bulk form containing an amount of multivalent anti-spike protein binding molecule suitable for multiple administrations. Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing multivalent anti-spike protein binding molecules of the desired purity with any pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically used in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to recipients at the dosages and concentrations employed.

[0187] Buffering agents help maintain pH in a range close to physiological conditions. They may be present in a wide variety of concentrations, but will typically be present at concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium glyconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium glyconate mixtures, etc.), Examples of buffers include oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (e.g., Tris) can be used.

[0188] Preservatives may be added to retard microbial growth and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalconium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity adjusting agents, sometimes known as "stabilizers," can be added to ensure the isotonicity of the liquid compositions of the present disclosure and include polyhydric sugar alcohols, such as trihydric or higher sugar alcohols (e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol). Stabilizers refer to a broad category of excipients that can range in function from bulking agents to additives, and serve to solubilize the therapeutic agent or prevent it from denaturing or adhering to the container wall.Typical stabilizers include polyhydric sugar alcohols (as listed above), amino acids (e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclitols such as inositol), polyethylene glycol, amino acid polymers, sulfur-containing reducing agents (e.g., urea, glutathione, thioctic acid, etc.), and the like. Stabilizers can be selected from the group consisting of: cellulose, cellulose acetate, cellulose acetate copolymer ...

[0189] Non-ionic surfactants or detergents (also known as "wetting agents") may be added to aid in solubilizing the glycoprotein and to protect it from agitation-induced aggregation, allowing the formulation to be exposed to stressful shear surfaces without denaturing the protein. Suitable non-ionic surfactants include polysorbates (e.g., 20, 80), poloxamers (e.g., 184, 188), and Pluronic® polyols. The non-ionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0190] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0191] The multivalent anti-spike protein binding molecules of the present disclosure can be formulated as a pharmaceutical composition comprising the multivalent anti-spike protein binding molecule and containing, for example, one or more pharmaceutically acceptable excipients or carriers. To prepare a pharmaceutical or sterile composition comprising the multivalent anti-spike protein binding molecule of the present disclosure, the multivalent anti-spike protein binding molecule preparation can be combined with one or more pharmaceutically acceptable excipients or carriers.

[0192] For example, a formulation of a multivalent anti-spike protein binding molecule can be prepared by mixing the multivalent anti-spike protein binding molecule with a physiologically acceptable carrier, excipient, or stabilizer, for example, in the form of a lyophilized powder, a slurry, an aqueous solution, a lotion, or a suspension (see, e.g., Hardman et al., 2001, Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro, 2000, Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.), 1993, Pharmaceutical Dosage Forms: General Medications, Marcel Dekker, NY; Lieberman, et al. (eds.), 1990, Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. al. (eds.), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie, 2000, Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0193] 6.9. Therapeutic Indications and Methods The present disclosure provides methods for using and applying the multivalent anti-spike protein binding molecules of the present disclosure.

[0194] In certain aspects, the present disclosure provides methods for preventing or treating diseases or conditions involving the interaction between the coronavirus RBD and cellular ACE2. In some embodiments, the disease or condition is prevented or treated by neutralizing the spike protein. In various embodiments, neutralizing the spike protein includes (a) inhibiting the ability of the spike protein to bind to a receptor such as ACE2, (b) inhibiting cleavage of the spike protein by a protease such as TMPRSS2, (c) inhibiting the spike protein from mediating (i) viral entry into a host cell or (ii) viral replication in a host cell, or (d) any combination of two, three, or all four of (a), (b), (c)(i), and (c)(ii).

[0195] Thus, in some embodiments, the multivalent anti-spike protein binding molecules and pharmaceutical compositions of the present disclosure can be used to inhibit the interaction between the RBD of a coronavirus and cellular ACE2. In some embodiments, the present disclosure provides methods for inhibiting the interaction between the RBDs of SARS-CoV. In other embodiments, the present disclosure provides methods for inhibiting the interaction between the RBDs of SARS-CoV-2. Thus, in some embodiments, the present disclosure provides a method for inhibiting the interaction between the RBD of a coronavirus and cellular ACE2, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule pharmaceutical composition described herein.

[0196] In some embodiments, the present disclosure provides methods of administering a multivalent anti-spike protein-binding molecule pharmaceutical composition described herein to a subject who has been exposed to a coronavirus but has not been diagnosed with an infection. In other embodiments, the subject is coronavirus-positive but asymptomatic. In yet other embodiments, the subject is coronavirus-positive and pre-symptomatic. In further embodiments, the subject is coronavirus-positive and symptomatic. In other embodiments, the subject develops COVID-19 or other coronavirus-mediated disease or condition.

[0197] In some embodiments, the present disclosure provides a method of reducing the severity of a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule pharmaceutical composition described herein.

[0198] In some other embodiments, the present disclosure provides methods of reducing coronavirus viral load, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule pharmaceutical composition described herein.

[0199] In a further embodiment, the present disclosure provides a method of preventing disease progression in a subject having a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule pharmaceutical composition described herein.

[0200] In some embodiments, the present disclosure provides a method of reducing the duration of a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule pharmaceutical composition described herein.

[0201] In other embodiments, the present disclosure provides a method of reducing the risk of severe disease or death in a subject having a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule pharmaceutical composition described herein.

[0202] 7. Numbered Embodiments While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). The present disclosure is exemplified by the numbered embodiments set forth below. Unless otherwise specified, any concept, aspect, and / or feature of any of the embodiments described in the above detailed description is applicable mutatis mutandis to any of the numbered embodiments below.

[0203] In the numbered embodiments below, the multimerizing moiety is preferably derived from a mammalian multimerizing moiety (e.g., a human Fc domain), the antigen-binding domain is preferably derived from a human or humanized antibody, and the subject is preferably a mammal (e.g., a human).

[0204] 1. A multivalent anti-spike protein binding molecule comprising at least four anti-spike protein antigen binding domains (ABDs) operably linked by one or more multimerization moieties. 2. The multivalent anti-spike protein binding molecule of embodiment 1, which is tetravalent.

[0205] 3. The multivalent anti-spike protein binding molecule of embodiment 1 or embodiment 2, wherein the antigen-binding domain (ABD) is human or humanized. 4. The multivalent anti-spike protein binding molecule of any one of embodiments 1-3, wherein one or more (or all) ABDs comprise a CDR sequence as set forth in any one of Tables 1-3.

[0206] 5. The multivalent anti-spike protein binding molecule of any one of embodiments 1-3, wherein one or more (or all) ABDs comprise a CDR sequence as set forth in any one of Tables 1-4.

[0207] 6. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of an antibody shown in Table 1.

[0208] 7. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of an antibody shown in Table 2.

[0209] 8. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of an antibody shown in Table 3.

[0210] 9. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of an antibody shown in Table 4.

[0211] 10. One or more (or all) ABDs (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 579, 580, and 581, respectively; (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 398, 372, and 583, respectively.

[0212] 11. One or more (or all) ABDs (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 507, 508, and 509, respectively; (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 511, 407, and 512, respectively.

[0213] 12. One or more (or all) ABDs (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 450, 451, and 452, respectively; (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 454, 415, and 455, respectively.

[0214] 13. The multivalent anti-spike protein binding molecule of any one of embodiments 1-3, wherein one or more (or all) ABDs comprise a VH sequence and a VL sequence as set forth in any one of Tables 1-3.

[0215] 14. The multivalent anti-spike protein binding molecule of any one of embodiments 1-3, wherein one or more (or all) ABDs comprise a VH sequence and a VL sequence as set forth in any one of Tables 1-4.

[0216] 15. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the VH and VL sequences of an antibody shown in Table 1.

[0217] 16. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the VH and VL sequences of an antibody shown in Table 2.

[0218] 17. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the VH and VL sequences of an antibody shown in Table 3.

[0219] 18. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise the VH and VL sequences of an antibody shown in Table 4.

[0220] 19. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise: (a) a VH 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 SEQ ID NO: 578; and (b) a VL 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 SEQ ID NO: 582.

[0221] 20. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise: (a) a VH comprising the amino acid sequence of SEQ ID NO: 578; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 582.

[0222] 21. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise: (a) a VH 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 SEQ ID NO: 506; and (b) a VL 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 SEQ ID NO: 510.

[0223] 22. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise: (a) a VH comprising the amino acid sequence of SEQ ID NO: 506; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 510.

[0224] 23. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise: (a) a VH 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 SEQ ID NO: 449; and (b) a VL 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 SEQ ID NO: 453.

[0225] 24. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise: (a) a VH comprising the amino acid sequence of SEQ ID NO: 449; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 453.

[0226] 25. The multivalent anti-spike protein binding molecule of any one of embodiments 1-24, wherein one or more (or all) ABDs are neutralizing. 26. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 25, which is capable of neutralizing SARS-CoV-2 variant BA.1.

[0227] 27. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 25, which is capable of neutralising SARS-CoV-2 variant BA.2. 28. The multivalent anti-spike protein binding molecule of any one of embodiments 1-27, wherein one or more multimerization moieties is an Fc domain.

[0228] 29. The multivalent anti-spike protein binding molecule of any one of embodiments 1 to 28, comprising two half antibodies, each comprising an Fc domain. 30. The multivalent anti-spike protein binding molecule of embodiment 28 or embodiment 29, wherein the Fc domain is an IgG domain.

[0229] 31. The multivalent anti-spike protein binding molecule of embodiment 30, wherein the IgG domain is an IgG1 domain. 32. The multivalent anti-spike protein binding molecule of embodiment 30, wherein the IgG domain is an IgG4 domain.

[0230] 33. The multivalent anti-spike protein binding molecule of any one of embodiments 29-32, wherein each half antibody comprises two ABDs. 34. (a) A first half antibody, (i) a first ABD, typically comprising a first VH and a first VL; (ii) an optional first linker; (iii) a second ABD, typically comprising a second VH and a second VL; (iv) an optional first hinge domain, and (v) a first half antibody comprising a first Fc domain; and (b) a second half antibody, (i) a third ABD, typically comprising a third VH and a third VL; (ii) an optional second linker; (iii) a fourth ABD, typically comprising a fourth VH and a fourth VL; (iv) an optional second hinge domain, and (v) a second half antibody comprising a second Fc domain.

[0231] 35. (a) A first half antibody, (i) a first ABD, typically comprising a first VH and a first VL; (ii) an optional first hinge domain; (iii) a first Fc domain; (iv) an optional first linker, and (v) a first half antibody comprising a second ABD, typically comprising a second VH and a second VL; (b) a second half antibody, (i) a third ABD, typically comprising a third VH and a third VL; (ii) an optional second hinge domain; (iii) a second Fc domain; (iv) an optional second linker, and (v) a second half antibody comprising a fourth ABD, typically comprising a fourth VH and a fourth VL.

[0232] 36. The multivalent anti-spike protein binding molecule of embodiment 34 or embodiment 35, wherein the first Fc domain and the second Fc domain form an Fc homodimer. 37. The multivalent anti-spike protein binding molecule of embodiment 34 or embodiment 35, wherein the first Fc domain and the second Fc domain form an Fc heterodimer.

[0233] 38. The multivalent anti-spike protein binding molecule of embodiment 37, wherein the first Fc domain or the second Fc domain comprises a knob mutation and the other Fc domain comprises a hole mutation.

[0234] 39. The multivalent anti-spike protein binding molecule of any one of embodiments 34 to 38, wherein the first Fc domain or the second Fc domain comprises a star mutation. 40. The multivalent anti-spike protein binding molecule of any one of embodiments 34-39, comprising a first hinge domain and a second hinge domain.

[0235] 41. The multivalent anti-spike protein binding molecule of embodiment 40, wherein the first hinge domain and the second hinge domain are IgG1 hinge domains. 42. The multivalent anti-spike protein binding molecule of embodiment 40, wherein the first hinge domain and the second hinge domain are IgG4 hinge domains.

[0236] 43. The multivalent anti-spike protein binding molecule of embodiment 40, wherein the first hinge domain and the second hinge domain are chimeric hinge domains. 44. The multivalent anti-spike protein binding molecule of any one of embodiments 34 to 43, lacking the first linker and the second linker.

[0237] 45. The multivalent anti-spike protein binding molecule of any one of embodiments 34 to 43, comprising a first linker and a second linker. 46. ​​The multivalent anti-spike protein binding molecule of embodiment 45, wherein the first linker and the second linker are each independently selected from (a) 1 to 60 amino acids in length, or (b) 1 to 40 amino acids in length, or (c) any range or value of linker lengths set forth in Section 6.6.

[0238] 47. The first linker and the second linker each comprise a glycine-serine sequence, and optionally, the glycine-serine sequence is n S) x (SEQ ID NO: 87), wherein n=0 to 5 and x=1 to 6.

[0239] 48. The multivalent anti-spike protein binding molecule of any one of embodiments 34-47, wherein the first, second, third, and fourth ABDs are the same. 49. The multivalent anti-spike protein binding molecule of any one of embodiments 34-48, wherein the first and second ABDs are the same.

[0240] 50. The multivalent anti-spike protein binding molecule of embodiment 49, wherein the third and fourth ABDs are the same. 51. The multivalent anti-spike protein binding molecule of embodiment 50, wherein the third and fourth ABDs are different from the first and second ABDs.

[0241] 52. The multivalent anti-spike protein binding molecule of any one of embodiments 34-47, wherein the first and third ABDs are the same. 53. The multivalent anti-spike protein binding molecule of embodiment 52, wherein the second and fourth ABDs are the same.

[0242] 54. The multivalent anti-spike protein binding molecule of embodiment 53, wherein the second and fourth ABDs are different from the first and third ABDs. 55. The multivalent anti-spike protein binding molecule of any one of embodiments 1-54, wherein at least two of the ABDs are Fab domains, and optionally the Fab domains are not single-chain Fab domains.

[0243] 56. The multivalent anti-spike protein binding molecule of any one of embodiments 1-54, wherein all ABDs are Fab domains, and optionally the Fab domains are not single-chain Fab domains.

[0244] 57. The multivalent anti-spike protein binding molecule of any one of embodiments 1-54, wherein at least two of the ABDs are scFvs. 58. The multivalent anti-spike protein binding molecule of any one of embodiments 1-54, wherein at least all of the ABDs are scFvs.

[0245] 59. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 58, which is monospecific. 60. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 58, which is multispecific.

[0246] 61. The multivalent anti-spike protein binding molecule of embodiment 60, which is bispecific. 62. A multivalent anti-spike protein binding molecule comprising at least four means for binding spike protein operably linked by one or more multimerization moieties.

[0247] 63. The multivalent anti-spike protein binding molecule of embodiment 62, wherein the means for binding spike protein is tetravalent. 64. A multivalent anti-spike protein binding molecule according to embodiment 62 or 63, capable of neutralizing SARS-CoV-2 variant BA.1.

[0248] 65. A multivalent anti-spike protein binding molecule according to embodiment 62 or 63, capable of neutralizing SARS-CoV-2 variant BA.2. 66. The multivalent anti-spike protein binding molecule of any one of embodiments 62-65, wherein one or more multimerization moieties is an Fc domain.

[0249] 67. The multivalent anti-spike protein binding molecule of any one of embodiments 62 to 65, comprising two half antibodies, each comprising an Fc domain. 68. The multivalent anti-spike protein binding molecule of embodiment 66 or embodiment 67, wherein the Fc domain is an IgG domain.

[0250] 69. The multivalent anti-spike protein binding molecule of embodiment 68, wherein the IgG domain is an IgG1 domain. 70. The multivalent anti-spike protein binding molecule of embodiment 68, wherein the IgG domain is an IgG4 domain.

[0251] 71. A multivalent anti-spike protein binding molecule according to any one of embodiments 67 to 70, wherein each half antibody comprises two means for binding spike protein. 72. (a) A first half antibody, (i) a first means for binding a spike protein; (ii) an optional first linker; (iii) a second means for binding the spike protein; (iv) an optional first hinge domain, and (v) a first half antibody comprising a first Fc domain; and (b) a second half antibody, (i) a third means for binding the spike protein; (ii) an optional second linker; (iii) a fourth means for binding the spike protein; (iv) an optional second hinge domain, and (v) a second half antibody comprising a second Fc domain.

[0252] 73. (a) A first half antibody, (i) a first means for binding a spike protein; (ii) an optional first hinge domain; (iii) a first Fc domain; (iv) an optional first linker, and (v) a first half antibody comprising a second means for binding the spike protein; and (b) a second half antibody, (i) a third means for binding the spike protein; (ii) an optional second hinge domain; (iii) a second Fc domain; (iv) an optional second linker, and (v) a second half antibody comprising a fourth means for binding spike protein.

[0253] 74. The multivalent anti-spike protein binding molecule of embodiment 72 or embodiment 73, wherein the first Fc domain and the second Fc domain form an Fc homodimer. 75. The multivalent anti-spike protein binding molecule of embodiment 72 or embodiment 73, wherein the first Fc domain and the second Fc domain form an Fc heterodimer.

[0254] 76. The multivalent anti-spike protein binding molecule of embodiment 75, wherein the first Fc domain or the second Fc domain comprises a knob mutation and the other Fc domain comprises a hole mutation.

[0255] 77. A multivalent anti-spike protein binding molecule according to any one of embodiments 72 to 76, wherein the first Fc domain or the second Fc domain comprises a star mutation. 78. A multivalent anti-spike protein binding molecule according to any one of embodiments 72 to 77, comprising a first hinge domain and a second hinge domain.

[0256] 79. The multivalent anti-spike protein binding molecule of embodiment 78, wherein the first hinge domain and the second hinge domain are IgG1 hinge domains. 80. The multivalent anti-spike protein binding molecule of embodiment 78, wherein the first hinge domain and the second hinge domain are IgG4 hinge domains.

[0257] 81. The multivalent anti-spike protein binding molecule of embodiment 78, wherein the first hinge domain and the second hinge domain are chimeric hinge domains. 82. The multivalent anti-spike protein binding molecule of any one of embodiments 72-81, lacking the first linker and the second linker.

[0258] 83. The multivalent anti-spike protein binding molecule of any one of embodiments 72-81, comprising a first linker and a second linker. 84. The multivalent anti-spike protein binding molecule of embodiment 83, wherein the first linker and the second linker are each independently selected from (a) 1 to 60 amino acids in length, or (b) 1 to 40 amino acids in length, or (c) any range or value of linker lengths set forth in Section 6.6.

[0259] 85. The first linker and the second linker each comprise a glycine-serine sequence, and optionally, the glycine-serine sequence is n S) x (SEQ ID NO: 87), wherein n=0 to 5 and x=1 to 6.

[0260] 86. The multivalent anti-spike protein binding molecule of any one of embodiments 62-85, wherein the first half antibody comprises a first Fab comprising a first means for binding spike protein.

[0261] 87. The multivalent anti-spike protein binding molecule of any one of embodiments 62 to 85, wherein the first half antibody comprises a first scFv comprising a first means for binding spike protein.

[0262] 88. The multivalent anti-spike protein binding molecule of any one of embodiments 62-87, wherein the first half antibody comprises a second Fab comprising a second means for binding spike protein.

[0263] 89. The multivalent anti-spike protein binding molecule of any one of embodiments 62-87, wherein the first half antibody comprises a second scFv comprising a second means for binding spike protein.

[0264] 90. The multivalent anti-spike protein binding molecule of any one of embodiments 62-89, wherein the second half antibody comprises a third Fab comprising a third means for binding spike protein.

[0265] 91. The multivalent anti-spike protein binding molecule of any one of embodiments 62-89, wherein the second half antibody comprises a third scFv comprising a third means for binding spike protein.

[0266] 92. The multivalent anti-spike protein binding molecule of any one of embodiments 62-91, wherein the second half antibody comprises a fourth scFv comprising a fourth means for binding spike protein.

[0267] 93. The multivalent anti-spike protein binding molecule of any one of embodiments 62-91, wherein the second half antibody comprises a fourth scFv comprising a fourth means for binding spike protein.

[0268] 94. A multivalent anti-spike protein binding molecule according to any one of embodiments 62 to 93, which is monospecific. 95. A multivalent anti-spike protein binding molecule according to any one of embodiments 62 to 93, which is multispecific.

[0269] 96. The multivalent anti-spike protein binding molecule of embodiment 95, which is bispecific. 97. A nucleic acid or a plurality of nucleic acids encoding a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 96.

[0270] 98. A host cell engineered to express a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 96 or a nucleic acid(s) according to embodiment 97. 99. A method for producing a multivalent anti-spike protein binding molecule described in any one of embodiments 1 to 96, comprising culturing a host cell described in embodiment 98 and recovering the multivalent anti-spike protein binding molecule expressed thereby.

[0271] 100. A pharmaceutical composition comprising a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 96, and an excipient. 101. A method for treating a coronavirus disease, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of embodiments 1 to 96 or a pharmaceutical composition of embodiment 100.

[0272] 102. A method for inhibiting the interaction between coronavirus RBD and cellular ACE2, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule described in any one of embodiments 1 to 96 or a pharmaceutical composition described in embodiment 100.

[0273] 103. A method for neutralizing coronavirus spike protein in vivo, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of embodiments 1 to 96 or a pharmaceutical composition of embodiment 100.

[0274] 104. A method for inhibiting protease-mediated cleavage (e.g., TMPRSS2-mediated cleavage) of coronavirus spike protein in vivo, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of embodiments 1 to 96 or a pharmaceutical composition of embodiment 100.

[0275] 105. A method for inhibiting viral entry of a coronavirus into host cells in a subject, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of embodiments 1 to 96 or a pharmaceutical composition of embodiment 100.

[0276] 106. A method for inhibiting reproduction of coronavirus spike protein in host cells in a subject, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of embodiments 1 to 96 or a pharmaceutical composition of embodiment 100.

[0277] 107. A method for reducing the severity of a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule described in any one of embodiments 1 to 96 or a pharmaceutical composition described in embodiment 100.

[0278] 108. A method for reducing coronavirus viral load, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule described in any one of embodiments 1 to 96 or a pharmaceutical composition described in embodiment 100.

[0279] 109. A method for preventing disease progression in a subject with a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of embodiments 1 to 96 or a pharmaceutical composition of embodiment 100.

[0280] 110. A method for reducing the duration of a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule described in any one of embodiments 1 to 96 or a pharmaceutical composition described in embodiment 100.

[0281] 111. A method for reducing the risk of severe disease or death in a subject with a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule described in any one of embodiments 1 to 96 or a pharmaceutical composition described in embodiment 100.

[0282] 112. The method of any one of embodiments 101 to 111, wherein the coronavirus is SARS-CoV. 113. The method of any one of embodiments 101 to 111, wherein the coronavirus is SARS-CoV-2. [Example]

[0283] 8. Working Example 8.1. Materials and Methods 8.1.1. Construction and Generation of Tetravalent Fab-Fc AF Antibodies Both monospecific and bispecific tetravalent 2x2 N-Fab AF antibodies were constructed by connecting two identical VH-CH1 fragments linked via a (G4S)x3 linker (SEQ ID NO: 74) to the N-terminus of an Fc fragment via the hinge region (Figures 1A and 1B). 2x2 C-Fab AF antibodies were constructed by connecting a VH-CH1 fragment to the N-terminus of an Fc fragment, which was then linked to another identical VH-CH1 fragment on the C-terminus via a (G4S)x3 linker (SEQ ID NO: 74) (Figures 1C and 1D). To achieve consistency in production, all AF antibodies incorporated "knob-in-hole" mutations in the Fc region to promote Fc heterodimer formation, a follow-up step to Red-Ox annealing assembly.

[0284] All antibodies were expressed separately in Expi293™ cells (ThermoFisher) as half antibodies designated "knob strand" and "hole* strand" by transient transfection according to the manufacturer's protocol. Antibodies were purified from the supernatant using Hitrap Protein G HP (Cytiva). After single-step elution, antibodies were neutralized and dialyzed into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80°C.

[0285] For Red-Ox annealing assembly, a solution containing 0.5 mg / mL knob strand, 0.5 mg / mL hole* strand, 50 mM Tris pH 8.0, 25 mM 2-MEA, and 50 mM L-arginine was made. The reaction was carried out at 37°C for 5 hours, then at 4°C overnight. The product was desalted using Zeba Spin Desalting Columns (ThermoFisher) into a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80°C.

[0286] The eluted fraction material was further refined to increase the purity of the desired species by SEC. Therefore, a Superdex 200 10 / 300GL column (Cytiva) was used at a flow rate of 0.75 mL / min in 1x DPBS, 5% glycerol, pH 7.4 running buffer. Fractions of interest were pooled and concentrated.

[0287] 8.1.2. SARS-CoV-2 Pseudovirus / Variant Neutralization Assays Vero cells were cultured in glutamine-free DMEM high-glucose medium containing sodium pyruvate, supplemented with 10% heat-inactivated FBS and penicillin / streptomycin / L-glutamine (complete DMEM), and seeded at 20,000 cells / well in 96-well black / clear-bottom cell culture plates. On the day of the assay, antibodies were diluted to 2x the assay concentration and serially diluted 3x to yield a total of 11 concentrations (e.g., 40 nM to 677.4 fM for the IgG control and all constructs in Table 10; concentrations used for constructs in Table 9 ranged from 13.3 nM to 225.8 fM). All dilutions were performed using infection medium consisting of glutamine-free DMEM high-glucose medium containing sodium pyruvate supplemented with sodium pyruvate, 0.2% IgG-free BSA, and gentamicin.

[0288] The pVSV-Luc-SARS-CoV2-S pseudovirus used herein is a non-replicating VSV-DG, expressing a dual GFP / firefly luciferase reporter in place of its native glycoprotein and pseudotyped with a SARS-CoV-2 spike. SARS-CoV-2 pseudovirus or variant was diluted 1:4 in infection medium and then combined 1:1 with antibody diluent at a final pseudovirus / variant dilution of 1:8 and final test article concentrations ranging from 20 nM to 338.7 fM for all IgG controls and constructs shown in Table 10, or at final test article concentrations ranging from 6.7 nM to 112.9 fM for constructs shown in Table 9, and incubated for 30 minutes at room temperature. The culture medium was then removed from the cells, and the combined antibody and pseudovirus / variant was added to wells at 100 μL / well in duplicate and then incubated at 37°C, 5% CO2 for 24 hours. At 24 hours post-infection, media was removed from the wells and cells were lysed using 100 μL / well of Glo-Lysis buffer (Promega). 100 μL of prepared Bright-Glo substrate (Promega) was added to the lysate immediately before reading luminescence on a Spectramax i3X plate reader. Results were exported to Microsoft Excel and % neutralization was calculated using the following equation: % neutralization = ((1 - (well value - media control) / (virus control - media control)) x 100. % neutralization was then plotted in GraphPad Prism and analyzed using a nonlinear regression of response: log(inhibitor) - variable slope (4 parameters) to calculate IC50 values.

[0289] 8.2. Example 1: Neutralizing Activity of Tetravalent 2x2 N-Fab AF Using five different Fab moieties (REGN10933, 10985, 10987, 14256, and 14315; containing the VH and VL domains from mAb10933, mAb10985, mAb10987, mAb14256, and mAb14315, respectively, as shown in Tables 3 and 4), a total of five monospecific and ten bispecific 2x2 N-Fab AF constructs were generated as described in Section 8.1.1 and listed in Table 7. Cell culture and virus neutralization assays were performed as described in Section 8.1.2.

[0290] [Table 8]

[0291] The SARS-CoV-2 pseudovirus or variant neutralization IC50 values ​​of the 2x2 N-Fab AF constructs were compared with the IC50 values ​​of an IgG mAb control with the same Fab moiety. In general, the monospecific 2x2 N-Fab AF constructs were more effective at neutralizing the SARS-CoV-2 pseudovirus D614G than the IgG mAb control (Figure 2A). This observation suggests that increased valency can help promote neutralization activity. Most bispecific 2x2 N-Fab AF constructs also performed better than their IgG mAb controls. However, the change in neutralization activity depended on which Fab arm was used to generate the construct.

[0292] The ability of monospecific and bispecific anti-SARS-CoV-2 2x2 N-Fab constructs to neutralize the SARS-CoV-2 BA.2 variant depended entirely on which Fab arm was used to generate the construct. For example, constructs lacking the Fab moieties REGN14315 and REGN10987 failed to neutralize the BA.2 variant. However, including either REGN14315 or REGN10987 in the construct enabled the construct to neutralize the BA.2 variant (Figure 2B).

[0293] 8.3. Example 2: Neutralizing Activity of Tetravalent 2x2 C-Fab AF The same five Fab portions were used to generate a total of five monospecific and ten bispecific 2x2 C-Fab AF constructs as done in Example 1, as described in Section 8.1.1 and listed in Table 8. Cell culture and virus neutralization assays were performed as described in Section 8.1.2.

[0294] [Table 9-1]

[0295] [Table 9-2]

[0296] The SARS-CoV-2 pseudovirus or variant neutralization IC50 values ​​of the 2x2 C-Fab AF constructs were compared to the IC50 values ​​of an IgG mAb control with the same Fab portion. In general, the monospecific 2x2 C-Fab AF constructs were more effective at neutralizing SARS-CoV-2 pseudoviruses than the IgG mAb control, except for COVAF36, which has the Fab portion REGN14356 (Figure 3A). As in Example 1, most bispecific 2x2 C-Fab AF constructs also performed better than their IgG mAb controls. Again, the change in neutralizing activity depended on which Fab arm was used to generate the construct.

[0297] The ability of the monospecific and bispecific 2x2 C-Fab constructs to neutralize the SARS-CoV-2 BA.2 variant again depended entirely on which Fab arm was used to generate the construct. Consistent with the observations in Example 1, constructs lacking the Fab moieties REGN14315 and REGN10987 failed to neutralize the BA.2 variant. Nevertheless, 2x2 N-Fab screening demonstrated that including either REGN14315 or REGN10987 in the construct enabled the 2x2 C-Fab AF construct to neutralize the BA.2 variant (Figure 3B).

[0298] Three selected 2x2 C-Fab leads, COVAF-40, 41, and 43, were further purified using size exclusion chromatography and tested for potency and breadth of coverage in SARS-CoV-2 pseudovirus neutralization assays using multiple Omicron variants, including BA.1, BA.2, BA.2.12.1, BA.4 / BA.5, and BA.4+BA.4.6 (Table 9). Compared to the parental IgG control, the 2x2 C-Fab COV-AF40 (10987x10987) showed enhanced broad neutralization potency against D614G, and all Omicron variants were tested against REGN10987 IgG. However, this enhancement is not sufficient to provide protection against individual omicron variants, as potency losses ranging from 18- to 576-fold are observed compared to the neutralization potency (IC50) of REGN10987 against D614G (Table 9).

[0299] [Table 10]

[0300] 8.1. Example 3: Neutralizing Activity of REGN14287-Based Tetravalent 2x2 AF Because REGN14287 (containing the VH and VL domains from mAb14287 shown in Table 4) targets a different epitope than the five previously tested antibodies, a second set of 12 AF molecules (COVAF46-57) was generated by pairing REGN14287 anchor arms in both 2x2 N-Fab and C-Fab formats with REGN10933, 10987, 10985, 14315, and 14256. For comparison, two 14287 x 14287 self-paired AF molecules were also generated (Tables 7 and 8). These AFs, the corresponding parental IgG, and the REGN10933 / 10987 combination were tested in pseudovirus neutralization assays using D614G and various Omicron variants, as described in section 8.1.2. Neutralization potency (IC50) and fold change of individual AF IC50 relative to REGN10933 / 10987 IC50 relative to D614G were determined.

[0301] Two tetravalent monospecific AFs, 2x2 N-Fab COVAF-51 and 2x2 C-Fab COVAF-57 (14287x14287), showed the broadest and most potent potency against all Omicron variants tested (BA.1, BA.2, BA.2.75, BA.4 / 5, IC50 ranged from 1.2 to 2.9 E -11 M range, which is within 1-2-fold of the potency of REGN10933 / 10987 against D614G (Table 10). All other tetravalent bispecific AFs showed much reduced (at least 4-fold) neutralization potency against at least one omicron variant (Table 10).

[0302] The neutralizing activity of COVAF-41–57 against the currently circulating predominant omicron variant BQ.1 was tested in a pseudovirus assay (Figures 4A and 4B). Both COVAF-51 2x2 N-Fab and COVAF-57 2x2 C-Fab, both bearing monospecific tetravalent REGN14287 arms, exhibited a neutralizing activity of 1.3E -11COVAF-51 and COVAF-57 demonstrated superior activity to other bispecific bivalent AFs with an IC50 of M (Figures 4A and 4B). The potency of COVAF-51 and COVAF-57 is slightly superior to the parental REGN14287 IgG control and similar to that of REGN10933 / 10987 against the D614G variant (IC50 = 1.2E -11 However, neutralization of this IgG combination was completely lost against BQ.1 (Figures 4A and 4B).

[0303] [Table 11-1]

[0304] [Table 11-2]

[0305] 9. Citation of References All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

Claims

1. A multivalent anti-spike protein binding molecule comprising at least four anti-spike protein antigen binding domains (ABDs) operably linked by one or more multimerization moieties.

2. The multivalent anti-spike protein binding molecule of claim 1, which is tetravalent.

3. (a) a first half antibody, (i) a first ABD comprising a first VH and a first VL; (ii) an optional first linker; (iii) a second ABD comprising a second VH and a second VL; (iv) an optional first hinge domain, and (v) a first half antibody comprising a first Fc domain; and (b) a second half antibody, (i) a third ABD comprising a third VH and a third VL; (ii) an optional second linker; (iii) a fourth ABD comprising a fourth VH and a fourth VL; (iv) an optional second hinge domain, and (v) a second half antibody comprising a second Fc domain.

4. (a) a first half antibody, (i) a first ABD, typically comprising a first VH and a first VL; (ii) an optional first hinge domain; (iii) a first Fc domain; (iv) an optional first linker, and (v) a first half antibody comprising a second ABD, typically comprising a second VH and a second VL; and (b) a second half antibody, (i) a third ABD, typically comprising a third VH and a third VL; (ii) an optional second hinge domain; (iii) a second Fc domain; (iv) an optional second linker, and (v) a second half antibody comprising a fourth ABD, typically comprising a fourth VH and a fourth VL.

5. 5. The multivalent anti-spike protein binding molecule of claim 3 or 4, wherein the first Fc domain and the second Fc domain form an Fc homodimer.

6. 5. The multivalent anti-spike protein binding molecule of claim 3 or 4, wherein the first Fc domain and the second Fc domain form an Fc heterodimer.

7. 7. The multivalent anti-spike protein binding molecule of claim 3, wherein the first, second, third, and fourth ABDs are the same.

8. The multivalent anti-spike protein binding molecule of any one of claims 3 to 6, wherein the first and second ABDs are the same.

9. 9. The multivalent anti-spike protein binding molecule of claim 8, wherein the third and fourth ABDs are the same.

10. 10. The multivalent anti-spike protein binding molecule of claim 9, wherein the third and fourth ABDs are different from the first and second ABDs.

11. The multivalent anti-spike protein binding molecule of any one of claims 3 to 10, wherein the first and third ABDs are the same.

12. 12. The multivalent anti-spike protein binding molecule of claim 11 , wherein the second and fourth ABDs are the same.

13. 13. The multivalent anti-spike protein binding molecule of claim 12, wherein the second and fourth ABDs are different from the first and third ABDs.

14. 14. The multivalent anti-spike protein binding molecule of any one of claims 1 to 13, wherein the antigen binding domain (ABD) is human or humanized.

15. 15. The multivalent anti-spike protein binding molecule of any one of claims 1 to 14, wherein one or more (or all) ABDs are neutralizing.

16. The multivalent anti-spike protein binding molecule of any one of claims 1 to 15, wherein the Fc domain is an IgG domain.

17. 17. The multivalent anti-spike protein binding molecule of claim 16, wherein the IgG domain is an IgG1 domain.

18. 17. The multivalent anti-spike protein binding molecule of claim 16, wherein the IgG domain is an IgG4 domain.

19. 19. The multivalent anti-spike protein binding molecule of any one of claims 1 to 18, lacking the first linker and the second linker.

20. The multivalent anti-spike protein binding molecule of any one of claims 1 to 18, comprising the first linker and the second linker.

21. 21. The multivalent anti-spike protein binding molecule of any one of claims 1 to 20, wherein one or more of the ABDs comprise the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences of an antibody shown in Table 4.

22. 21. The multivalent anti-spike protein binding molecule of any one of claims 1 to 20, wherein one or more of the ABDs comprise the VH and VL sequences of an antibody shown in Table 4.

23. One or more of the ABDs are (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 579, 580, and 581, respectively; (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs: 398, 372, and 583, respectively.

24. 24. The multivalent anti-spike protein binding molecule of any one of claims 1 to 23, wherein at least two of the ABDs are Fab domains, and optionally the Fab domains are not single-chain Fab domains.

25. 24. The multivalent anti-spike protein binding molecule of any one of claims 1 to 23, wherein all the ABDs are Fab domains, and optionally the Fab domains are not single-chain Fab domains.

26. 24. The multivalent anti-spike protein binding molecule of any one of claims 1 to 23, wherein at least two of the ABDs are scFvs.

27. 24. The multivalent anti-spike protein binding molecule of any one of claims 1 to 23, wherein at least all of the ABDs are scFvs.

28. 28. The multivalent anti-spike protein binding molecule of any one of claims 1 to 27, which is monospecific.

29. 28. The multivalent anti-spike protein binding molecule of any one of claims 1 to 27, which is multispecific.

30. 30. The multivalent anti-spike protein binding molecule of claim 29, which is bispecific.

31. A nucleic acid or a plurality of nucleic acids encoding the multivalent anti-spike protein binding molecule of any one of claims 1 to 30.

32. 32. A host cell engineered to express the multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or the nucleic acid(s) of claim 31.

33. 31. A method of producing a multivalent anti-spike protein binding molecule according to any one of claims 1 to 30, comprising culturing a host cell according to claim 32 and recovering the multivalent anti-spike protein binding molecule expressed thereby.

34. A pharmaceutical composition comprising the multivalent anti-spike protein binding molecule of any one of claims 1 to 30 and an excipient.

35. 36. A method of treating a coronavirus disease, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

36. 36. A method for inhibiting the interaction between coronavirus RBD and cellular ACE2, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

37. 36. A method of neutralizing coronavirus spike protein in vivo, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

38. 36. A method of inhibiting protease-mediated cleavage (e.g., TMPRSS2-mediated cleavage) of coronavirus spike protein in vivo, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

39. 36. A method of inhibiting viral entry of a coronavirus into a host cell in a subject, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

40. 36. A method of inhibiting reproduction of coronavirus spike protein in host cells in a subject, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

41. 36. A method of reducing the severity of a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

42. 36. A method of reducing coronavirus viral load, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

43. 36. A method of preventing disease progression in a subject having a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

44. 36. A method of reducing the duration of a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

45. 36. A method of reducing the risk of severe disease or death in a subject having a coronavirus infection, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule of any one of claims 1 to 30 or a pharmaceutical composition of claim 34.

46. 46. ​​The method of any one of claims 35 to 45, wherein the coronavirus is SARS-CoV.

47. 46. ​​The method of any one of claims 35 to 45, wherein the coronavirus is SARS-CoV-2.

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