Multivalent anti-spike protein binding molecules and uses thereof

Abstract

The present disclosure provides multivalent anti-spike protein binding molecules. 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 claims the benefit of priority to U.S. Provisional Application No. 63 / 487,424 (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-029WO_SL.xml, and is 641,288 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 in the Betacoronavirus genus, which also includes SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-OC43, and HCoV-HKU1 (Jackson et al., 2022, Nat Rev Mol Cell Biol. 23(1):3-20). SARS-CoV-2 causes COVID-19, a potentially life-threatening disease that was first characterized in late 2019 and expanded 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 cell types.

[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, prior SARS-CoV-2 infection does not appear to confer complete immunity against 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 reemerges (Callaway, Nature (News), 11 August 2022). 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 treatments that are effective against SARS-CoV-2. Summary of the Invention

[0007] 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 and are suitable for inhibiting interactions between coronaviruses and host cells. Multivalent anti-spike protein binding molecules of the present disclosure typically comprise multiple spike protein antigen-binding domains (ABDs) (e.g., 10 or 12 spike protein ABDs) operably linked by one or more multimerization moieties (e.g., 5 or 6 multimerization moieties). Multivalent anti-spike protein binding molecules of the present disclosure are described in numbered embodiments 1-102.

[0008] Multivalent anti-spike protein binding molecules of the present disclosure are typically decavalent or dodecavalent and include ABDs, e.g., in the form of Fabs or scFvs. In some embodiments, 10 or 12 ABDs are spike protein ABDs. 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, optionally all having the same sequence) or multispecific (e.g., at least two of the ABDs bind to different regions or variants of the spike protein, differ in sequence, or bind to the spike protein and another target). 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 6.2 and 6.3, and numbered embodiments 4-27, 36, and 37.

[0009] Multivalent anti-spike protein binding molecules of the present disclosure comprise one or more multimerization moieties. Typically, multivalent anti-spike protein binding molecules of the present disclosure comprise five or six multimerization moieties (e.g., five or six dimeric IgM Fc domains), each comprising or consisting of an Fc dimer. In certain aspects, the multivalent anti-spike protein binding molecules further comprise a J chain connecting the IgM Fc domains, the inclusion of which typically provides a molecule comprising 10 ABDs (as opposed to 12 ABDs in molecules that do not include a J chain connecting the IgM Fc domains). In some embodiments, the J chain is operably linked to an Fc domain (which can be a dimerizing IgG domain, a non-dimerizing Fc domain, or an Fc 1.5 domain), e.g., an IgG Fc domain. Multimerization moieties suitable for incorporation into multivalent anti-spike protein binding molecules of the present disclosure are described in Section 6.4 and numbered embodiments 35, and 38-66.

[0010] 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 6.5.

[0011] 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 6.6 and numbered embodiments 103-105.

[0012] 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 6.7 and numbered embodiment 106. Methods of use of the multivalent anti-spike protein binding molecules are described in Section 6.8 and numbered embodiments 107-119.

[0013] 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]

[0014] 5. Brief description of the drawings [Figure 1A] 1 shows the structures of pentameric and hexameric constructs of exemplary IgM alternating format ("AF") antibodies of the present disclosure, where (1) represents the Fab portion, (2) represents the Cμ2 domain of IgM-Fc, (3) represents the Cμ3 domain of IgM-Fc, (4) represents the Cμ4 domain of IgM-Fc, (5) represents the J chain region of IgM, (6) represents the J chain operably linked to a dimerized IgG Fc domain, (7) represents the J chain operably linked to an Fc1.5 domain, and (8) represents the J chain operably linked to a non-dimerized Fc domain. [Figure 1B] 1 shows the structures of pentameric and hexameric constructs of exemplary IgM alternating format ("AF") antibodies of the present disclosure, where (1) represents the Fab portion, (2) represents the Cμ2 domain of IgM-Fc, (3) represents the Cμ3 domain of IgM-Fc, (4) represents the Cμ4 domain of IgM-Fc, (5) represents the J chain region of IgM, (6) represents the J chain operably linked to a dimerized IgG Fc domain, (7) represents the J chain operably linked to an Fc1.5 domain, and (8) represents the J chain operably linked to a non-dimerized Fc domain. [Figure 1C]1 shows the structures of pentameric and hexameric constructs of exemplary IgM alternating format ("AF") antibodies of the present disclosure, where (1) represents the Fab portion, (2) represents the Cμ2 domain of IgM-Fc, (3) represents the Cμ3 domain of IgM-Fc, (4) represents the Cμ4 domain of IgM-Fc, (5) represents the J chain region of IgM, (6) represents the J chain operably linked to a dimerized IgG Fc domain, (7) represents the J chain operably linked to an Fc1.5 domain, and (8) represents the J chain operably linked to a non-dimerized Fc domain. [Figure 1D] 1 shows the structures of pentameric and hexameric constructs of exemplary IgM alternating format ("AF") antibodies of the present disclosure, where (1) represents the Fab portion, (2) represents the Cμ2 domain of IgM-Fc, (3) represents the Cμ3 domain of IgM-Fc, (4) represents the Cμ4 domain of IgM-Fc, (5) represents the J chain region of IgM, (6) represents the J chain operably linked to a dimerized IgG Fc domain, (7) represents the J chain operably linked to an Fc1.5 domain, and (8) represents the J chain operably linked to a non-dimerized Fc domain. [Figure 1E] 1 shows the structures of pentameric and hexameric constructs of exemplary IgM alternating format ("AF") antibodies of the present disclosure, where (1) represents the Fab portion, (2) represents the Cμ2 domain of IgM-Fc, (3) represents the Cμ3 domain of IgM-Fc, (4) represents the Cμ4 domain of IgM-Fc, (5) represents the J chain region of IgM, (6) represents the J chain operably linked to a dimerized IgG Fc domain, (7) represents the J chain operably linked to an Fc1.5 domain, and (8) represents the J chain operably linked to a non-dimerized Fc domain. [Figure 2] Two representative reducing SDS-PAGE gels of six exemplary IgM constructs with distinct anti-SARS-CoV-2 S-protein Fab portions are shown before (left gel) or after affinity purification (right gel). [Figure 3A]Figure 3 shows size exclusion chromatography (SEC) profiles of six exemplary IgM constructs with distinct anti-SARS-CoV-2 S-protein Fab portions. Figure 3A shows the SEC profile of IgM construct 10933-IgM. Figure 3B shows the SEC profile of IgM construct 14256-IgM. Figure 3C shows the SEC profile of IgM construct 10987-IgM. Figure 3D shows the SEC profile of IgM construct 14315-IgM. Figure 3E shows the SEC profile of IgM construct 10985-IgM. Figure 3F shows the SEC profile of IgM construct 10989-IgM. [Figure 3B] Figure 3 shows size exclusion chromatography (SEC) profiles of six exemplary IgM constructs with distinct anti-SARS-CoV-2 S-protein Fab portions. Figure 3A shows the SEC profile of IgM construct 10933-IgM. Figure 3B shows the SEC profile of IgM construct 14256-IgM. Figure 3C shows the SEC profile of IgM construct 10987-IgM. Figure 3D shows the SEC profile of IgM construct 14315-IgM. Figure 3E shows the SEC profile of IgM construct 10985-IgM. Figure 3F shows the SEC profile of IgM construct 10989-IgM. [Figure 3C] Figure 3 shows size exclusion chromatography (SEC) profiles of six exemplary IgM constructs with distinct anti-SARS-CoV-2 S-protein Fab portions. Figure 3A shows the SEC profile of IgM construct 10933-IgM. Figure 3B shows the SEC profile of IgM construct 14256-IgM. Figure 3C shows the SEC profile of IgM construct 10987-IgM. Figure 3D shows the SEC profile of IgM construct 14315-IgM. Figure 3E shows the SEC profile of IgM construct 10985-IgM. Figure 3F shows the SEC profile of IgM construct 10989-IgM. [Figure 3D]Figure 3 shows size exclusion chromatography (SEC) profiles of six exemplary IgM constructs with distinct anti-SARS-CoV-2 S-protein Fab portions. Figure 3A shows the SEC profile of IgM construct 10933-IgM. Figure 3B shows the SEC profile of IgM construct 14256-IgM. Figure 3C shows the SEC profile of IgM construct 10987-IgM. Figure 3D shows the SEC profile of IgM construct 14315-IgM. Figure 3E shows the SEC profile of IgM construct 10985-IgM. Figure 3F shows the SEC profile of IgM construct 10989-IgM. [Figure 3E] Figure 3 shows size exclusion chromatography (SEC) profiles of six exemplary IgM constructs with distinct anti-SARS-CoV-2 S-protein Fab portions. Figure 3A shows the SEC profile of IgM construct 10933-IgM. Figure 3B shows the SEC profile of IgM construct 14256-IgM. Figure 3C shows the SEC profile of IgM construct 10987-IgM. Figure 3D shows the SEC profile of IgM construct 14315-IgM. Figure 3E shows the SEC profile of IgM construct 10985-IgM. Figure 3F shows the SEC profile of IgM construct 10989-IgM. [Figure 3F] Figure 3 shows size exclusion chromatography (SEC) profiles of six exemplary IgM constructs with distinct anti-SARS-CoV-2 S-protein Fab portions. Figure 3A shows the SEC profile of IgM construct 10933-IgM. Figure 3B shows the SEC profile of IgM construct 14256-IgM. Figure 3C shows the SEC profile of IgM construct 10987-IgM. Figure 3D shows the SEC profile of IgM construct 14315-IgM. Figure 3E shows the SEC profile of IgM construct 10985-IgM. Figure 3F shows the SEC profile of IgM construct 10989-IgM. [Figure 4A]Figure 4 shows the degree of neutralization of the SARS-CoV-2 pseudovirus D614G and the BA.1 and BA.2 variants by exemplary IgMs targeting different RBDs. Figure 4A shows the degree of neutralization of the pseudovirus D614G. Figures 4B and 4C show the degree of neutralization of the BA.1 and BA.2 variants, respectively. In all evaluations, REGEN-COV (REGN10987 / REGN10933) was included as a control. [Figure 4B] Figure 4 shows the degree of neutralization of the SARS-CoV-2 pseudovirus D614G and the BA.1 and BA.2 variants by exemplary IgMs targeting different RBDs. Figure 4A shows the degree of neutralization of the pseudovirus D614G. Figures 4B and 4C show the degree of neutralization of the BA.1 and BA.2 variants, respectively. In all evaluations, REGEN-COV (REGN10987 / REGN10933) was included as a control. [Figure 4C] Figure 4 shows the degree of neutralization of the SARS-CoV-2 pseudovirus D614G and the BA.1 and BA.2 variants by exemplary IgMs targeting different RBDs. Figure 4A shows the degree of neutralization of the pseudovirus D614G. Figures 4B and 4C show the degree of neutralization of the BA.1 and BA.2 variants, respectively. In all evaluations, REGEN-COV (REGN10987 / REGN10933) was included as a control. [Figure 5A] Figure 5 shows the neutralization potency of 10933-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10933 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 5A shows the degree of neutralization of D614G. Figure 5B shows the degree of neutralization of the BA.1 variant, and Figure 5C shows the degree of neutralization of the BA.2 variant. [Figure 5B]Figure 5 shows the neutralization potency of 10933-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10933 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 5A shows the degree of neutralization of D614G. Figure 5B shows the degree of neutralization of the BA.1 variant, and Figure 5C shows the degree of neutralization of the BA.2 variant. [Figure 5C] Figure 5 shows the neutralization potency of 10933-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10933 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 5A shows the degree of neutralization of D614G. Figure 5B shows the degree of neutralization of the BA.1 variant, and Figure 5C shows the degree of neutralization of the BA.2 variant. [Figure 6A] Figure 6 shows the neutralization potency of 10989-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, the REGN10989 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 6A shows the degree of neutralization of D614G. Figure 6B shows the degree of neutralization of the BA.1 variant, and Figure 6C shows the degree of neutralization of the BA.2 variant. Figure 6C shows the degree of neutralization of the BA.2 variant. [Figure 6B] Figure 6 shows the neutralization potency of 10989-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, the REGN10989 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 6A shows the degree of neutralization of D614G. Figure 6B shows the degree of neutralization of the BA.1 variant, and Figure 6C shows the degree of neutralization of the BA.2 variant. Figure 6C shows the degree of neutralization of the BA.2 variant. [Figure 6C]Figure 6 shows the neutralization potency of 10989-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10989 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 6A shows the degree of neutralization of D614G. Figure 6B shows the degree of neutralization of the BA.1 variant, and Figure 6C shows the degree of neutralization of the BA.2 variant. [Figure 7A] Figure 7 shows the neutralization potency of 10987-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10987 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 7A shows the degree of neutralization of D614G. Figure 7B shows the degree of neutralization of the BA.1 variant, and Figure 7C shows the degree of neutralization of the BA.2 variant. [Figure 7B] Figure 7 shows the neutralization potency of 10987-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10987 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 7A shows the degree of neutralization of D614G. Figure 7B shows the degree of neutralization of the BA.1 variant, and Figure 7C shows the degree of neutralization of the BA.2 variant. [Figure 7C] Figure 7 shows the neutralization potency of 10987-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10987 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 7A shows the degree of neutralization of D614G. Figure 7B shows the degree of neutralization of the BA.1 variant, and Figure 7C shows the degree of neutralization of the BA.2 variant. [Figure 8A]Figure 8 shows the neutralization potency of 10985-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10985 parental IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 8A shows the degree of neutralization of D614G. Figure 8B shows the degree of neutralization of the BA.1 variant, and Figure 8C shows the degree of neutralization of the BA.2 variant. [Figure 8B] Figure 8 shows the neutralization potency of 10985-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10985 parental IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 8A shows the degree of neutralization of D614G. Figure 8B shows the degree of neutralization of the BA.1 variant, and Figure 8C shows the degree of neutralization of the BA.2 variant. [Figure 8C] Figure 8 shows the neutralization potency of 10985-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN10985 parental IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 8A shows the degree of neutralization of D614G. Figure 8B shows the degree of neutralization of the BA.1 variant, and Figure 8C shows the degree of neutralization of the BA.2 variant. [Figure 9A] Figure 9 shows the neutralization potency of 14315-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN14315 parental IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 9A shows the degree of neutralization of D614G. Figure 9B shows the degree of neutralization of the BA.1 variant, and Figure 9C shows the degree of neutralization of the BA.2 variant. [Figure 9B]Figure 9 shows the neutralization potency of 14315-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN14315 parental IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 9A shows the degree of neutralization of D614G. Figure 9B shows the degree of neutralization of the BA.1 variant, and Figure 9C shows the degree of neutralization of the BA.2 variant. [Figure 9C] Figure 9 shows the neutralization potency of 14315-IgM against the SARS-CoV-2 pseudovirus D614G and two Omicron variants. For comparison, REGN14315 parental IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 9A shows the degree of neutralization of D614G. Figure 9B shows the degree of neutralization of the BA.1 variant, and Figure 9C shows the degree of neutralization of the BA.2 variant. [Figure 10A] Figure 10 shows the neutralization potency of 14287-IgM against the SARS-CoV-2 pseudovirus D614G and four Omicron variants. For comparison, REGN14287 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 10A shows the degree of neutralization of D614G. Figure 10B shows the degree of neutralization of the BA.1 variant. Figure 10C shows the degree of neutralization of the BA.2 variant. Figure 10D shows the degree of neutralization of the BA.2.75 variant. Figure 10E shows the degree of neutralization of the BA.4 / BA.5 variant. [Figure 10B] Figure 10 shows the neutralization potency of 14287-IgM against the SARS-CoV-2 pseudovirus D614G and four Omicron variants. For comparison, REGN14287 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 10A shows the degree of neutralization of D614G. Figure 10B shows the degree of neutralization of the BA.1 variant. Figure 10C shows the degree of neutralization of the BA.2 variant. Figure 10D shows the degree of neutralization of the BA.2.75 variant. Figure 10E shows the degree of neutralization of the BA.4 / BA.5 variant. [Figure 10C]Figure 10 shows the neutralization potency of 14287-IgM against the SARS-CoV-2 pseudovirus D614G and four Omicron variants. For comparison, REGN14287 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 10A shows the degree of neutralization of D614G. Figure 10B shows the degree of neutralization of the BA.1 variant. Figure 10C shows the degree of neutralization of the BA.2 variant. Figure 10D shows the degree of neutralization of the BA.2.75 variant. Figure 10E shows the degree of neutralization of the BA.4 / BA.5 variant. [Figure 10D] Figure 10 shows the neutralization potency of 14287-IgM against the SARS-CoV-2 pseudovirus D614G and four Omicron variants. For comparison, REGN14287 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 10A shows the degree of neutralization of D614G. Figure 10B shows the degree of neutralization of the BA.1 variant. Figure 10C shows the degree of neutralization of the BA.2 variant. Figure 10D shows the degree of neutralization of the BA.2.75 variant. Figure 10E shows the degree of neutralization of the BA.4 / BA.5 variant. [Figure 10E] Figure 10 shows the neutralization potency of 14287-IgM against the SARS-CoV-2 pseudovirus D614G and four Omicron variants. For comparison, REGN14287 parent IgG and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 10A shows the degree of neutralization of D614G. Figure 10B shows the degree of neutralization of the BA.1 variant. Figure 10C shows the degree of neutralization of the BA.2 variant. Figure 10D shows the degree of neutralization of the BA.2.75 variant. Figure 10E shows the degree of neutralization of the BA.4 / BA.5 variant. [Figure 11A]Figure 11 shows the neutralization potency of 14315-IgM and 14287-IgM against the currently circulating Omicron variant BQ.1. Figure 11A shows the neutralization of BQ.1 by 14315-IgM, 14287-IgM, 10933-IgM, and 10985-IgM. REGEN-COV was included as a control. Figure 11B shows a comparison of BQ.1 neutralization between 14315-IgM and REGN14315 IgG. Figure 11C shows a comparison of BQ.1 neutralization between 14287-IgM and REGN14287 IgG. [Figure 11B] Figure 11 shows the neutralization potency of 14315-IgM and 14287-IgM against the currently circulating Omicron variant BQ.1. Figure 11A shows the neutralization of BQ.1 by 14315-IgM, 14287-IgM, 10933-IgM, and 10985-IgM. REGEN-COV was included as a control. Figure 11B shows a comparison of BQ.1 neutralization between 14315-IgM and REGN14315 IgG. Figure 11C shows a comparison of BQ.1 neutralization between 14287-IgM and REGN14287 IgG. [Figure 11C] Figure 11 shows the neutralization potency of 14315-IgM and 14287-IgM against the currently circulating Omicron variant BQ.1. Figure 11A shows the neutralization of BQ.1 by 14315-IgM, 14287-IgM, 10933-IgM, and 10985-IgM. REGEN-COV was included as a control. Figure 11B shows a comparison of BQ.1 neutralization between 14315-IgM and REGN14315 IgG. Figure 11C shows a comparison of BQ.1 neutralization between 14287-IgM and REGN14287 IgG. [Figure 12A] Figure 12 shows Protein A binding and size-exclusion chromatography (SEC) of multivalent anti-spike protein binding molecules containing IgG Fc-linked J chains. Figure 12A shows a representative SDS-PAGE gel of three exemplary IgM constructs after Protein A incubation. Figure 12B shows the SEC profile of the same three exemplary IgM constructs shown in Figure 12A. [Figure 12B]Figure 12 shows Protein A binding and size-exclusion chromatography (SEC) of multivalent anti-spike protein binding molecules containing IgG Fc-linked J chains. Figure 12A shows a representative SDS-PAGE gel of three exemplary IgM constructs after Protein A incubation. Figure 12B shows the SEC profile of the same three exemplary IgM constructs shown in Figure 12A. [Figure 13A] Figure 13 shows the neutralization potency of 14287-IgM, which contains an IgG Fc-linked J chain, against the SARS-CoV-2 pseudovirus D614G and XBB1.5 variants. For comparison, REGN14287 parent IgG, 14287-IgM, and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 13A shows the degree of neutralization of D614G. Figure 13B shows the degree of neutralization of the XBB1.5 variant. [Figure 13B] Figure 13 shows the neutralization potency of 14287-IgM, which contains an IgG Fc-linked J chain, against the SARS-CoV-2 pseudovirus D614G and XBB1.5 variants. For comparison, REGN14287 parent IgG, 14287-IgM, and REGEN-COV (REGN10987 / REGN10933) were also included. Figure 13A shows the degree of neutralization of D614G. Figure 13B shows the degree of neutralization of the XBB1.5 variant. DETAILED DESCRIPTION OF THE INVENTION

[0015] 6. Detailed Description 6.1.Definition As used herein, the following terms are intended to have the following meanings:

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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).

[0020] 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).

[0021] 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 6.4.1).

[0022] Bispecific: As used herein, the term "bispecific" refers to an antigen-binding molecule that comprises two or more different ABDs. For example, the ABDs in a bispecific molecule can bind to two different portions of the same target antigen (or, in the case of a viral protein, different variants of the same target antigen), or each ABD can bind to a different target antigen.

[0023] 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.

[0024] 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 VH 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.

[0025] 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.

[0026] Decavalent: The term "decavalent" is used herein with reference to an antigen-binding molecule comprising 10 ABDs. Decavalent anti-spike protein binding molecules of the present disclosure can be monospecific or multispecific, e.g., bispecific. In some embodiments, a decavalent anti-spike protein binding molecule refers to an anti-spike protein binding molecule comprising 10 spike protein ABDs. The 10 spike protein ABDs can be the same (i.e., the antigen-binding molecule is monospecific) or different (i.e., the antigen-binding molecule is multispecific and binds to different regions and / or variants of the spike protein). In some embodiments, a decavalent anti-spike protein binding molecule is a pentameric assembly of five IgM Fc dimers, each containing a spike protein ABD at its N-terminus and connected via a J chain. In other embodiments, a decavalent anti-spike protein binding molecule refers to an anti-spike protein comprising multiple spike protein ABDs and multiple ABDs that bind to different target molecules (i.e., the antigen-binding molecule is multispecific). For example, in some embodiments, the decavalent anti-spike protein binding molecule is bispecific and comprises five anti-spike protein ABDs and five ABDs that bind to additional targets.

[0027] Dodecavalent: The term "dodecavalent" is used herein with reference to an antigen-binding molecule comprising 12 ABDs. The dodecavalent anti-spike protein-binding molecules of the present disclosure can be monospecific or multispecific, e.g., bispecific. In some embodiments, a dodecavalent anti-spike protein-binding molecule refers to an anti-spike protein-binding molecule comprising 12 spike protein ABDs. The 12 spike protein ABDs can be the same (i.e., the antigen-binding molecule is monospecific) or different (i.e., the antigen-binding molecule is multispecific and binds to different regions and / or variants of the spike protein). In some embodiments, a dodecavalent spike protein-binding molecule is a hexameric assembly of six IgM Fc dimers, each IgM Fc comprising a spike protein ABD at its N-terminus and lacking a J-chain connection. In other embodiments, a dodecavalent anti-spike protein-binding molecule refers to an anti-spike protein comprising multiple spike protein ABDs and multiple ABDs that bind to different target molecules (i.e., the antigen-binding molecule is multispecific). For example, in some embodiments, the dodecavalent anti-spike protein binding molecule is bispecific and comprises six anti-spike protein ABDs and six ABDs that bind to additional targets.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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).

[0036] 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.

[0037] Multispecific: As used herein, the term "multispecific" refers to an antigen-binding molecule that comprises two or more ABDs. For example, the ABDs in a multispecific molecule can bind to two or more different portions of the same target antigen (or, in the case of viral proteins, different variants of the same target antigen, such as the spike protein), or each ABD can bind to a different target antigen.

[0038] 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.

[0039] 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 domain, J chain and IgG Fc domain, etc.) 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.

[0040] Polypeptide, Peptide, and Protein: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0041] 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.

[0042] 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.

[0043] 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 are typically arranged N-terminally to C-terminally in the order VH-VL or VL-VH, separated by a linker.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 6.2. Spike Protein Antigen-Binding Domain The present disclosure relates to multivalent anti-spike protein binding molecules of the present disclosure that comprise multiple spike protein antigen binding domains (ABDs).

[0048] In some embodiments, a multivalent anti-spike protein binding molecule of the disclosure comprises 10 or 12 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 anti-spike protein ABDs are identical.

[0049] 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.

[0050] 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.

[0051] Suitable spike protein ABD formats are described in Section 6.3. 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 6.3.1 or an scFv as described in Section 6.3.2.

[0052] 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.

[0053] [Table 1-1]

[0054] [Table 1-2]

[0055] [Table 1-3]

[0056] [Table 1-4]

[0057] [Table 1-5]

[0058] 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.

[0059] [Table 2-1]

[0060] [Table 2-2]

[0061] [Table 2-3]

[0062] [Table 2-4]

[0063] [Table 2-5]

[0064] [Table 2-6]

[0065] [Table 2-7]

[0066] [Table 2-8]

[0067] [Table 2-9]

[0068] [Table 2-10]

[0069] 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.

[0070] [Table 3-1]

[0071] [Table 3-2]

[0072] [Table 3-3]

[0073] [Table 3-4]

[0074] 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.

[0075] [Table 4-1]

[0076] [Table 4-2]

[0077] [Table 4-3]

[0078] 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.

[0079] 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.

[0080] 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.

[0081] [Table 5]

[0082] 6.3. 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 cleavage) or engineered fragment of an immunoglobulin. Antibody fragments include, but are not limited to, VH (or VH) fragments, VL (or VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0083] In some embodiments, the spike protein ABD is in the form of a Fab or scFv. 6.3.1.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.

[0084] 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.

[0085] 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:

[0086] [Table 6]

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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).

[0093] 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).

[0094] 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.

[0095] 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).

[0096] 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.

[0097] 6.3.2.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 6.5.

[0098] 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.

[0099] 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 6.5 (typically, a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4-Ser)3), 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).

[0100] 6.4. Multimerization moiety In some embodiments, multivalent anti-spike protein binding molecules of the present disclosure comprise one or more multimerization moieties, e.g., one or more multimerization moieties that are or comprise Fc domains. In specific embodiments, multivalent anti-spike protein binding molecules of the present disclosure comprise a single multimerization moiety (e.g., a single Fc domain) and / or 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 ACE fusion protein is a pentamer or hexamer of five or six dimeric Fc regions from IgM, e.g., as described in Section 6.4.1.

[0101] Fc Domain The multivalent anti-spike protein binding molecules of the present disclosure can include an Fc domain, or a pair of Fc domains that associate to form an Fc region, from any suitable species operably linked to an ACE2 moiety. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the ACE2 moiety is fused to an IgM Fc domain.

[0102] 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 IgM.

[0103] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (Cμ2 and Cμ3), while the domain of IgE and IgM consists of three heavy chain constant domains (Cμ2, Cμ3, and Cμ4), which dimerize to create the Fc region.

[0104] 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.

[0105] IgM Fc Domain In some embodiments, the multivalent anti-spike protein binding molecule of the present disclosure comprises an Fc domain derived from IgM. IgM naturally exists in humans as a covalent multimer of heavy (H) and light (L) chains that form a common H2L2 antibody unit. In addition to heavy and light chains, IgM also has a third chain known as the connecting (J) chain (Keyt et al., 2020, Antibodies. 9(4):53). IgM exists as a pentamer when the J chain is incorporated and as a hexamer when the J chain is absent.

[0106] Heavy chain constant domains for use in generating IgM 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. In one example, the Fc regions of the present disclosure comprise at least one constant domain that differs in sequence from the wild-type constant domain. It will be understood that variant constant domains may be longer or shorter than their corresponding wild-type constant domains.

[0107] The heavy chain of IgM has an 18-amino acid extension to the C-terminal constant domain known as the tail. The tail contains cysteine ​​residues that form disulfide bonds between heavy chains within the polymer and is thought to play an important role in polymerization. The tail also contains glycosylation sites. In certain embodiments, the multivalent anti-spike protein binding molecules of the present disclosure comprise a tail.

[0108] IgM assembly typically begins with the association of heavy (H) and light (L) chains in an HL configuration, which then dimerizes to form the H2L2 subunit. A key site for this intrasubunit assembly is Cys337, which forms a disulfide bond between the two Cμ2 domains and stabilizes the H2L2 domain. These subunits then join together through disulfide bridges to form multimers. A residue involved in this multimerization is Cys575 on the tail domain of Cμ4, which forms a disulfide bond and enables noncovalent Cμ4 interactions. Another key residue is Cys414 on Cμ3, which further connects the two Cμ3 domains of adjacent H2L2 subunits in tandem with the disulfide bond between the Cys337 residues of Cμ2. In the presence of J chain, IgM assembly results in a pentamer in which the Cys337 disulfide bond is in tandem with both the Cys414 and Cys575 disulfide bonds (Pasalic et al., 2017, Proc. Nat'l Acad. Sci USA 114(41)E8575-E8584; Keyt et al., 2020, Antibodies. 9(4):53; Casali, 1998. Encyclopedia of Immunology (2nd Ed), p1212-1217). For IgM assembly to include a J chain, the J chain polypeptide must be coexpressed with a polypeptide encoding the H2L2 subunit domain.

[0109] In certain embodiments, the multimerizing moieties provided by the present disclosure are pentameric or hexameric binding molecules comprising a dimeric IgM heavy chain constant region or a multimerizing fragment thereof. An exemplary sequence of a full-length human IgM heavy chain constant domain is reproduced below.

[0110] [ka]

[0111] Without wishing to be bound by theory, assembly of dimeric IgM Fc regions into pentameric or hexameric structures is thought to involve at least the Cμ4 and / or tail (TP) domains (Braathen, R., et al., 2002. J. Biol. Chem. 277:42755-42762). Thus, multimerization moieties based on IgM Fc domains typically contain at least the Cμ4 and / or TP domain sequences.

[0112] An IgM heavy chain constant domain can further comprise a Cμ3 domain or a fragment thereof, a Cμ2 domain or a fragment thereof, and / or other IgM or other immunoglobulin heavy chain domains.

[0113] Exemplary sequences of human IgM heavy chain constant domains are reproduced in Table 6 below.

[0114] [Table 7]

[0115] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ4 domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.

[0116] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ4 and tail domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0117] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ3 and Cμ4 domains of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:6.

[0118] In some embodiments, the Fc domain comprises the amino acid sequence of the Cμ3 and Cμ4 and tail domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0119] In further embodiments, the Fc domain comprises the amino acid sequence of the Cμ2, Cμ3, and Cμ4 domains of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0120] In still further embodiments, the Fc domain comprises the amino acid sequence of the Cμ2, Cμ3, Cμ4 and tail domain of IgM, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the Fc domain comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0121] 6.4.3.J chain The J chain is a small 137-residue polypeptide that associates with IgM by forming a disulfide bond with the Cμ4 tail. Incorporation of the J chain into pentameric IgM closes the ring structure by bridging the first and fifth monomer units, thereby precluding the addition of a sixth IgM monomer.

[0122] An exemplary amino acid sequence of a human mature wild-type J chain is reproduced below.

[0123] [ka]

[0124] In some embodiments, the engineered J chain is incorporated into an IgM pentamer. An exemplary amino acid sequence of an engineered human mature J chain is reproduced below.

[0125] [ka]

[0126] Multivalent anti-spike protein binding molecules (e.g., pentameric multivalent anti-spike protein binding molecules) of the present disclosure may further comprise a J chain polypeptide associated with the CH4 tail. In various embodiments, the J chain polypeptide comprises the amino acid sequence of a mature naturally occurring or engineered J chain polypeptide, or an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the J chain polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 93%, at least 95%, or at least 98% sequence identity, at least 99% sequence identity, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0127] 6.4.3.1.IgG Fc-linked J chain In some embodiments, a multivalent anti-spike protein binding molecule of the present disclosure comprises a J chain polypeptide operably linked to an IgG Fc domain, optionally via a polypeptide linker. Examples of linkers suitable for connecting the J chain and IgG Fc domains are those identified in Section 6.5. In some embodiments, the IgG Fc domain is attached to the N-terminus of the J chain polypeptide. In some embodiments, the IgG Fc domain is attached to the C-terminus of the J chain polypeptide. Examples of such multivalent anti-spike protein binding molecules are shown in Figures 1C-1E.

[0128] In one embodiment, the IgG Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc IgG domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0129] Exemplary sequences of IgG Fc domains from IgG1, IgG2, IgG3, and IgG4 are provided in Table Y-1 below.

[0130] [Table 8]

[0131] In some embodiments, the IgG Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:10.

[0132] In some embodiments, the IgG Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:11.

[0133] In some embodiments, the IgG Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:12.

[0134] In some embodiments, the IgG Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:13.

[0135] In some embodiments, the IgG Fc domain is a non-dimerizing (or "monomeric") Fc domain, which refers to an Fc domain that has a reduced ability to self-associate compared to a wild-type Fc domain, or that completely lacks the ability to self-associate, as described, for example, in Helm et al., 1996, J. Biol. Chem. 271:7494-7500 or Ying et al., 2012, J. Biol. Chem. 287(23):19399-19408. One example of a non-dimerizing Fc domain contains amino acid substitutions at positions corresponding to T366 and / or Y407 (Kabat EU index numbering) in CH3, as described in U.S. Patent Publication No. 2019 / 0367611, incorporated herein by reference. Specific amino acid substitutions that may be included in non-dimerizing Fc domains include, for example, L351S, T366R, L368H, P395K, L242C, K334C, L351S, P343C, A431C, L351Y, T366Y, L368A, P395R, F405R, Y407M, K409A, F405E, Y407K, L351K, T366S, P395V, Y407A, and K409Y (numbering according to the Kabat EU index). The non-dimerizing Fc domains of the present disclosure may include any one, two, three, four, five, six, seven, eight, nine, or ten or more of the above substitutions.

[0136] Exemplary sequences of non-dimerizing Fc domains are provided below in Table Y-2. Bolded residues indicate the positions of amino acid substitutions relative to the wild-type human IgG sequence.

[0137] [Table 9]

[0138] In some embodiments, the non-dimerizing Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the non-dimerizing Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the non-dimerizing Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the non-dimerizing Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the non-dimerizing Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the non-dimerizing Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 19.

[0139] In some embodiments, an IgG Fc domain, in addition to a CH2 domain and a CH3 domain, further comprises an additional CH3 domain connected to the first CH3 domain via a linker (e.g., a linker described in Section 6.5). An Fc domain comprising such a configuration (CH2-CH3-linker-CH3) may be conveniently referred to herein as an "Fc1.5 domain" or simply "Fc1.5." The linker between the first and second CH3 domains of the Fc1.5 domain is preferably sufficiently long and flexible to allow dimerization between the first and second CH3 domains. Thus, in some embodiments, the Fc1.5 domain comprises a linker at least 5, at least 10, at least 15, or at least 20 amino acids in length connecting the first and second CH3 domains.

[0140] Exemplary sequences of the Fc1.5 domain are provided in Table Y-3 below.

[0141] [Table 10-1]

[0142] [Table 10-2]

[0143] In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:21. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 22. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 23. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 25.In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 26. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 27.

[0144] Exemplary sequences of J chains linked to IgG Fc domains are provided in Table Y-4 below.

[0145] [Table 11-1]

[0146] [Table 11-2]

[0147] In some embodiments, the IgG Fc-linked J chain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 29. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 30. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the Fc1.5 domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to SEQ ID NO:32.

[0148] 6.4.3.1.1. IgG Fc Domains with Altered Effector Functions In some embodiments, the IgG Fc domain of an IgG Fc-linked J chain of the present disclosure comprises one or more amino acid substitutions that alter (eg, reduce) binding to an Fc receptor and / or effector function.

[0149] 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.

[0150] In one embodiment, the Fc domain (e.g., the Fc domain of an IgG Fc-linked J chain) comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331, and P329 (numbering according to Kabat EU index). In a more particular embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235, and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. 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 Kabat EU index). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and an additional 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 additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a specific embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA," "PGLALA," or "LALAPG").

[0151] 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).

[0152] 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.

[0153] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to Fc receptors. An exemplary IgG4 Fc domain with reduced binding to Fc receptors may comprise an amino acid sequence selected from Table H below. In some embodiments, the Fc domain comprises only the bolded portion of the sequence shown below.

[0154] [Table 12-1]

[0155] [Table 12-2]

[0156] 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.

[0157] Hinge domain The IgG Fc-linked J chain of the present disclosure can comprise an Fc domain comprising a hinge domain at its N-terminus. The hinge region can be a natural hinge region or a modified hinge region. The hinge region is typically found at the N-terminus of the Fc region. The term "hinge domain," unless otherwise indicated by context, refers to a naturally occurring or non-naturally occurring hinge sequence that is a monomeric hinge domain in the context of a single or monomeric polypeptide chain, and can comprise two associated hinge sequences on separate polypeptide chains (e.g., an Fc region formed by the association of two Fc domains). Sometimes, the two associated hinge sequences are referred to as "hinge regions." In certain embodiments of IgG Fc-linked J chains, additional repeats of the hinge region may be incorporated into the polypeptide sequence.

[0158] 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.

[0159] 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.

[0160] In one embodiment, the IgG Fc linked J chain comprises an Fc region in which one or both Fc domains have an intact hinge domain 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.

[0161] In some embodiments, the IgG Fc-linked J chain 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).

[0162] In one embodiment, the IgG Fc-linked J chain comprises an Fc region in which each Fc domain has an intact hinge domain at its N-terminus, and each Fc domain and hinge domain is derived from IgG4, with each hinge domain containing the modified sequence CPPC. The core hinge region of human IgG4 contains the sequence CPSC, compared to IgG1, which contains the sequence CPPC. 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 proline 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.

[0163] 6.4.3.1.2.1. Chimeric Hinge Sequence The hinge domain can be a chimeric hinge domain. A "chimeric" hinge domain describes a hinge domain that includes a first region from a first type of IgG (e.g., IgG1, IgG2, IgG3, or IgG4) and a second region from a second, different type of IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0164] For example, a chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region.

[0165] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (previously disclosed as SEQ ID NO: 8 in WO2014 / 121087, incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (previously disclosed as SEQ ID NO: 9 in WO2014 / 121087). Such chimeric hinge sequences can be suitably linked to an IgG4 CH2 region (e.g., by incorporation into an IgG4 Fc domain, e.g., a human Fc domain or a murine Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, e.g., as described in Section 6.4.3.1.1).

[0166] 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.

[0167] Peptide linkers can range from 2 to 60 or more amino acids, and in certain embodiments, peptide linkers can range from 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids in length.

[0168] In certain embodiments, the peptide linker is at least 5, at least 6, or at least 7 amino acids in length, and optionally up to 30, up to 40, up to 50, or up to 60 amino acids in length.

[0169] In some of the aforementioned embodiments, the 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 of the aforementioned embodiments, the 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 still other of the aforementioned embodiments, the 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.

[0170] In some embodiments, the linker is a G4S linker. In some embodiments, the linker comprises two consecutive G4S sequences, three consecutive G4S sequences, four consecutive G4S sequences, five consecutive G4S sequences, or six consecutive G4S sequences.

[0171] 6.6. 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.

[0172] 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 more than two 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.

[0173] 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).

[0174] 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.

[0175] 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).

[0176] 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 flaviviruses.

[0177] 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.

[0178] 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.

[0179] 6.6.2.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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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 ...

[0188] 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).

[0189] Additional miscellaneous excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0190] 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.

[0191] 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).

[0192] 6.8. Treatment Indications and Methods The present disclosure provides methods for using and applying the multivalent anti-spike protein binding molecules of the present disclosure.

[0193] 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).

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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. In the following numbered embodiments, 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).

[0202] 1. A multivalent anti-spike protein binding molecule comprising at least five 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, comprising at least 10 anti-spike protein ABDs.

[0203] 3. The multivalent anti-spike protein binding molecule of embodiment 1 or embodiment 2, which is decavalent or dodecavalent. 4. The multivalent anti-spike protein binding molecule of any one of embodiments 1 to 3, wherein the antigen binding domain (ABD) is human or humanized.

[0204] 5. The multivalent anti-spike protein binding molecule of any one of embodiments 1 to 4, wherein the antigen binding domain (ABD) is a Fab. 6. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise a CDR sequence as set forth in any one of Tables 1-3.

[0205] 7. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, wherein one or more (or all) ABDs comprise a CDR sequence as set forth in any one of Tables 1-4.

[0206] 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 1.

[0207] 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 2.

[0208] 10. 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.

[0209] 11. 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 as set forth in Table 4.

[0210] 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: 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.

[0211] 13. 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.

[0212] 14. 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.

[0213] 15. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, 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.

[0214] 16. The multivalent anti-spike protein binding molecule of any one of embodiments 1-5, 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.

[0215] 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 1.

[0216] 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 2.

[0217] 19. 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.

[0218] 20. 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.

[0219] 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: 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.

[0220] 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: 578; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 582.

[0221] 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: 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.

[0222] 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: 506; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 510.

[0223] 25. 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.

[0224] 26. 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.

[0225] 27. The multivalent anti-spike protein binding molecule of any one of embodiments 1-26, wherein one or more (or all) ABDs are neutralizing. 28. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 26, which is capable of neutralizing SARS-CoV-2 variant BA.1.

[0226] 29. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 28, which is capable of neutralising SARS-CoV-2 variant BA.2. 30. The multivalent anti-spike protein binding molecule of any one of embodiments 1 to 29, which is monospecific.

[0227] 31. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 30, wherein the antigen binding domains (ABDs) are all the same. 32. A multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 29, which is multispecific.

[0228] 33. The multivalent anti-spike protein binding molecule of embodiment 32, which is bispecific. 34. The multivalent anti-spike protein binding molecule of embodiment 32 or embodiment 33, comprising two types of ABD.

[0229] 35. The multivalent anti-spike protein binding molecule of any one of embodiments 1-34, wherein one or more multimerization moieties comprises an Fc domain. 36. The multivalent anti-spike protein binding molecule of embodiment 34, wherein both types of ABD bind to the spike protein.

[0230] 37. The multivalent anti-spike protein binding molecule of embodiment 34, wherein one type of ABD binds to the spike protein and the other type of ABD binds to a different target. 38. The multivalent anti-spike protein binding molecule of embodiment 35, wherein the Fc domain is an IgM Fc domain.

[0231] 39. The multivalent anti-spike protein binding molecule of embodiment 38, wherein the Fc domain comprises a Cμ3 domain and a Cμ4 domain. 40. The multivalent anti-spike protein binding molecule of embodiment 38 or embodiment 39, wherein the Fc domain comprises a Cμ2 domain.

[0232] 41. The multivalent anti-spike protein binding molecule of any one of embodiments 38-40, which is a pentamer. 42. The multivalent anti-spike protein binding molecule of embodiment 41, which is a pentamer of five dimers, each dimer comprising two polypeptides, each polypeptide comprising an anti-spike protein ABD and an IgM Fc domain.

[0233] 43. The multivalent anti-spike protein binding molecule of embodiment 41 or embodiment 42, which is a homopentamer. 44. A multivalent anti-spike protein binding molecule according to any one of embodiments 41 to 43, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

[0234] 45. A multivalent anti-spike protein binding molecule according to any one of embodiments 41 to 44, comprising a J chain. 46. ​​The multivalent anti-spike protein binding molecule of embodiment 45, wherein the J chain is operably linked to the IgG Fc domain.

[0235] 47. The multivalent anti-spike protein binding molecule of embodiment 46, wherein the IgG Fc domain is N-terminal to the J chain. 48. The multivalent anti-spike protein binding molecule of embodiment 46, wherein the IgG Fc domain is C-terminal to the J chain.

[0236] 49. The multivalent anti-spike protein binding molecule of any one of embodiments 46 to 48, wherein the IgG Fc domain and the J chain are connected via a linker. 50. The multivalent anti-spike protein binding molecule of embodiment 49, wherein the linker is or comprises the amino acid sequence G4S.

[0237] 51. The multivalent anti-spike protein binding molecule of any one of embodiments 46-50, wherein the IgG Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0238] 52. The multivalent anti-spike protein binding molecule of any one of embodiments 46 to 50, wherein the IgG Fc domain is an IgG1 domain. 53. The multivalent anti-spike protein binding molecule of any one of embodiments 46-50, wherein the IgG Fc domain is an IgG4 domain.

[0239] 54. The multivalent anti-spike protein binding molecule of embodiment 33 or embodiment 53, wherein the Fc domain comprises a hinge at its N-terminus. 55. The multivalent anti-spike protein binding molecule of embodiment 54, wherein the hinge is a chimeric hinge.

[0240] 56. A multivalent anti-spike protein binding molecule according to any one of embodiments 46 to 55, wherein the IgG Fc domain is a non-dimerizing Fc domain. 57. The multivalent anti-spike protein binding molecule of embodiment 56, wherein the non-dimerizing Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 14-19.

[0241] 58. The multivalent anti-spike protein binding molecule of any one of embodiments 46-51, wherein the IgG Fc domain is an Fc 1.5 domain. 59. The multivalent anti-spike protein binding molecule of embodiment 58, wherein the Fc 1.5 domain comprises the amino acid sequence of any one of SEQ ID NOs: 20-27.

[0242] 60. The multivalent anti-spike protein binding molecule of any one of embodiments 46-59, wherein the J chain operably linked to the IgG Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 28-32.

[0243] 61. Optionally, a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 60, wherein the multivalent anti-spike protein binding molecule has the configuration shown in FIG. 1A.

[0244] 62. The multivalent anti-spike protein binding molecule of any one of embodiments 38-40, which is a hexamer. 63. The multivalent anti-spike protein binding molecule of embodiment 62, which is a hexamer of six dimers, each dimer comprising two polypeptides, each polypeptide comprising an anti-spike protein ABD and an IgM Fc domain.

[0245] 64. The multivalent anti-spike protein binding molecule of embodiment 62 or embodiment 63, which is a homohexamer. 65. A multivalent anti-spike protein binding molecule according to any one of embodiments 62 to 64, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

[0246] 66. The multivalent anti-spike protein binding molecule of any one of embodiments 62 to 65, which lacks a J chain. 67. Optionally, a multivalent anti-spike protein binding molecule according to any one of embodiments 1-40 and 62-66, wherein the multivalent anti-spike protein binding molecule has the configuration shown in FIG. 1B.

[0247] 68. Optionally, a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 61, wherein the multivalent anti-spike protein binding molecule has the configuration shown in FIG. 1C.

[0248] 69. Optionally, a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 61, wherein the multivalent anti-spike protein binding molecule has the configuration shown in FIG. 1D.

[0249] 70. Optionally, a multivalent anti-spike protein binding molecule of any one of embodiments 1-61, wherein the multivalent anti-spike protein binding molecule has the configuration shown in FIG. 1E.

[0250] 71. A multivalent anti-spike protein binding molecule comprising at least five means for binding spike protein operably linked by one or more multimerization moieties. 72. The multivalent anti-spike protein binding molecule of embodiment 71, comprising at least 10 means for binding spike proteins.

[0251] 73. The multivalent anti-spike protein binding molecule of embodiment 71 or 72, which is decavalent or dodecavalent for the means for binding spike protein. 74. A multivalent anti-spike protein binding molecule according to any one of embodiments 71 to 73, comprising at least five Fabs, each comprising a means for binding a spike protein.

[0252] 75. A multivalent anti-spike protein binding molecule according to any one of embodiments 71 to 74, which is monospecific. 76. The multivalent anti-spike protein binding molecule of embodiment 75, wherein at least five means for binding spike protein are the same.

[0253] 77. A multivalent anti-spike protein binding molecule according to any one of embodiments 71 to 73, which is multispecific. 78. The multivalent anti-spike protein binding molecule of embodiment 77, which is bispecific.

[0254] 79. The multivalent anti-spike protein binding molecule of any one of embodiments 71-78, wherein one or more multimerization moieties comprises an Fc domain. 80. The multivalent anti-spike protein binding molecule of embodiment 79, wherein the Fc domain is an IgM Fc domain.

[0255] 81. The multivalent anti-spike protein binding molecule of embodiment 80, wherein the Fc domain comprises a Cμ3 domain and a Cμ4 domain. 82. A multivalent anti-spike protein binding molecule according to embodiment 80 or embodiment 81, wherein the Fc domain comprises a Cμ2 domain.

[0256] 83. The multivalent anti-spike protein binding molecule of any one of embodiments 80-82, which is a pentamer. 84. The multivalent anti-spike protein binding molecule of embodiment 83, which is a pentamer of five dimers, each dimer comprising two polypeptides, each polypeptide comprising a means for binding spike protein and an IgM Fc domain.

[0257] 85. The multivalent anti-spike protein binding molecule of embodiment 83 or embodiment 84, which is a homopentamer. 86. A multivalent anti-spike protein binding molecule according to any one of embodiments 83 to 85, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

[0258] 87. A multivalent anti-spike protein binding molecule according to any one of embodiments 83 to 86, comprising a J chain. 88. The multivalent anti-spike protein binding molecule of embodiment 87, wherein the J chain is operably linked to the IgG Fc domain.

[0259] 89. The multivalent anti-spike protein binding molecule of embodiment 87, wherein the IgG Fc domain is N-terminal to the J chain. 90. The multivalent anti-spike protein binding molecule of embodiment 87, wherein the IgG Fc domain is C-terminal to the J chain.

[0260] 91. The multivalent anti-spike protein binding molecule of any one of embodiments 87-90, wherein the IgG Fc domain and the J chain are connected via a linker. 92. The multivalent anti-spike protein binding molecule of embodiment 91, wherein the linker is or comprises the amino acid sequence G4S.

[0261] 93. The multivalent anti-spike protein binding molecule of any one of embodiments 88-92, wherein the IgG Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0262] 94. The multivalent anti-spike protein binding molecule of any one of embodiments 88-93, wherein the IgG Fc domain is an IgG1 domain. 95. The multivalent anti-spike protein binding molecule of any one of embodiments 88-94, wherein the IgG Fc domain is an IgG4 domain.

[0263] 96. The multivalent anti-spike protein binding molecule of embodiment 33 or embodiment 95, wherein the Fc domain comprises a hinge at its N-terminus. 97. The multivalent anti-spike protein binding molecule of embodiment 96, wherein the hinge is a chimeric hinge.

[0264] 98. The multivalent anti-spike protein binding molecule of any one of embodiments 88-97, wherein the IgG Fc domain is a non-dimerizing Fc domain. 99. The multivalent anti-spike protein binding molecule of embodiment 98, wherein the non-dimerizing Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 14 to 19.

[0265] 100. The multivalent anti-spike protein binding molecule of any one of embodiments 88-97, wherein the IgG Fc domain is an Fc 1.5 domain. 101. The multivalent anti-spike protein binding molecule of embodiment 100, wherein the Fc 1.5 domain comprises the amino acid sequence of any one of SEQ ID NOs: 20-27.

[0266] 102. The multivalent anti-spike protein binding molecule of any one of embodiments 88-101, wherein the J chain operably linked to the IgG Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 28-32.

[0267] 103. 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 102. 104. A host cell engineered to express a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 102 or a nucleic acid(s) according to embodiment 103.

[0268] 105. A method for producing a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 102, comprising culturing a host cell according to embodiment 104 and recovering the multivalent anti-spike protein binding molecule expressed thereby.

[0269] 106. A pharmaceutical composition comprising a multivalent anti-spike protein binding molecule according to any one of embodiments 1 to 102 and an excipient. 107. 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 102 or a pharmaceutical composition of embodiment 106.

[0270] 108. 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 102 or a pharmaceutical composition described in embodiment 106.

[0271] 109. 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 102 or a pharmaceutical composition of embodiment 106.

[0272] 110. 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 102 or a pharmaceutical composition of embodiment 106.

[0273] 111. 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 102 or a pharmaceutical composition of embodiment 106.

[0274] 112. 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 102 or a pharmaceutical composition of embodiment 106.

[0275] 113. 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 102 or a pharmaceutical composition described in embodiment 106.

[0276] 114. A method for reducing the viral load of a coronavirus, comprising administering to a subject in need thereof a multivalent anti-spike protein binding molecule described in any one of embodiments 1 to 102 or a pharmaceutical composition described in embodiment 106.

[0277] 115. 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 102 or a pharmaceutical composition of embodiment 106.

[0278] 116. 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 102 or a pharmaceutical composition described in embodiment 106.

[0279] 117. 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 102 or a pharmaceutical composition described in embodiment 106.

[0280] 118. The method of any one of embodiments 107 to 117, wherein the coronavirus is SARS-CoV. 119. The method of any one of embodiments 107 to 117, wherein the coronavirus is SARS-CoV-2. [Example]

[0281] 8. Working Example 8.1. Materials and Methods 8.1.1. Construction and Generation of Anti-SARS-CoV-2 IgM Constructs The IgM heavy chain construct was designed as a DNA fragment containing the following components from the 5' to 3' end: the mROR1 signal sequence, the heavy chain variable region, and the IgM heavy chain constant region (Uniprot ID: P01871). All IgM antibodies were expressed in FreeStyle™ 293-F cells (ThermoFisher) by transient transfection according to the manufacturer's protocol, whereby 250 mL of cells were transfected at a 1:1:1 ratio for each IgM antibody construct with three chains (H, L, J).

[0282] Antibodies were purified from the supernatant using POROS Capture Select IgM Affinity Matrix (ThermoFisher). First, the column was equilibrated with 5 column volumes (CV) of PBS. The sterile-filtered supernatant containing the fusion protein was then loaded onto the pre-equilibrated column at a flow rate of approximately 2.0 mL / min. Any nonspecifically bound material was washed off the column using 50 mM Tris-HCl, 500 mM NaCl, pH 7.5 at a flow rate of 2.0 mL / min for 5 CV. The affinity-bound fusion protein was eluted from the column using Pierce™ IgG Elution Buffer (pH 2.8, ThermoFisher). After elution, the protein was neutralized using 1 / 10 (v / v) of 1 M Tris-HCl, pH 8.0. The eluted fraction material was further refined to increase the purity of the desired species by SEC. Therefore, a Superose 6 10 / 300GL column (Cytiva) was used in 1x DPBS, pH 7.1 running buffer at a flow rate of 0.75 mL / min. The desired fractions were pooled and concentrated. Each fraction pool was analyzed by UV-Vis to determine protein concentration. Each fraction pool was further analyzed by SE-UPLC to determine the relative purity of the desired species. Proteins isolated from each fraction pool were analyzed under denaturing conditions using SDS-PAGE. Samples were then run on a 4-20% Tris-Glycine gel loaded with 2 μg of sample per well at a constant voltage of 200 V for 1 hour.

[0283] 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., for the IgG control, 40 nM to 677.4 fM was obtained. For IgM molecules, the concentrations used were 13.3 nM to 225.8 fM for the experiments in Table 7 and 1.3 nM to 22.5 fM for the experiments in Tables 8 and 9). 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.

[0284] The pVSV-Luc-SARS-CoV-2-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 pseudoviruses or variants were diluted 1:4 in infection medium and then combined 1:1 with antibody diluents, with a final pseudovirus / variant dilution of 1:8 and final test article concentrations of 20 nM to 338.7 fM for the IgG control, concentrations of 6.7 nM to 112.9 fM for the IgM molecules in Table 7, and concentrations of 2.0 nM to 33.8 fM for the IgM molecules in Tables 8 and 9. The combined antibody and pseudovirus / variant were incubated at room temperature for 30 minutes. Culture medium was then removed from the cells, and the combined antibodies and 100 μL / well of pseudovirus / variant were added to wells in duplicate, followed by incubation at 37°C and 5% CO2 for 24 hours. At 24 hours post-infection, medium was removed from the wells, and cells were lysed using 100 μL / well of Glo-Lysis buffer (Promega). Immediately before reading luminescence on a Spectramax i3X plate reader, 100 μL of prepared Bright-Glo substrate (Promega) was added to the lysate. Results were exported to Microsoft Excel, and % neutralization was calculated using the following equation: % neutralization = ((1 - (well value - medium control) / (virus control - medium control)) × 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.

[0285] 8.2. Example 1: Generation of anti-SARS-CoV-2 IgM constructs A total of seven anti-SARS-CoV-2 IgMs (REGN10933, 10985, 10987, 10989, 14256, 14315, and 14287; containing the VH and VL domains from mAb10985, mAb10987, mAb10989, mAb14256, mAb14315, and mAb14287, respectively, as shown in Tables 3 and 4) were generated using different Fab fragments against the SARS-CoV-2 S protein and purified as described in Section 8.1.1. Affinity purification correlated with a thicker band on non-reducing SDS-PAGE gels corresponding to the expected product size (Figure 2). Furthermore, SEC purification profiles of the six anti-SARS-CoV-2 IgM constructs showed distinct major peaks with varying levels of high molecular weight species (Figure 3).

[0286] 8.3. Example 2: Neutralizing Activity of Anti-SARS-CoV-2 IgM Targeting the RBD A series of virus neutralization assays were performed as described in Section 8.1.2 to compare the percent neutralization activity of all six anti-SARS-CoV-2 IgMs characterized in Example 1 against SARS-CoV2 pseudovirus D614G and Omicron variants BA.1 and BA.2 compared to REGEN-COV (REGN10987 / REGN10933).

[0287] All anti-SARS-CoV2 IgM molecules possessed neutralizing activity against the pseudovirus D614G variant, albeit with different potencies (Figure 4A and Table 7). Nevertheless, all IgM molecules were associated with a higher neutralization potency against the pseudovirus than against REGEN-COV.

[0288] REGEN-COV completely lost neutralizing activity against BA.1 and only weakly neutralized against BA.2 (Figures 4B and 4C and Table 7). However, the neutralizing activity of individual anti-SARS-CoV-2 IgMs against the BA.1 and BA.2 variants varied. For example, REGN10933-based IgMs exhibited enhanced neutralization of the D614G, BA.1, and BA.2 variants compared with the parental IgG and REGEN-COV. However, 10933 IgMs failed to restore full potency (defined as the IC50 of REGEN-COV against D614G) against BA.1 and BA.2 (Figures 5A, 5B, and 5C). 10989-IgMs exhibited enhanced neutralization of D614G compared with the parental IgG and REGEN-COV. 10989-IgM completely lost activity against BA.1 and BA.2 (Figures 6A, 6B, and 6C). 10987-IgM had better neutralization than the parental IgG and REGEN-COV against the D614G and BA.2 variants, but had no activity against BA.1 (Figures 7A, 7B, and 7C). 10985-IgM showed increased neutralization of the D614G and BA.1 variants compared to the parental IgG and REGEN-COV, but neutralization activity was completely lost against BA.2 (Figures 8A, 8B, and 8C).

[0289] 14315-IgM was the only RBD-based IgM associated with high neutralization potency against both variants with IC50s superior to the full potency of REGEN-COV (against D614G) (Figures 9A, 9B, and 9C, and Table 7).

[0290] [Table 13]

[0291] 8.4. Example 3: Neutralizing Activity of Non-RBD Targeted Anti-SARS-CoV-2 IgMs One of the seven anti-SARS-CoV-2 IgMs generated in Example 1, 14287-IgM, was derived from a parent antibody that binds to a non-RBD epitope on the spike protein. It was generated and tested in virus neutralization assays as described in Sections 8.1.1 and 8.1.2, respectively. For the pseudovirus-based neutralization assays, REGN14287 was included along with REGEN-COV as a parent IgG control with the same Fab portion.

[0292] 14287-IgM exhibited the desired broad and potent neutralizing activity across D614G and Omicron BA.1, BA.2, BA.2.75, and BA.4 / BA.5, with IC50 values ​​ranging from 3.2 to 9.6E. -12 M and was 2-5 fold more potent than REGEN-COV against D614G, and exhibits a 10-fold potency enhancement over the parental REGN14287 IgG (Figures 10A-10E, and Table 8).

[0293] [Table 14]

[0294] 8.5. Example 4: Neutralizing Activity of 14315-IgM and 14287-IgM Against the Currently Circulating Omicron Variant BQ.1 To determine whether lead IgMs with the desired breadth and potency could neutralize currently circulating variants, the efficacy of 14315-IgM and 14287-IgM bearing the Omicron BQ.1 variant was tested using the neutralization assay described in Section 8.1.2. In addition, 10933-IgM, 10985-IgM, 14287-IgG (REGN14287), 14315-IgG (REGN14315), and REGEN-COV were included as controls. The two broad-spectrum IgM neutralizers, 14315-IgM and 14287-IgM, demonstrated potent inhibition of pseudoviral Omicron BQ.1 variants, with IC50 values ​​of 1.2E, respectively. -11 and 3.3E -12The parental REGN14315 and REGN14287 IgGs were 4.0 E, but not 10933-IgM or 10985-IgM. -10 M and 2.4E -11 The 14315-IgM neutralized the BQ.1 variant with potency values ​​of 34-fold and 2-fold weaker than those of REGEN-COV against D614G (Figures 11B and 11C). The corresponding 14315-IgM enhanced the activity to the full potency of REGEN-COV, and 14287-IgM showed 3- to 4-fold greater activity than REGEN-COV (Figures 11A, 11B, and 11C). In addition, both IgMs were 3- to 12-fold more potent than the corresponding parental IgG against BQ.1 (Figures 11B, 11C and Table 9).

[0295] These findings suggest that the combination of multivalency and targeting epitopes on the SARS-CoV2 spike protein in an IgM format plays an important role in achieving higher potency and broader protection than the parent IgG with the corresponding Fab portion. Increasing valency alone for an IgG with weakened or lost neutralizing capacity does not guarantee enhanced activity against a specific variant. Even when increased activity is observed, it may not fully restore the desired potency (e.g., IC50 of REGEN-CoV against D614G).

[0296] [Table 15]

[0297] 8.6. Example 5: SARS-CoV-2 Virus Neutralization Activity of 14287-IgM Containing an IgG Fc-Linked J Chain Using 14287-IgM as a starting point, three anti-SARS-CoV-2 IgMs containing IgG Fc-linked J chains were generated and purified as described in Section 8.1.1. Construct 14287-IgM J-Fc contained a J chain operably linked to a dimerized IgG Fc domain, construct 14287-IgM J-Fc1.5 contained a J chain operably linked to an Fc1.5 domain, and construct 14287-IgM J-mFc contained a J chain operably linked to a non-dimerized Fc domain. The purified constructs were able to bind to Protein A (Figure 12A). SEC purification profiles of all three constructs showed distinct major peaks (Figure 12B).

[0298] IgM constructs containing IgG Fc-linked J chain, 14287-IgM J-Fc, 14287-IgM J-mFc, and 14287-IgM J-Fc1.5 were evaluated in virus neutralization assays as described in Section 8.1.2. For comparison, REGN14287-IgG, 14287-IgM, and REGEN-COV (REGN10987 / REGN10933) were also included. Results showed that all three IgM constructs containing IgG Fc-linked J chain exhibited varying degrees of potency against SARS-CoV-2 pseudoviruses (Figure 13A and Table 10) and the XBB1.5 variant (Figure 13B and Table 10).

[0299] [Table 16]

[0300] 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 disclosure are intended.

Claims

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

2. 2. The multivalent anti-spike protein binding molecule of claim 1, comprising at least 10 anti-spike protein ABDs.

3. The multivalent anti-spike protein binding molecule of claim 1 or 2, which is decavalent or dodecavalent.

4. 4. The multivalent anti-spike protein binding molecule of claim 1, wherein the antigen binding domain (ABD) is human or humanized.

5. The multivalent anti-spike protein binding molecule of any one of claims 1 to 4, wherein the antigen binding domain (ABD) is a Fab.

6. 6. The multivalent anti-spike protein binding molecule of claim 1, wherein one or more (or all) ABDs are neutralizing.

7. 7. The multivalent anti-spike protein binding molecule of any one of claims 1 to 6, which is monospecific.

8. The multivalent anti-spike protein binding molecule of any one of claims 1 to 7, wherein the antigen binding domains (ABDs) are all the same.

9. 7. The multivalent anti-spike protein binding molecule of claim 1, which is multispecific.

10. 10. The multivalent anti-spike protein binding molecule of claim 9, which is bispecific.

11. 11. The multivalent anti-spike protein binding molecule of claim 9 or 10, comprising two types of ABD.

12. 12. The multivalent anti-spike protein binding molecule of claim 1, wherein the one or more multimerization moieties comprises an Fc domain.

13. 12. The multivalent anti-spike protein binding molecule of claim 11, wherein both types of ABD bind to the spike protein.

14. 12. The multivalent anti-spike protein binding molecule of claim 11, wherein one type of ABD binds to the spike protein and the other type of ABD binds to a different target.

15. 13. The multivalent anti-spike protein binding molecule of claim 12, wherein the Fc domain is an IgM Fc domain.

16. 16. The multivalent anti-spike protein binding molecule of claim 15, wherein the Fc domain comprises a Cμ3 domain and a Cμ4 domain.

17. 17. The multivalent anti-spike protein binding molecule of claim 15 or 16, wherein the Fc domain comprises a Cμ2 domain.

18. 18. The multivalent anti-spike protein binding molecule of any one of claims 15 to 17, which is a pentamer.

19. 20. The multivalent anti-spike protein binding molecule of claim 18, which is a pentamer of five dimers, each dimer comprising two polypeptides, each polypeptide comprising an anti-spike protein ABD and an IgM Fc domain.

20. 20. The multivalent anti-spike protein binding molecule of claim 18 or 19, which is a homopentamer.

21. 21. The multivalent anti-spike protein binding molecule of any one of claims 18 to 20, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

22. 22. The multivalent anti-spike protein binding molecule of any one of claims 18 to 21, comprising a J chain.

23. 23. The multivalent anti-spike protein binding molecule of claim 22, wherein the J chain is operably linked to an IgG Fc domain.

24. 24. The multivalent anti-spike protein binding molecule of claim 23, wherein the IgG Fc domain and the J chain are connected via a linker.

25. 25. The multivalent anti-spike protein binding molecule of claim 23 or 24, wherein the IgG Fc domain is a non-dimerizing Fc domain.

26. 25. The multivalent anti-spike protein binding molecule of claim 23 or 24, wherein the IgG Fc domain is an Fc 1.5 domain.

27. 18. The multivalent anti-spike protein binding molecule of any one of claims 15 to 17, which is a hexamer.

28. 28. The multivalent anti-spike protein binding molecule of claim 27, which is a hexamer of six dimers, each dimer comprising two polypeptides, each polypeptide comprising an anti-spike protein ABD and an IgM Fc domain.

29. 29. The multivalent anti-spike protein binding molecule of claim 27 or 28, which is a homohexamer.

30. 30. The multivalent anti-spike protein binding molecule of any one of claims 27 to 29, wherein some or all of the Cμ3 and / or Cμ4 domains are disulfide-linked.

31. 31. The multivalent anti-spike protein binding molecule of any one of claims 27 to 30, which lacks a J chain.

32. 32. The multivalent anti-spike protein binding molecule of any one of claims 1 to 31, 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.

33. 32. The multivalent anti-spike protein binding molecule of any one of claims 1 to 31, wherein one or more (or all) ABDs comprise the VH and VL sequences of an antibody shown in Table 4.

34. One or more (or all) 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.

35. A nucleic acid or a plurality of nucleic acids encoding the multivalent anti-spike protein binding molecule of any one of claims 1 to 34.

36. 36. A host cell engineered to express the multivalent anti-spike protein binding molecule of any one of claims 1 to 34 or the nucleic acid(s) of claim 35.

37. 35. A method of producing a multivalent anti-spike protein binding molecule according to any one of claims 1 to 34, comprising culturing a host cell according to claim 36 and recovering the multivalent anti-spike protein binding molecule expressed thereby.

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

39. 39. 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 34 or a pharmaceutical composition of claim 38.

40. 39. 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 34 or a pharmaceutical composition of claim 38.

41. 39. 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 34 or a pharmaceutical composition of claim 38.

42. 39. 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 34 or a pharmaceutical composition of claim 38.

43. 39. 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 34 or a pharmaceutical composition of claim 38.

44. 39. 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 34 or a pharmaceutical composition of claim 38.

45. 40. 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 34 or a pharmaceutical composition of claim 38.

46. 40. 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 34 or a pharmaceutical composition of claim 38.

47. 40. A method of 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 claims 1 to 34 or a pharmaceutical composition of claim 38.

48. 40. 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 34 or a pharmaceutical composition of claim 38.

49. 40. 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 34 or a pharmaceutical composition of claim 38.

50. 50. The method of any one of claims 39 to 49, wherein the coronavirus is SARS-CoV-2.

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