Modified Multabody Constructs, Compositions, and Methods Targeting SARS-CoV-2

Abstract

1. A self-assembled polypeptide complex comprising: (a) one or more fusion proteins comprising a nanocage monomer or subunit thereof linked to an Fc polypeptide; (b) one or more fusion proteins comprising a nanocage monomer or subunit thereof linked to a SARS-CoV-2 binding moiety; Multiple fusion proteins self-assemble to form a nanocage.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 256,565, filed October 16, 2021, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to polypeptides. In particular, the present invention relates to modified Multabody constructs, compositions and methods targeting SARS-CoV-2. [Background technology]

[0003] Nanoparticles have contributed to advances in various fields: their use allows targeted delivery, allows design of ordered microarrays, sustained release, and caged microenvironments for catalytic processes.

[0004] Protein self-assembly is an attractive method for producing nanoparticles containing sensitive and metastable proteins. Indeed, self-assembled nanoparticles form under physiological conditions through non-covalent interactions, reliably generating uniform and often symmetrical nanocapsules or nanocages.

[0005] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a coronavirus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COVID-19 pandemic.

[0006] There is a need for improved compositions and methods for treating and / or preventing SARS-CoV-2. Summary of the Invention

[0007] According to one aspect, a self-assembled polypeptide complex is provided, the complex comprising: (a) one or more fusion proteins comprising a nanocage monomer or subunit thereof linked to an Fc polypeptide; (b) one or more fusion proteins comprising a nanocage monomer or subunit thereof linked to a SARS-CoV-2 binding moiety; Multiple fusion proteins self-assemble to form a nanocage.

[0008] In one embodiment, the Fc polypeptide does not bind to an Fcγ receptor.

[0009] In one embodiment, the Fc polypeptide comprises an IgG4 Fc chain having mutations at one or more of positions 228, 234, 235, 237, and 238 according to EU numbering.

[0010] In one embodiment the IgG4 Fc chain comprises mutations at positions 234 and 235.

[0011] In one aspect, the IgG4 Fc chain comprises a F234A mutation and a L235A mutation.

[0012] In one embodiment the IgG4 Fc chain comprises a mutation at position 228.

[0013] In one embodiment, the IgG4 Fc chain comprises a S228P mutation.

[0014] In one embodiment the IgG4 Fc chain comprises mutations at positions 237 and 238.

[0015] In one embodiment, the IgG4 Fc chain comprises a G237A mutation and a P238S mutation.

[0016] In one embodiment, the IgG4 Fc chain does not comprise a mutation at G237 or P238.

[0017] In one embodiment, the IgG4 Fc chain comprises an S228P mutation, an F234A mutation and an L235A mutation.

[0018] In one embodiment, the IgG4 Fc chain comprises a S228P mutation, a F234A mutation, a L235A mutation, a G237A mutation and a P238S mutation.

[0019] In one embodiment, the IgG4 Fc chain comprises a F234A mutation, a L235A mutation, a G237A mutation and a P238S mutation.

[0020] In one embodiment the IgG4 Fc chain does not comprise a mutation at S228.

[0021] In one embodiment, the nanocage monomer or subunit thereof is a ferritin monomer or subunit thereof.

[0022] In one embodiment, the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

[0023] In one embodiment, the ferritin monomer or subunit thereof is human ferritin or a subunit thereof.

[0024] In one aspect, the ferritin monomer or subunit thereof is a ferritin monomer subunit.

[0025] In one embodiment, the ferritin monomer subunit is C-half ferritin.

[0026] In one embodiment, the Fc polypeptide is linked to the N-terminus of the C-half ferritin.

[0027] In one embodiment, the Fc polypeptide is linked to the N-terminus of the C-half ferritin via an amino acid linker.

[0028] In one aspect, the amino acid linker is n S) m Includes a linker.

[0029] In one embodiment, (G n S) m The linker is (GGGGS) mIt is a linker.

[0030] In one embodiment, the Fc polypeptide comprises a single chain Fc (scFc) comprising two Fc chains, wherein the two Fc chains are linked via an amino acid linker.

[0031] In one embodiment, the amino acid linker linking the two Fc chains is n S) m Includes a linker.

[0032] In one embodiment, (G n S) m The linker is (GGGGS) m It is a linker.

[0033] In one embodiment, the SARS-CoV-2 binding moiety targets the SARS-CoV-2 S glycoprotein.

[0034] In one embodiment, the SARS-CoV-2 binding moieties decorate the inner and / or outer surface of the assembled nanocage, preferably the outer surface.

[0035] In one embodiment, the SARS-CoV-2 binding moiety comprises an antibody or fragment thereof.

[0036] In one embodiment, the antibody or fragment thereof is a Fab fragment.

[0037] In one embodiment, the antibody or fragment thereof comprises an scFab fragment, an scFv fragment, an sdAb fragment, a VHH domain, or a combination thereof.

[0038] In one embodiment, the antibody or fragment thereof comprises the heavy and / or light chain of a Fab fragment.

[0039] In one embodiment, the SARS-CoV-2 binding portion comprises the single variable domains VHH-72, BD23 and / or 4A8.

[0040] In one embodiment, the SARS-CoV-2 binding moiety comprises a mAb listed in Table 4.

[0041] In one embodiment, the SARS-CoV-2 binding portion comprises mAb 298, 324, 46, 80, 52, 82, or 236 in Table 4, or a variant thereof.

[0042] In one embodiment, the SARS-CoV-2 binding portion comprises mAb 298, 80, and 52 in Table 4, or variants thereof.

[0043] In one embodiment, the SARS-CoV-2 binding moiety is linked to the N-terminus or C-terminus of the nanocage monomer, or there is a first SARS-CoV-2 binding moiety linked to the N-terminus and a second SARS-CoV-2 binding moiety linked to the C-terminus of the nanocage monomer, where the first and second SARS-CoV-2 binding moieties are the same or different.

[0044] In one embodiment, the nanocage monomer comprises a first nanocage monomer subunit linked to a SARS-CoV-2 binding moiety, and the first nanocage monomer subunit self-assembles with a second nanocage monomer subunit to form a nanocage monomer.

[0045] In one embodiment, the SARS-CoV-2 binding moiety is linked to the N-terminus or C-terminus of the first nanocage monomer, or there is a first SARS-CoV-2 binding moiety linked to the N-terminus and a second SARS-CoV-2 binding moiety linked to the C-terminus of the first nanocage monomer subunit, wherein the first and second SARS-CoV-2 binding moieties are the same or different.

[0046] In one embodiment, the self-assembled polypeptide complex exhibits binding to hFcRn.

[0047] In one embodiment, the self-assembled polypeptide complex exhibits binding to hFcRn substantially similar to the binding of IgG, such as IgG1 or IgG4, to hFcRn.

[0048] In one embodiment, the self-assembled polypeptide complex does not exhibit binding to at least one human Fcγ receptor as determined by an in vitro assay.

[0049] In one aspect, the self-assembled polypeptide complex does not exhibit binding to one or more human Fcγ receptors selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof, as determined by an in vitro assay.

[0050] In one embodiment, the self-assembled polypeptide complex exhibits no binding to hFcγRI, hFcγRIIa and hFcγRIIb as determined by in vitro assays.

[0051] In one embodiment, the self-assembled polypeptide complex exhibits substantially no IgG4 effector function.

[0052] In one embodiment, the self-assembled polypeptide complex exhibits binding to at least one human Fcγ receptor as determined in an in vitro assay.

[0053] In one aspect, the self-assembled polypeptide complex of claim 42 exhibits binding to one or more human Fcγ receptors selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof, as determined by an in vitro assay.

[0054] In one embodiment, the self-assembled polypeptide complex exhibits binding to hFcγRI, hFcγRIIa and hFcγRIIb as determined in an in vitro assay.

[0055] In one embodiment, the self-assembled polypeptide complex exhibits an antibody effector function, such as an IgG effector function.

[0056] In one embodiment, the self-assembled polypeptide complex exhibits IgG4 effector function.

[0057] According to one aspect, a composition is provided that includes a plurality of the self-assembled polypeptide complexes described herein.

[0058] In one embodiment, the composition comprises a mixture of different self-assembled polypeptide complexes.

[0059] According to one aspect, a SARS-CoV-2 therapeutic or prophylactic composition is provided comprising a self-assembled polypeptide complex as described herein.

[0060] According to one aspect, a method for treating and / or preventing SARS-CoV-2 is provided, the method comprising administering a self-assembling polypeptide complex described herein to a subject in need thereof.

[0061] According to one aspect, there is provided a use of the self-assembling polypeptide complex described herein for the treatment and / or prevention of SARS-CoV-2.

[0062] In one aspect, the self-assembled polypeptide complex is for use in the treatment and / or prevention of SARS-CoV-2.

[0063] According to one aspect, there is provided a fusion protein comprising a nanocage monomer or a subunit thereof linked to an Fc polypeptide, the Fc polypeptide comprises an IgG4 Fc chain having a mutation at one or more of positions 228, 234, 235, 237, and 238 according to EU numbering; Multiple fusion proteins self-assemble to form a nanocage.

[0064] In one embodiment the IgG4 Fc chain comprises mutations at positions 234 and 235.

[0065] In one aspect, the IgG4 Fc chain comprises a F234A mutation and a L235A mutation.

[0066] In one embodiment the IgG4 Fc chain comprises a mutation at position 228.

[0067] In one embodiment, the IgG4 Fc chain comprises a S228P mutation.

[0068] In one embodiment the IgG4 Fc chain comprises mutations at positions 237 and 238.

[0069] In one embodiment, the IgG4 Fc chain comprises a G237A mutation and a P238S mutation.

[0070] In one embodiment, the IgG4 Fc chain comprises an S228P mutation, an F234A mutation and an L235A mutation.

[0071] In one embodiment, the IgG4 Fc chain does not comprise a mutation at G237 or P238.

[0072] In one embodiment, the IgG4 Fc chain comprises a S228P mutation, a F234A mutation, a L235A mutation, a G237A mutation and a P238S mutation.

[0073] In one embodiment, the IgG4 Fc chain comprises a F234A mutation, a L235A mutation, a G237A mutation and a P238S mutation.

[0074] In one embodiment the IgG4 Fc chain does not comprise a mutation at S228.

[0075] In one embodiment, the nanocage monomer or subunit thereof is a ferritin monomer or subunit thereof.

[0076] In one embodiment, the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

[0077] In one embodiment, the ferritin monomer or subunit thereof is human ferritin or a subunit thereof.

[0078] In one aspect, the ferritin monomer or subunit thereof is a ferritin monomer subunit.

[0079] In one embodiment, the ferritin monomer subunit is C-half ferritin.

[0080] In one embodiment, the Fc polypeptide is linked to the N-terminus of the C-half ferritin.

[0081] In one embodiment, the Fc polypeptide is linked to the N-terminus of the C-half ferritin via an amino acid linker.

[0082] In one aspect, the amino acid linker is n S) m Includes a linker.

[0083] In one embodiment, (G n S) m The linker is (GGGGS) m It is a linker.

[0084] In one embodiment, the Fc polypeptide comprises a single chain Fc (scFc) comprising two Fc chains, wherein the two Fc chains are linked via an amino acid linker.

[0085] In one embodiment, the amino acid linker linking the two Fc chains is n S) m Includes a linker.

[0086] In one embodiment, (G n S) m The linker is (GGGGS) m It is a linker.

[0087] According to one aspect, a self-assembled polypeptide complex is provided, the complex comprising: (a) one or more first fusion polypeptides, each of which is a fusion polypeptide according to any one of claims 1 to 22; (b) one or more second fusion polypeptides, each second fusion polypeptide comprising an antigen-binding portion linked to a nanocage monomer or a subunit thereof.

[0088] In one embodiment, the nanocage monomer or subunit thereof of each second fusion polypeptide is a ferritin monomer or subunit thereof.

[0089] In one embodiment, the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

[0090] In one embodiment, the ferritin monomer or subunit thereof is human ferritin or a subunit thereof.

[0091] In one embodiment, the self-assembled polypeptide complex does not include any ferritin heavy chain or subunits of a ferritin heavy chain.

[0092] In one embodiment, the antigen-binding moiety is linked to a nanocage monomer or subunit thereof within each second fusion polypeptide via an amino acid linker.

[0093] In one aspect, the amino acid linker is n S) m Includes a linker.

[0094] In one embodiment, (G n S) m The linker is (GGGGS) m It is a linker.

[0095] In one embodiment, the antigen-binding portion of each second fusion polypeptide is linked to the N-terminus of a nanocage monomer or a subunit thereof.

[0096] In one embodiment, the antigen-binding portion of each second fusion polypeptide is a Fab fragment.

[0097] In one embodiment, each second fusion polypeptide does not contain any antibody CH2 or CH3 domains.

[0098] In one embodiment, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each of which comprises an antigen-binding portion linked to a nanocage monomer or a subunit thereof, and the third fusion polypeptide is different from the second fusion polypeptide.

[0099] In one embodiment, the antigen-binding portion of each third fusion polypeptide is a Fab fragment.

[0100] In one embodiment, each third fusion polypeptide does not contain any antibody CH2 or CH3 domains.

[0101] In one embodiment, the nanocage monomer or subunit thereof of each first fusion polypeptide and each second fusion polypeptide is a ferritin monomer or subunit thereof; a. each first fusion polypeptide comprises a C-half ferritin and each second fusion polypeptide comprises an N-half ferritin, or b. Each first fusion polypeptide contains an N-half ferritin and each second fusion polypeptide contains a C-half ferritin.

[0102] In one embodiment, the self-assembled polypeptide complex is characterized by a 1:1 ratio of the first fusion polypeptide to the second fusion polypeptide.

[0103] In one embodiment, the self-assembled polypeptide complex comprises a total of 24 to 48 fusion polypeptides.

[0104] In one embodiment, the self-assembled polypeptide complex comprises a total of at least 24 fusion polypeptides.

[0105] In one embodiment, the self-assembled polypeptide complex comprises a total of at least 32 fusion polypeptides.

[0106] In one embodiment, the self-assembled polypeptide complex has a total of about 32 fusion polypeptides.

[0107] In one embodiment, the self-assembled polypeptide complex exhibits binding to hFcRn.

[0108] In one embodiment, the self-assembled polypeptide complex exhibits binding to hFcRn substantially similar to the binding of IgG, such as IgG1 or IgG4, to hFcRn.

[0109] In one embodiment, the self-assembled polypeptide complex does not exhibit binding to at least one human Fcγ receptor as determined by an in vitro assay.

[0110] In one aspect, the self-assembled polypeptide complex does not exhibit binding to one or more human Fcγ receptors selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof, as determined by an in vitro assay.

[0111] In one embodiment, the self-assembled polypeptide complex exhibits no binding to hFcγRI, hFcγRIIa and hFcγRIIb as determined by in vitro assays.

[0112] In one embodiment, the self-assembled polypeptide complex exhibits substantially no IgG4 effector function.

[0113] In one embodiment, the self-assembled polypeptide complex exhibits binding to at least one human Fcγ receptor as determined in an in vitro assay.

[0114] In one aspect, the self-assembled polypeptide complex exhibits binding to one or more human Fcγ receptors selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof, as determined by an in vitro assay.

[0115] In one embodiment, the self-assembled polypeptide complex exhibits binding to hFcγRI, hFcγRIIa and hFcγRIIb as determined in an in vitro assay.

[0116] In one embodiment, the self-assembled polypeptide complex exhibits an antibody effector function, such as an IgG effector function.

[0117] In one embodiment, the self-assembled polypeptide complex exhibits IgG4 effector function.

[0118] According to one aspect, a composition is provided that includes a plurality of the self-assembled polypeptide complexes described herein.

[0119] In one embodiment, the composition comprises a mixture of different self-assembled polypeptide complexes.

[0120] According to one aspect, a method is provided that includes administering to a mammalian subject a composition that includes a self-assembled polypeptide complex as described herein.

[0121] In one embodiment, the subject is a human.

[0122] In one aspect, the subject has or is at risk of developing cancer.

[0123] In one aspect, the subject is suffering from or at risk of developing an autoimmune disease.

[0124] In one aspect, the subject is suffering from or at risk of developing an infectious disease.

[0125] In one aspect, the subject is suffering from or at risk of developing a metabolic disease.

[0126] In one embodiment, the method comprises administration by a systemic route.

[0127] In one embodiment, the systemic route includes subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

[0128] According to one aspect, there is provided a use of a composition comprising a self-assembled polypeptide complex as described herein for administration to a mammalian subject.

[0129] In some embodiments, the subject is a human.

[0130] In one aspect, the subject has or is at risk of developing cancer.

[0131] In one aspect, the subject is suffering from or at risk of developing an autoimmune disease.

[0132] In one aspect, the subject is suffering from or at risk of developing an infectious disease.

[0133] In one aspect, the use is for administration by a systemic route.

[0134] In one embodiment, the systemic route includes subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

[0135] According to one aspect, there is provided a composition comprising a self-assembled polypeptide complex as described herein for use in administration to a mammalian subject.

[0136] In one embodiment, the subject is a human.

[0137] In one aspect, the subject has or is at risk of developing cancer.

[0138] In one aspect, the subject is suffering from or at risk of developing an autoimmune disease.

[0139] In one aspect, the subject is suffering from or at risk of developing an infectious disease.

[0140] In one embodiment, the composition is for administration by a systemic route.

[0141] In one embodiment, the systemic route includes subcutaneous, intravenous, or intramuscular injection, inhalation, or intranasal administration.

[0142] The novel features of the present invention will become apparent to those skilled in the art upon review of the following detailed description of the invention. However, while the detailed description and specific examples set forth set forth certain aspects of the present invention, they are provided for illustrative purposes only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the detailed description of the invention and the claims that follow.

[0143] The invention will be better understood from the following description, taken in conjunction with the drawings, in which: [Brief description of the drawings]

[0144] [Figure 1]Avidity drives VHH binding and neutralization against SARS-CoV-2. a Schematic of monomeric VHH domains and their multimerization with conventional Fc (dark red) scaffold or human apoferritin (grey). b Size-exclusion chromatography and SDS-PAGE of apoferritin alone (grey) and VHH-72 apoferritin particles (gold). c Negative staining electron micrographs of VHH-72 apoferritin particles. (Scale bar 50 nm, representative of two independent experiments). d Comparison of avidity (apparent KD) of VHH-72 to SARS-CoV-2 S protein when displayed in bivalent (dark red) or 24-mer (gold) format. Bars indicate the mean of n = 2 biologically independent experiments. Apparent KDs lower than 10-12 M (dashed line) are beyond the detection limit of the instrument. Neutralization potency against eSARS-CoV-2 PsV (color coding as in (d)). One representative of two biologically independent replicates with similar results is shown. The mean ± SD of the two technical replicates is shown on the plots. The median IC50 values ​​of the two biologically independent replicates are shown. [Figure 2a]Binding interface of Fab52 and 298 with RBD. The interaction of Fab298 (a) and 52 (b) with RBD (core region in light green, RBM region in dark green) is mediated by complementarity determining regions (CDRs) heavy chain (H)1 (yellow), H2 (orange), H3 (red), kappa light chain (K)1 (light blue), and K3 (purple). Key binding residues are shown as sticks (insets). Hydrogen bonds and salt bridges are represented by dashed black lines. The Fab light and heavy chains are shown in tan and white, respectively. c) Bottom and side views of ACE2 (left) and Fab298 (right) bound to RBD. RBD side chains that are part of the binding interface of the ACE2-RBD and Fab298-RBD complexes are shown in pink, and RBD side chains that are unique to a particular interface are shown in yellow. The surface of ACE2, the variable regions of Fab298HC and Fab298KC are shown in white, grey and tan, respectively. The RBD is color coded as in (a). d) Superposition of Fab46 (light pink) and Fab52 (dark pink) bound to the RBD (green) reveals the different angled approaches of the two mAbs. Stereo images of the composite omit-map electron density contoured at 1.3 sigma at the e) 298-RBD and f) 52-RBD interfaces. [Figure 2b]Binding interface of Fab52 and 298 with RBD. The interaction of Fab298 (a) and 52 (b) with RBD (core region in light green, RBM region in dark green) is mediated by complementarity determining regions (CDRs) heavy chain (H)1 (yellow), H2 (orange), H3 (red), kappa light chain (K)1 (light blue), and K3 (purple). Key binding residues are shown as sticks (insets). Hydrogen bonds and salt bridges are represented by dashed black lines. The Fab light and heavy chains are shown in tan and white, respectively. c) Bottom and side views of ACE2 (left) and Fab298 (right) bound to RBD. RBD side chains that are part of the binding interface of the ACE2-RBD and Fab298-RBD complexes are shown in pink, and RBD side chains that are unique to a particular interface are shown in yellow. The surface of ACE2, the variable regions of Fab298HC and Fab298KC are shown in white, grey and tan, respectively. The RBD is color coded as in (a). d) Superposition of Fab46 (light pink) and Fab52 (dark pink) bound to the RBD (green) reveals the different angled approaches of the two mAbs. Stereo images of the composite omit-map electron density contoured at 1.3 sigma at the e) 298-RBD and f) 52-RBD interfaces. [Figure 2c]Binding interface of Fab52 and 298 with RBD. The interaction of Fab298 (a) and 52 (b) with RBD (core region in light green, RBM region in dark green) is mediated by complementarity determining regions (CDRs) heavy chain (H)1 (yellow), H2 (orange), H3 (red), kappa light chain (K)1 (light blue), and K3 (purple). Key binding residues are shown as sticks (insets). Hydrogen bonds and salt bridges are represented by dashed black lines. The Fab light and heavy chains are shown in tan and white, respectively. c) Bottom and side views of ACE2 (left) and Fab298 (right) bound to RBD. RBD side chains that are part of the binding interface of the ACE2-RBD and Fab298-RBD complexes are shown in pink, and RBD side chains that are unique to a particular interface are shown in yellow. The surface of ACE2, the variable regions of Fab298HC and Fab298KC are shown in white, grey and tan, respectively. The RBD is color coded as in (a). d) Superposition of Fab46 (light pink) and Fab52 (dark pink) bound to the RBD (green) reveals the different angled approaches of the two mAbs. Stereo images of the composite omit-map electron density contoured at 1.3 sigma at the e) 298-RBD and f) 52-RBD interfaces. [Figure 2d]Binding interface of Fab52 and 298 with RBD. The interaction of Fab298 (a) and 52 (b) with RBD (core region in light green, RBM region in dark green) is mediated by complementarity determining regions (CDRs) heavy chain (H)1 (yellow), H2 (orange), H3 (red), kappa light chain (K)1 (light blue), and K3 (purple). Key binding residues are shown as sticks (insets). Hydrogen bonds and salt bridges are represented by dashed black lines. The Fab light and heavy chains are shown in tan and white, respectively. c) Bottom and side views of ACE2 (left) and Fab298 (right) bound to RBD. RBD side chains that are part of the binding interface of the ACE2-RBD and Fab298-RBD complexes are shown in pink, and RBD side chains that are unique to a particular interface are shown in yellow. The surface of ACE2, the variable regions of Fab298HC and Fab298KC are shown in white, grey and tan, respectively. The RBD is color coded as in (a). d) Superposition of Fab46 (light pink) and Fab52 (dark pink) bound to the RBD (green) reveals the different angled approaches of the two mAbs. Stereo images of the composite omit-map electron density contoured at 1.3 sigma at the e) 298-RBD and f) 52-RBD interfaces. [Figure 2e]Binding interface of Fab52 and 298 with RBD. The interaction of Fab298 (a) and 52 (b) with RBD (core region in light green, RBM region in dark green) is mediated by complementarity determining regions (CDRs) heavy chain (H)1 (yellow), H2 (orange), H3 (red), kappa light chain (K)1 (light blue), and K3 (purple). Key binding residues are shown as sticks (insets). Hydrogen bonds and salt bridges are represented by dashed black lines. The Fab light and heavy chains are shown in tan and white, respectively. c) Bottom and side views of ACE2 (left) and Fab298 (right) bound to RBD. RBD side chains that are part of the binding interface of the ACE2-RBD and Fab298-RBD complexes are shown in pink, and RBD side chains that are unique to a particular interface are shown in yellow. The surface of ACE2, the variable regions of Fab298HC and Fab298KC are shown in white, grey and tan, respectively. The RBD is color coded as in (a). d) Superposition of Fab46 (light pink) and Fab52 (dark pink) bound to the RBD (green) reveals the different angled approaches of the two mAbs. Stereo images of the composite omit-map electron density contoured at 1.3 sigma at the e) 298-RBD and f) 52-RBD interfaces. [Figure 2f]Binding interface of Fab52 and 298 with RBD. The interaction of Fab298 (a) and 52 (b) with RBD (core region in light green, RBM region in dark green) is mediated by complementarity determining regions (CDRs) heavy chain (H)1 (yellow), H2 (orange), H3 (red), kappa light chain (K)1 (light blue), and K3 (purple). Key binding residues are shown as sticks (insets). Hydrogen bonds and salt bridges are represented by dashed black lines. The Fab light and heavy chains are shown in tan and white, respectively. c) Bottom and side views of ACE2 (left) and Fab298 (right) bound to RBD. RBD side chains that are part of the binding interface of the ACE2-RBD and Fab298-RBD complexes are shown in pink, and RBD side chains that are unique to a particular interface are shown in yellow. The surface of ACE2, the variable regions of Fab298HC and Fab298KC are shown in white, grey and tan, respectively. The RBD is color coded as in (a). d) Superposition of Fab46 (light pink) and Fab52 (dark pink) bound to the RBD (green) reveals the different angled approaches of the two mAbs. Stereo images of the composite omit-map electron density contoured at 1.3 sigma at the e) 298-RBD and f) 52-RBD interfaces. [Diagram 3]Bioavailability, Biodistribution, and Immunogenicity of Mouse Surrogate Multabody. a Binding kinetics of WT and Fc-modified (LALAP mutant) MB to mouse FcγRI (left) and mouse FcRn at endosomal pH (middle) and physiological pH (right) compared to parental IgG. Two-fold dilution series from 100 to 3 nM (IgG) and 10 to 0.3 nM (MB) were used. Red lines represent raw data, black lines represent global fits. b Serum concentrations of surrogate mouse MB, Fc-modified MB (LALAP mutant), and parental mouse IgG (IgG1 and IgG2a subtypes) were evaluated after subcutaneous administration of 5 mg / kg using five male C57BL / 6 mice per group. c MB and IgG2a samples were labeled with Alexa-647 for visualization of biodistribution by live non-invasive 2D whole-body imaging after subcutaneous injection into three male BALB / c mice / group. Fluorescently labeled gold nanoparticles (GNPs) of 15 nm with similar Rh values ​​as the Multabody are shown for comparison. d Five male C57BL / 6 mice per group were used to evaluate the anti-drug antibody responses elicited by the mouse surrogate Multabody in comparison to parental IgG and a species-mismatched malarial PfCSP peptide fused to Helicobacter pylori ferritin (HpFerr). Mean values ​​± SD from n=5 mice are shown in (b) and (d). [Figure 4]The 3D biodistribution of the surrogate mouse Multabody is comparable to its parental IgG. Biodistribution of 15 nm gold nanoparticles (GNPs), MB, and IgG samples labeled with Alexa-647 was visualized by live non-invasive 3D whole-body imaging after subcutaneous injection in BALB / c mice. a) Representative 3D-rendered fluorescence images overlaid with CT scans of PBS-injected controls. b) Depiction of localization of major mouse organs overlaid with CT scans. c) 3D-rendered fluorescence images overlaid with CT scans 1 hour (1H), 2 days (D2), 8 days (D8), and 11 days (D11) after subcutaneous injection of gold nanoparticles (top), MB (middle three panels), or IgG (bottom panel). Each 3D image set is shown, with dorsal views overlaid with CT scans (right), and frontal (top left), medial (middle), and transverse (bottom left) views selected based on the location of the signal. The 3D fluorescence images were mapped to a rainbow lookup table (LUT) and the minimum of the color scale was set to the background and the maximum to 50 pmol M-1 cm-1 (GNP) or 1000 pmol M-1 cm-1 (MB and IgG). [Diagram 5] Protein engineering to multimerize IgG-like particles against SARS-CoV-2. a Schematic of human apoferritin split design. b Negative stain electron micrograph of MB. (Scale bar 50 nm, representative of two independent experiments). c Hydrodynamic radius (Rh) of MB. d Avidity effect of 4A8 (purple) and BD23 (grey) on binding (apparent KD) to SARS-CoV-2 spike. e Sensorgrams of BD23 IgG and MB with different Fc sequence variants binding to FcγRI (top), FcRn at endosomal pH (middle), and FcRn at physiological pH (bottom). Red lines represent raw data, black lines represent global fits. f Neutralization of SARS-CoV-2 PsV by 4A8 and BD23 IgG and MB. Representative data of three biologically independent samples. Mean ± SD of two technical replicates is displayed for each neutralization plot. The median IC50 values ​​of three biologically independent replicates are shown. [Figure 6]Multabodies enhance the potency of phage display-derived human mAbs. a Workflow to identify potent anti-SARS-CoV-2 neutralizers using MB technology. Generated with Biorender. b Comparison of neutralization potency of IgG (cyan) and MB (pink) displaying the same human Fab sequence derived from phage display. c IC50 values ​​are multiplied by 2 upon multimerization. d Apparent affinity (KD), on-co (k), and off-co (koff) rates of the most potent neutralizing MB (pink) compared to its IgG counterpart (cyan) for binding to SARS-CoV-2 S protein. Three biological replicates and their average as IC50 values ​​are shown in (b) and (c). [Figure 7a-1] Neutralization of Multabody and its parental IgG targeting SARS-CoV-2 RBD. a) Representative neutralization titration curves of 20 antibodies against SARS-CoV-2 PsV when displayed as IgG (black) and MB (dark red). For comparison, the mean IC50 values ​​of three biological replicates are displayed. The mean ± SD of two technical replicates is displayed for each neutralization plot. b) Neutralization profile of selected IgG and MB against SARS-CoV-2 PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (grey) target cells. The mean IC50 values ​​and individual IC50 values ​​of three and two biological replicates are shown for 293T-ACE2 and HeLa-ACE2 cells, respectively. c) Neutralization titration curves of three biological replicates (different grey shades) against the authentic SARS-CoV-2 / SB2-P4-PB strain. The mean IC50 is shown. The neutralization potency of recombinant mAbs REGN10933 (red) and REGN10987 (blue) are shown in (a) and (c) as benchmarks for comparison. [Figure 7a-2]Neutralization of Multabody and its parental IgG targeting SARS-CoV-2 RBD. a) Representative neutralization titration curves of 20 antibodies against SARS-CoV-2 PsV when displayed as IgG (black) and MB (dark red). For comparison, the mean IC50 values ​​of three biological replicates are displayed. The mean ± SD of two technical replicates is displayed for each neutralization plot. b) Neutralization profile of selected IgG and MB against SARS-CoV-2 PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (grey) target cells. The mean IC50 values ​​and individual IC50 values ​​of three and two biological replicates are shown for 293T-ACE2 and HeLa-ACE2 cells, respectively. c) Neutralization titration curves of three biological replicates (different grey shades) against the authentic SARS-CoV-2 / SB2-P4-PB strain. The mean IC50 is shown. The neutralization potency of recombinant mAbs REGN10933 (red) and REGN10987 (blue) are shown in (a) and (c) as benchmarks for comparison. [Figure 7a-3] Neutralization of Multabody and its parental IgG targeting SARS-CoV-2 RBD. a) Representative neutralization titration curves of 20 antibodies against SARS-CoV-2 PsV when displayed as IgG (black) and MB (dark red). For comparison, the mean IC50 values ​​of three biological replicates are displayed. The mean ± SD of two technical replicates is displayed for each neutralization plot. b) Neutralization profile of selected IgG and MB against SARS-CoV-2 PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (grey) target cells. The mean IC50 values ​​and individual IC50 values ​​of three and two biological replicates are shown for 293T-ACE2 and HeLa-ACE2 cells, respectively. c) Neutralization titration curves of three biological replicates (different grey shades) against the authentic SARS-CoV-2 / SB2-P4-PB strain. The mean IC50 is shown. The neutralization potency of recombinant mAbs REGN10933 (red) and REGN10987 (blue) are shown in (a) and (c) as benchmarks for comparison. [Figure 7b]Neutralization of Multabody and its parental IgG targeting SARS-CoV-2 RBD. a) Representative neutralization titration curves of 20 antibodies against SARS-CoV-2 PsV when displayed as IgG (black) and MB (dark red). For comparison, the mean IC50 values ​​of three biological replicates are displayed. The mean ± SD of two technical replicates is displayed for each neutralization plot. b) Neutralization profile of selected IgG and MB against SARS-CoV-2 PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (grey) target cells. The mean IC50 values ​​and individual IC50 values ​​of three and two biological replicates are shown for 293T-ACE2 and HeLa-ACE2 cells, respectively. c) Neutralization titration curves of three biological replicates (different grey shades) against the authentic SARS-CoV-2 / SB2-P4-PB strain. The mean IC50 is shown. The neutralization potency of recombinant mAbs REGN10933 (red) and REGN10987 (blue) are shown in (a) and (c) as benchmarks for comparison. [Figure 7c] Neutralization of Multabody and its parental IgG targeting SARS-CoV-2 RBD. a) Representative neutralization titration curves of 20 antibodies against SARS-CoV-2 PsV when displayed as IgG (black) and MB (dark red). For comparison, the mean IC50 values ​​of three biological replicates are displayed. The mean ± SD of two technical replicates is displayed for each neutralization plot. b) Neutralization profile of selected IgG and MB against SARS-CoV-2 PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (grey) target cells. The mean IC50 values ​​and individual IC50 values ​​of three and two biological replicates are shown for 293T-ACE2 and HeLa-ACE2 cells, respectively. c) Neutralization titration curves of three biological replicates (different grey shades) against the authentic SARS-CoV-2 / SB2-P4-PB strain. The mean IC50 is shown. The neutralization potency of recombinant mAbs REGN10933 (red) and REGN10987 (blue) are shown in (a) and (c) as benchmarks for comparison. [Figure 8]Expression yield and homogeneity of SARS-CoV-2 RBD targeting Multabodies. a) Yields (mg / L) of the seven most potent IgGs (white) and their respective MBs (dark red). Mean values ​​± SD of two biologically independent samples. b) Comparison of aggregation temperatures (Tagg, °C) as in (a). The solid line indicates the mean Tagg value of two biologically independent samples. c) SEC chromatograms of 298IgG (top, black) and 298MB (bottom, dark red) from three independent expression and purification runs. In both cases, samples were purified using Protein A affinity chromatography prior to SEC. Arrows indicate the peaks from each batch that were used to perform the PsV neutralization assay. IC50 values ​​(μg / mL) are recorded. Mean values ​​± SD of two technical replicates are displayed for each neutralization plot. [Figure 9-1] Binding profiles of IgG and MB. Sensorgrams of IgG and MB binding to the RBD (left) and S protein (right) of SARS-CoV-2 immobilized on a Ni-NTA biosensor. A two-fold dilution series was used from 125 to 4 nM (IgG) and 16 to 0.5 nM (MB). The red line represents the raw data and the black line represents the global fit. [Figure 9-2] Binding profiles of IgG and MB. Sensorgrams of IgG and MB binding to the RBD (left) and S protein (right) of SARS-CoV-2 immobilized on a Ni-NTA biosensor. A two-fold dilution series was used from 125 to 4 nM (IgG) and 16 to 0.5 nM (MB). The red line represents the raw data and the black line represents the global fit. [Figure 10]Epitope delineation of the strongest mAb specificities. Surface and diagrammatic representation of aRBD (core in light green, RBM in dark green) and ACE266 (light brown) binding. Heatmap showing binding competition experiment. High signal response (red) indicates low competition, low signal response (white) indicates high competition. Epitope bins are highlighted with dashed boxes. 15.0 Å filtered cryo-EM reconstructions of spike (grey) in complex with bFab80 (yellow), 298 (orange), and 324 (red). RBD and NTD are shown in green and blue, respectively. Cryo-EM reconstruction of cFab46 (pink) in complex with RBD (green). Representation of the secondary structure of RBD66 fitted to the partial density observed for the RBD. Crystal structure of the ternary complex formed by dFab52 (purple), Fab298 (orange), and RBD (green). e Composite image showing side and top views of the unliganded (PDB6XM4) and antibody-bound SARS-CoV-2 spike with available PDB or EMD entries. Inset: Close-up view of antibodies targeting different antigenic sites on the RBD. The mAb with the lowest reported IC50 value against SARS-CoV-2 PsV was selected as the representative antibody for the bin (highlighted in bold), and antibodies with similar binding epitopes are listed below in the same colors (color coding for spike, NTD, and RBD as in (b)). Individual protomers in the unliganded spike are shown in white, pink, and purple. [Figure 11] Epitope binning. mAb binding competition experiment against His-tagged RBD measured by Biolayer Interferometry (BLI). mAb1 was incubated at 50 μg / ml for 3 min, followed by mAb2 at 50 μg / ml for 5 min. [Figure 12a]Cryo-EM analysis of Fab-spike and Fab-RBD complexes. Representative cryo-EM micrographs (scale bar 50 nm, top left), selected 2D class averages (top right), Fourier shell correlation curves from the final 3D non-uniform refinement (bottom left), and local resolutions (Å) plotted on the surface of the cryo-EM maps (bottom right) are shown for Fab80-spike (a), Fab298-spike (b), Fab324-spike (c), and Fab46-RBD (d). [Figure 12b] Cryo-EM analysis of Fab-spike and Fab-RBD complexes. Representative cryo-EM micrographs (scale bar 50 nm, top left), selected 2D class averages (top right), Fourier shell correlation curves from the final 3D non-uniform refinement (bottom left), and local resolutions (Å) plotted on the surface of the cryo-EM maps (bottom right) are shown for Fab80-spike (a), Fab298-spike (b), Fab324-spike (c), and Fab46-RBD (d). [Figure 12c] Cryo-EM analysis of Fab-spike and Fab-RBD complexes. Representative cryo-EM micrographs (scale bar 50 nm, top left), selected 2D class averages (top right), Fourier shell correlation curves from the final 3D non-uniform refinement (bottom left), and local resolutions (Å) plotted on the surface of the cryo-EM maps (bottom right) are shown for Fab80-spike (a), Fab298-spike (b), Fab324-spike (c), and Fab46-RBD (d). [Figure 12d] Cryo-EM analysis of Fab-spike and Fab-RBD complexes. Representative cryo-EM micrographs (scale bar 50 nm, top left), selected 2D class averages (top right), Fourier shell correlation curves from the final 3D non-uniform refinement (bottom left), and local resolutions (Å) plotted on the surface of the cryo-EM maps (bottom right) are shown for Fab80-spike (a), Fab298-spike (b), Fab324-spike (c), and Fab46-RBD (d). [Figure 13a] Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 13b]Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 13c]Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 13d]Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 13e]Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 13f]Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 13g]Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 13h]Multabodies overcome sequence diversity of SARS-CoV-2. a Diagrammatic representation of the RBD showing the four naturally occurring mutations as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative epitopes for each bin. b Comparison of affinity change between WT and mutant RBD and PsV. c Comparison of IC50 fold change between WT and mutant RBD and PsV. d Neutralization potency of IgG (grey bars) and MB (dark red bars) against SARS-CoV-2 PsV mutants compared to WT PsV. e Comparison of neutralization potency of two IgG cocktails (3 IgG), a monospecific MB cocktail (3 MB), and a trispecific MB against WT SARS-CoV-2 PsV and mutants. mAbs sensitive to one or more PsV mutants (d) were selected to generate the cocktail and trispecific MB. f Neutralization potency of trispecific 298-80-52 MB against SARS-CoV-2 B.1.351 PsV mutant. g IC50 values ​​for PsV (y-axis) and replication-competent SARS-CoV-2 viruses (SB2-P4-PB: x-axis) show the ability of trispecific MB (red) to enhance potency across a broad range of mAb signatures (blue and black). h IC50 values ​​double upon multimerization. The average of three biological replicates is shown in (bh). [Figure 14a]MBs potently overcome sequence variability of SARS-CoV-2. a) Comparison of neutralization potency of selected IgGs and MBs against WT PsV (dark red) and the more infectious D614GPsV (grey). b) Schematic of trispecific MBs generated by combining three Fab specificities and Fc fragments using MB split design. c) Cocktails and trispecific MBs combining mAbs 298, 80, 52 or 298, 324, 46 specificities were generated and tested against WT PsV. Mean ± SD of two technical replicates are displayed for each representative neutralization plot. Source data are provided as source data files. d) Change in neutralization potency of cocktails and trispecific MBs against pseudotyped SARS-CoV-2 mutants compared to WT PsV. PsV mutants sensitive to individual antibodies in the cocktail were selected. The area within the dotted line represents a 3-fold change in IC50 values. This threshold was established as the cutoff for increased sensitivity (upward bars) or increased resistance (downward bars). e) Neutralization titration curves showing three biological replicates of the cocktail and trispecific MBs against the authentic SARS-CoV-2 / SB2-P4-PB strain. The median IC50 values ​​of the three biologically independent replicates are shown. [Figure 14b]MBs potently overcome sequence variability of SARS-CoV-2. a) Comparison of neutralization potency of selected IgGs and MBs against WT PsV (dark red) and the more infectious D614GPsV (grey). b) Schematic of trispecific MBs generated by combining three Fab specificities and Fc fragments using MB split design. c) Cocktails and trispecific MBs combining mAbs 298, 80, 52 or 298, 324, 46 specificities were generated and tested against WT PsV. Mean ± SD of two technical replicates are displayed for each representative neutralization plot. Source data are provided as source data files. d) Change in neutralization potency of cocktails and trispecific MBs against pseudotyped SARS-CoV-2 mutants compared to WT PsV. PsV mutants sensitive to individual antibodies in the cocktail were selected. The area within the dotted line represents a 3-fold change in IC50 values. This threshold was established as the cutoff for increased sensitivity (upward bars) or increased resistance (downward bars). e) Neutralization titration curves showing three biological replicates of the cocktail and trispecific MBs against the authentic SARS-CoV-2 / SB2-P4-PB strain. The median IC50 values ​​of the three biologically independent replicates are shown. [Figure 14c]MBs potently overcome sequence variability of SARS-CoV-2. a) Comparison of neutralization potency of selected IgGs and MBs against WT PsV (dark red) and the more infectious D614GPsV (grey). b) Schematic of trispecific MBs generated by combining three Fab specificities and Fc fragments using MB split design. c) Cocktails and trispecific MBs combining mAbs 298, 80, 52 or 298, 324, 46 specificities were generated and tested against WT PsV. Mean ± SD of two technical replicates are displayed for each representative neutralization plot. Source data are provided as source data files. d) Change in neutralization potency of cocktails and trispecific MBs against pseudotyped SARS-CoV-2 mutants compared to WT PsV. PsV mutants sensitive to individual antibodies in the cocktail were selected. The area within the dotted line represents a 3-fold change in IC50 values. This threshold was established as the cutoff for increased sensitivity (upward bars) or increased resistance (downward bars). e) Neutralization titration curves showing three biological replicates of the cocktail and trispecific MBs against the authentic SARS-CoV-2 / SB2-P4-PB strain. The median IC50 values ​​of the three biologically independent replicates are shown. [Figure 14d]MBs potently overcome sequence variability of SARS-CoV-2. a) Comparison of neutralization potency of selected IgGs and MBs against WT PsV (dark red) and the more infectious D614GPsV (grey). b) Schematic of trispecific MBs generated by combining three Fab specificities and Fc fragments using MB split design. c) Cocktails and trispecific MBs combining mAbs 298, 80, 52 or 298, 324, 46 specificities were generated and tested against WT PsV. Mean ± SD of two technical replicates are displayed for each representative neutralization plot. Source data are provided as source data files. d) Change in neutralization potency of cocktails and trispecific MBs against pseudotyped SARS-CoV-2 mutants compared to WT PsV. PsV mutants sensitive to individual antibodies in the cocktail were selected. The area within the dotted line represents a 3-fold change in IC50 values. This threshold was established as the cutoff for increased sensitivity (upward bars) or increased resistance (downward bars). e) Neutralization titration curves showing three biological replicates of the cocktail and trispecific MBs against the authentic SARS-CoV-2 / SB2-P4-PB strain. The median IC50 values ​​of the three biologically independent replicates are shown. [Figure 14e]MBs potently overcome sequence variability of SARS-CoV-2. a) Comparison of neutralization potency of selected IgGs and MBs against WT PsV (dark red) and the more infectious D614GPsV (grey). b) Schematic of trispecific MBs generated by combining three Fab specificities and Fc fragments using MB split design. c) Cocktails and trispecific MBs combining mAbs 298, 80, 52 or 298, 324, 46 specificities were generated and tested against WT PsV. Mean ± SD of two technical replicates are displayed for each representative neutralization plot. Source data are provided as source data files. d) Change in neutralization potency of cocktails and trispecific MBs against pseudotyped SARS-CoV-2 mutants compared to WT PsV. PsV mutants sensitive to individual antibodies in the cocktail were selected. The area within the dotted line represents a 3-fold change in IC50 values. This threshold was established as the cutoff for increased sensitivity (upward bars) or increased resistance (downward bars). e) Neutralization titration curves showing three biological replicates of the cocktail and trispecific MBs against the authentic SARS-CoV-2 / SB2-P4-PB strain. The median IC50 values ​​of the three biologically independent replicates are shown. [Figure 15] The N92T mutation in the VL of mAb52 did not affect potency either as IgG or as monospecific MB in the WT pseudovirus neutralization assay. [Figure 16] The 298-80-52 trispecific MB (T10 MB), containing the N92T mutation in the VL of mAb52, was screened in a P.1PsV neutralization assay and confirmed to have no reduced potency compared to the parent trispecific MB. [Figure 17] The trispecific MB298-80-52 demonstrated superior potency across the variants of interest in pseudoviral neutralization assays. [Figure 18] T10 MB shows comparable neutralization in pseudovirus and authentic virus assays. [Figure 19a]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19b]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19c]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19d]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19e]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19f]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19g]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19h]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 19i]In vivo protection by T10 MB in SARS-CoV-2 challenge studies. See Table 17 for MB nomenclature. (a) The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding IgG cocktail. (bc) Binding studies revealed that both the trispecific MB* and IgG4* antibody cocktails bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (FcγR), in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control. (d) Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktails to bind Fc receptors, whereas the IgG1 antibody cocktail significantly internalized beads coated with SARS-CoV-2 spike. (e) The trispecific MB* provided significantly better protection (60% survival) compared to the IgG4* cocktail. (f) The improved protection was associated with a significant reduction in lung virus titers, especially in animals that survived the challenge. (g) Comparable in vivo protection was achieved when the trispecific MB* was administered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg). (h) The difference in dosage could be observed in the circulating serum concentrations of the administered molecules at day 2 post-challenge. (i) Pulmonary MB and IgG titers were also assessed and trispecific MB* was detected in the lungs at the endpoint, highlighting the ability of MB to enter the lungs. [Figure 20a] (a) The IgG4 Fc MB mutants showed comparable binding to hFcRn but distinct binding profiles to hFcγR. (b) Binding of T10.A, T10.B, and T10.G to human FcRn and FcγRI showed similar trends for CynoFcRn and FcγRI. (c) No binding to mouse FcγRI was detected for T10.A, T10.B, and T10.G. [Figure 20b](a) The IgG4 Fc MB mutants showed comparable binding to hFcRn but distinct binding profiles to hFcγR. (b) Binding of T10.A, T10.B, and T10.G to human FcRn and FcγRI showed similar trends for CynoFcRn and FcγRI. (c) No binding to mouse FcγRI was detected for T10.A, T10.B, and T10.G. [Figure 20c] (a) The IgG4 Fc MB mutants showed comparable binding to hFcRn but distinct binding profiles to hFcγR. (b) Binding of T10.A, T10.B, and T10.G to human FcRn and FcγRI showed similar trends for CynoFcRn and FcγRI. (c) No binding to mouse FcγRI was detected for T10.A, T10.B, and T10.G. [Figure 21a] (a) Both T10.B and T10.G MB achieved 75% survival at D12 compared to the IgG negative control. (b) In vivo protection was accompanied by reduced weight loss in surviving mice over the course of the experiment. (c) In vivo protection was accompanied by a reduction in viral titers in the lungs of surviving mice at D12 at the detection limit of the assay. [Figure 21b] (a) Both T10.B and T10.G MB achieved 75% survival at D12 compared to the IgG negative control. (b) In vivo protection was accompanied by reduced weight loss in surviving mice over the course of the experiment. (c) In vivo protection was accompanied by a reduction in viral titers in the lungs of surviving mice at D12 at the detection limit of the assay. [Figure 21c] (a) Both T10.B and T10.G MB achieved 75% survival at D12 compared to the IgG negative control. (b) In vivo protection was accompanied by reduced weight loss in surviving mice over the course of the experiment. (c) In vivo protection was accompanied by a reduction in viral titers in the lungs of surviving mice at D12 at the detection limit of the assay. [Figure 22a] (a) T10.G MB exerted a 75% protective effect in homozygous hFcRn / hACE2 transgenic mice compared to the negative control IgG group. (b) The in vivo protective effect was accompanied by a reduced weight loss throughout the experimental period in surviving mice. [Figure 22b] (a) T10.G MB exerted a 75% protective effect in homozygous hFcRn / hACE2 transgenic mice compared to the negative control IgG group. (b) The in vivo protective effect was accompanied by a reduced weight loss throughout the experimental period in surviving mice. [Figure 23] T10.BMB achieved the expected maximum serum concentration (Cmax) and was detectable in the circulation for several weeks following administration to NHPs. [Figure 24-1] (a) Generation of trispecific MB molecules using a modified apoferritin split design. [Figure 24-2] (b) Analysis of cryo-electron micrographs reveals the formation of highly decorated, homogenous nanocage-like particles. Consistent with the presence of flexible (GGS)x linkers connecting the scFab and scFc components to the apoferritin scaffold, (c) the density of these antibody fragments is not well resolved in 2D class and (d) 3D reconstructions of trispecific MBs. [Diagram 25] To gain molecular insight into the assembly of the multibody design, we characterized this trispecific MB by cryo-electron microscopy (cryoEM). [Figure 26] (a)-(i) Proper assembly of Fab and Fc components on MB confirmed at approximately 7 Å resolution. [Figure 27] The 3D reconstruction of the apoferritin scaffold in MB reached a resolution of 2.4 Å and 2.1 Å when (a)(d) no symmetry (C1) or (e)–(h) octahedral symmetry (O) were applied, respectively. [Figure 28] Crystal structure of 80Fab in complex with the RBD at 3.1 Å resolution. [Figure 29a](a) mAb80 inhibits SARS-CoV-2 infection by blocking the receptor, preventing the interaction of ACE2 with the receptor-binding motif. (b)-(c) Residues S477 and T478 of the RBD form hydrogen bonds with Y92 and D100D of the antibody, burying 124 Å2 of its surface area and accounting for 15% of the total buried surface area of ​​the RBD (BSA). (d)-(e) These residues are mutated in several mAbs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to Omicron BA.1 RBD. The interaction of mAb80 with the mutated Omicron BA.1 RBD has a high apparent binding affinity with no detectable off-rate, (g) which may contribute to its strong neutralization potency against Omicron BA.1. (f) The potency of 80MB against OmicronBA.2 was further confirmed using replication-competent virus, and as expected, it was observed that 80mAb had significantly reduced potency against OmicronBA.2 live virus, while the MB format retained high neutralization potency. [Figure 29b] (a) mAb80 inhibits SARS-CoV-2 infection by blocking the receptor, preventing the interaction of ACE2 with the receptor-binding motif. (b)-(c) Residues S477 and T478 of the RBD form hydrogen bonds with Y92 and D100D of the antibody, burying 124 Å2 of its surface area and accounting for 15% of the total buried surface area of ​​the RBD (BSA). (d)-(e) These residues are mutated in several mAbs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to Omicron BA.1 RBD. The interaction of mAb80 with the mutated Omicron BA.1 RBD has a high apparent binding affinity with no detectable off-rate, (g) which may contribute to its strong neutralization potency against Omicron BA.1. (f) The potency of 80MB against OmicronBA.2 was further confirmed using replication-competent virus, and as expected, it was observed that 80mAb had significantly reduced potency against OmicronBA.2 live virus, while the MB format retained high neutralization potency. [Figure 29c](a) mAb80 inhibits SARS-CoV-2 infection by blocking the receptor, preventing the interaction of ACE2 with the receptor-binding motif. (b)-(c) Residues S477 and T478 of the RBD form hydrogen bonds with Y92 and D100D of the antibody, burying 124 Å2 of its surface area and accounting for 15% of the total buried surface area of ​​the RBD (BSA). (d)-(e) These residues are mutated in several mAbs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to Omicron BA.1 RBD. The interaction of mAb80 with the mutated Omicron BA.1 RBD has a high apparent binding affinity with no detectable off-rate, (g) which may contribute to its strong neutralization potency against Omicron BA.1. (f) The potency of 80MB against OmicronBA.2 was further confirmed using replication-competent virus, and as expected, it was observed that 80mAb had significantly reduced potency against OmicronBA.2 live virus, while the MB format retained high neutralization potency. [Figure 29d] (a) mAb80 inhibits SARS-CoV-2 infection by blocking the receptor, preventing the interaction of ACE2 with the receptor-binding motif. (b)-(c) Residues S477 and T478 of the RBD form hydrogen bonds with Y92 and D100D of the antibody, burying 124 Å2 of its surface area and accounting for 15% of the total buried surface area of ​​the RBD (BSA). (d)-(e) These residues are mutated in several mAbs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to Omicron BA.1 RBD. The interaction of mAb80 with the mutated Omicron BA.1 RBD has a high apparent binding affinity with no detectable off-rate, (g) which may contribute to its strong neutralization potency against Omicron BA.1. (f) The potency of 80MB against OmicronBA.2 was further confirmed using replication-competent virus, and as expected, it was observed that 80mAb had significantly reduced potency against OmicronBA.2 live virus, while the MB format retained high neutralization potency. [Figure 29e](a) mAb80 inhibits SARS-CoV-2 infection by blocking the receptor, preventing the interaction of ACE2 with the receptor-binding motif. (b)-(c) Residues S477 and T478 of the RBD form hydrogen bonds with Y92 and D100D of the antibody, burying 124 Å2 of its surface area and accounting for 15% of the total buried surface area of ​​the RBD (BSA). (d)-(e) These residues are mutated in several mAbs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to Omicron BA.1 RBD. The interaction of mAb80 with the mutated Omicron BA.1 RBD has a high apparent binding affinity with no detectable off-rate, (g) which may contribute to its strong neutralization potency against Omicron BA.1. (f) The potency of 80MB against OmicronBA.2 was further confirmed using replication-competent virus, and as expected, it was observed that 80mAb had significantly reduced potency against OmicronBA.2 live virus, while the MB format retained high neutralization potency. [Fig. 29f] (a) mAb80 inhibits SARS-CoV-2 infection by blocking the receptor, preventing the interaction of ACE2 with the receptor-binding motif. (b)-(c) Residues S477 and T478 of the RBD form hydrogen bonds with Y92 and D100D of the antibody, burying 124 Å2 of its surface area and accounting for 15% of the total buried surface area of ​​the RBD (BSA). (d)-(e) These residues are mutated in several mAbs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to Omicron BA.1 RBD. The interaction of mAb80 with the mutated Omicron BA.1 RBD has a high apparent binding affinity with no detectable off-rate, (g) which may contribute to its strong neutralization potency against Omicron BA.1. (f) The potency of 80MB against OmicronBA.2 was further confirmed using replication-competent virus, and as expected, it was observed that 80mAb had significantly reduced potency against OmicronBA.2 live virus, while the MB format retained high neutralization potency. [Figure 29g](a) mAb80 inhibits SARS-CoV-2 infection by blocking the receptor, preventing the interaction of ACE2 with the receptor-binding motif. (b)-(c) Residues S477 and T478 of the RBD form hydrogen bonds with Y92 and D100D of the antibody, burying 124 Å2 of its surface area and accounting for 15% of the total buried surface area of ​​the RBD (BSA). (d)-(e) These residues are mutated in several mAbs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to Omicron BA.1 RBD. The interaction of mAb80 with the mutated Omicron BA.1 RBD has a high apparent binding affinity with no detectable off-rate, (g) which may contribute to its strong neutralization potency against Omicron BA.1. (f) The potency of 80MB against OmicronBA.2 was further confirmed using replication-competent virus, and as expected, it was observed that 80mAb had significantly reduced potency against OmicronBA.2 live virus, while the MB format retained high neutralization potency. [Diagram 30] (a)-(b) The heavy chain of mAb80 is primarily responsible for interactions with the RBD, accounting for 10 of the 11 hydrogen bonds at the binding interface. (c) The interaction of F54 of the antibody heavy chain with Y489 of the RBD leads to the formation of a novel 3-fold pi-stacking between residues Y473, F456, and Y421 in the RBD structure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0145] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0146] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Numerous patent applications, patents, and publications are referenced herein in order to understand the described aspects. Each of these references is incorporated herein by reference in its entirety.

[0147] For purposes of understanding the scope of this application, the articles "a," "an," "the," and "said" shall mean the presence of one or more elements. Furthermore, as used herein, the term "comprising" and its derivatives are intended to be open-ended terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but not excluding the presence of other unstated features, elements, components, groups, integers, and / or steps. The above also applies to words of similar meaning, such as terms such as "including," "having," and their derivatives.

[0148] An embodiment described as "comprising" a particular component may also "consist" or "consist essentially of," where "consisting" has a restrictive or limiting meaning, and "consisting essentially of" means including the specified component, but excluding materials present as impurities, unavoidable materials present as a result of the process used to provide the component, and components other than components added for purposes other than achieving the technical effect of the invention. For example, a composition defined using the phrase "consisting essentially of" includes any known acceptable additives, excipients, diluents, carriers, and the like. Typically, a composition consisting essentially of a set of components contains less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight, and even more typically less than 0.1% by weight of the unspecified component(s).

[0149] It is understood that any component defined as included herein may be expressly excluded from the claimed invention by disclaimer or negative limitation. For example, in some embodiments, the nanocages and / or fusion proteins described herein may exclude ferritin heavy chains and / or exclude iron binding components.

[0150] Moreover, all ranges set forth herein include the ends of the ranges as well as intermediate range points, whether or not expressly stated.

[0151] As used herein, terms of degree such as "substantially," "about," "approximately," and the like, refer to reasonable deviations of the modified term such that the end result is not significantly altered. These terms of degree should be interpreted as including deviations of at least ±5% of the modified term, if such deviations do not negate the meaning of the word they modify. For example, the term "about" may encompass values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0152] The abbreviation "eg" is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the terms "for example" or "such as." The word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0153] As used herein, the term "subject" refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, zoo, sports, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is a human.

[0154] The terms "protein nanoparticle", "nanocage" and "multabody" are used interchangeably herein and refer to protein-based polyhedral shaped structures composed of multiple subunits, each of which is composed of a protein or polypeptide (e.g., a glycosylated polypeptide) and, optionally, one or more of the following features: nucleic acids, prosthetic groups, organic compounds and inorganic compounds. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci., 12:5406-5421, 2011, incorporated herein by reference), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct, and Mol. Biol., 15:939-947, 2008, incorporated herein by reference), sulfur oxidase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, incorporated herein by reference), lumazine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306:1099-1114, 2001), or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., J. Mol. Biol., 306:1099-1114, 2001). al., PNAS96:1240-1245, 1999, incorporated herein by reference). Ferritin, apoferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble into globular protein complexes that may consist of 24, 60, 24, 60, and 60 protein subunits, respectively. Ferritin and apoferritin are generally referred to synonymously herein, and both are understood to be suitable for use in the fusion proteins, nanocages, and methods described herein. Carboxysomes, vault proteins, GroEL, heat shock proteins, E2P, and MS2 coat proteins also generate nanocages and are contemplated for use herein. Additionally, fully or partially synthetic self-assembling monomers are also contemplated for use herein.

[0155] It will be understood that each nanocage monomer can be split into two or more subunits that self-assemble into functional nanocage monomers. For example, ferritin or apoferritin can be split into N and C subunits, e.g., by splitting full-length ferritin substantially in half, with each subunit separately attached to a SARS-CoV-2 binding moiety or a biologically active moiety, and then self-assembled into nanocage monomers and then nanocages. Each subunit can, in some embodiments, be attached to a SARS-CoV-2 binding moiety and / or a biologically active moiety at both the same or different termini. By "functional nanocage monomer," it is intended that the nanocage monomer can self-assemble with other such monomers into nanocages, as described herein.

[0156] The terms "ferritin" and "apoferritin" are used interchangeably herein and generally refer to a polypeptide (e.g., a ferritin chain) that can be assembled into a ferritin complex, typically comprising 24 protein subunits. It will be understood that the ferritin can be from any species. Typically, the ferritin is human ferritin. In some embodiments, the ferritin is wild-type ferritin. For example, the ferritin can be wild-type human ferritin. In some embodiments, a ferritin light chain is used as a nanocage monomer and / or a subunit of the ferritin light chain is used as a nanocage monomer subunit. In some embodiments, the assembled nanocage does not include a ferritin heavy chain or other ferritin components that can bind iron.

[0157] The term "multispecificity" as used herein refers to the characteristic of having at least two binding sites to which at least two different binding partners, e.g., antigens or receptors (e.g., Fc receptors), can bind. For example, a nanocage comprising at least two Fab fragments, each of which can bind a different antigen, is "multispecific." As an additional example, a nanocage comprising an Fc fragment (capable of binding to an Fc receptor) and a Fab fragment (capable of binding to an antigen) is "multispecific."

[0158] As used herein, the term "multivalent" refers to the characteristic of having at least two binding sites to which a binding partner, e.g., an antigen or a receptor (e.g., an Fc receptor), can bind. The binding partners capable of binding to the at least two binding sites can be the same or different.

[0159] The term "antibody" as used herein, also referred to in the art as "immunoglobulin" (Ig), refers to a protein constructed from pairs of heavy and light polypeptide chains, of which there are various Ig isotypes, including IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4, etc.), and IgM. It will be understood that antibodies may be derived from any species, including human, mouse, rat, monkey, llama, shark, etc. When an antibody is properly folded, each chain folds into a number of different globular domains connected by a more linear polypeptide sequence. For example, in the case of IgG, the immunoglobulin light chain is made up of variable (V L ) and constant (CL) domains, and the heavy chains fold into variable (V H ) domain and three constant (C H , C H2 , C H3 ) domain. The heavy and light chain variable domains (V H and V L ) interact to form the antigen-binding domain (Fv). Each domain has a well-defined structure that is well known to those skilled in the art.

[0160] The variable regions of the light and heavy chains are responsible for binding to target antigens and therefore may exhibit a large degree of sequence diversity among antibodies. The constant regions are less diverse in sequence and are responsible for binding to many natural proteins to trigger important immunological events. The variable regions of an antibody contain the antigen-binding determinants of the molecule, thereby determining the specificity of the antibody for its target antigen. The majority of sequence variation occurs in six hypervariable regions, three per variable heavy and light chain, which combine to form the antigen-binding site and contribute to the binding and recognition of antigenic determinants. The specificity and affinity of an antibody for its antigen are determined by the structure of the hypervariable regions and the size, shape, and chemistry of the surface it presents to the antigen.

[0161] An "antibody fragment" as referred to herein may include any suitable antigen-binding antibody fragment known in the art. Antibody fragments may be naturally occurring antibody fragments or may be obtained by manipulation of naturally occurring antibodies or by using recombinant methods. For example, antibody fragments include Fv, single chain Fv (scFv; V linked by a peptide linker), and the like. L and V H Fc, single-chain Fc, Fab, single-chain Fab, F(ab')2, single domain antibody (sdAb; a molecule consisting of a single V L or V H and multivalent representations of any of these.

[0162] The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA technology. This term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0163] The term "epitope" refers to an antigenic determinant. An epitope is a particular chemical group or peptide sequence on a molecule that is antigenic, i.e., that elicits a particular immune response. An antibody specifically binds to a particular antigenic epitope on, for example, a polypeptide. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically contains at least 3, more usually at least 5, about 9, about 11, or about 8-12 amino acids in a unique spatial structure. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0164] As used herein, the term "antigen" is defined as a molecule that elicits an immune response. This immune response may involve either antibody production or activation of cells with a particular immunological capability, or both. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode the term "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the embodiments described herein include, but are not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is apparent that an antigen can be synthesized or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, cells, or bodily fluids.

[0165] The term "encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0166] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0167] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide would be "isolated" if it was partially or completely separated from the coexisting materials in its natural state. An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell.

[0168] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can include introns, to the extent that a nucleotide sequence encoding a protein may in some versions contain an intron(s).

[0169] As used herein, the term "modulation" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of the treatment or compound and / or compared to the level of the response in an otherwise identical subject not receiving the treatment. The term also refers to perturbing and / or affecting an innate signal or response, thereby mediating a beneficial therapeutic response in a subject, usually a human.

[0170] The term "operably linked" refers to the functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are adjacent in reading frame, and, if necessary, to link two protein coding regions.

[0171] "Parenteral" administration of the compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques. Inhalation and intranasal administration are also included.

[0172] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, nucleic acid and polynucleotide are synonymous. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, any nucleic acid sequence obtained by any means available in the art, including, but not limited to, by recombinant means, i.e., by cloning a nucleic acid sequence from a recombinant library or a cell genome using conventional cloning techniques and PCR, etc., and by synthetic means.

[0173] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. A polypeptide includes a peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and longer chains, also commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0174] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind that antigen from one or more species. However, such cross-species reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" are used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure, rather than proteins in general. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or unlabeled free A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0175] As used herein, the phrases "unbound", "unbound" or "no binding" between two entities, or similar phrases, refer to 1) no detectable binding, or 2) binding below a set threshold that corresponds to no binding in an appropriate assay (e.g., an in vitro binding assay such as Biolayer Interferometry). For example, in some embodiments, a maximum association binding response of less than 0.1 nm after 180 seconds to a biosensor loaded with 0.8 nm target when a test substance is present at a concentration of 20 nM in an in vitro Biolayer Interferometry assay is classified as "unbound".

[0176] The terms "therapeutically effective amount," "effective amount," or "sufficient amount" refer to an amount sufficient to achieve a desired result, e.g., an amount effective to elicit a protective immune response, when administered to a subject, including a mammal (e.g., a human). The effective amount of the compounds described herein may vary depending on factors such as the molecule, age, sex, species, weight, etc. of the subject. Dosage or treatment may be adjusted to provide an optimal therapeutic response, as understood by one of skill in the art. For example, administration of a therapeutically effective amount of the fusion proteins described herein is sufficient in some embodiments to treat and / or prevent COVID-19.

[0177] Furthermore, a treatment regimen for a subject with a therapeutically effective amount may consist of a single administration or, alternatively, a series of applications. The frequency and length of the treatment period will vary depending on various factors, such as the molecule, the age of the subject, the concentration of the drug, the patient's responsiveness to the drug, or a combination thereof. It will also be understood that the effective dosage of the drug used for treatment may increase or decrease over the course of a particular treatment regimen. Modifications in dosage may be evident by standard diagnostic assays well known in the art. The fusion proteins described herein may, in some embodiments, be administered before, during, or after treatment with conventional therapies for the disease or disorder in question. For example, the fusion proteins described herein may find particular use in combination with conventional therapies for viral infections.

[0178] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0179] As used herein, the phrase "under transcriptional control" or "operably linked" means that a promoter is in the correct location and orientation relative to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0180] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0181] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0182] The term "pharmaceutical acceptable" means that a compound or combination of compounds is compatible with the remaining ingredients of a formulation for use as a pharmaceutical and generally safe for administration to humans according to established governmental standards, including those promulgated by the U.S. Food and Drug Administration.

[0183] The term "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and / or absorption delaying agents, etc. The uses of pharmaceutically acceptable carriers are well known.

[0184] A "variant" is a biologically active fusion protein, antibody, or fragment thereof that has an amino acid sequence that differs from the reference sequence by the insertion, deletion, modification, and / or substitution of one or more amino acid residues in the reference sequence. A variant generally has less than 100% sequence identity with the reference sequence. However, a biologically active variant usually has an amino acid sequence that has at least about 70% amino acid sequence identity with the reference sequence, such as at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Variants include peptide fragments of at least 10 amino acids that retain some biological activity of the reference sequence. Variants also include polypeptides in which one or more amino acid residues have been added to the N-terminus or C-terminus of the comparison sequence, or within the comparison sequence. Variants also include polypeptides in which a number of amino acid residues have been deleted and / or optionally substituted with one or more amino acid residues. Variants may also be covalently modified, for example, by substituting non-naturally occurring amino acids or by modifying amino acid residues to produce non-naturally occurring amino acids.

[0185] As used herein, "percent amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the residues of a sequence of interest, such as a polypeptide of the present invention, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Neither N-terminal, C-terminal, nor internal extensions, deletions, or insertions in the candidate sequence should be construed as affecting sequence identity or homology. Methods and computer programs for alignment are well known in the art, such as "BLAST".

[0186] For purposes of this specification, "Active" or "Activity" refers to the biological and / or immunological activity of the fusion proteins described herein, and "biological" activity refers to a biological function, either inhibitory or stimulatory, exerted by the fusion protein.

[0187] The fusion proteins described herein may include modifications. Such modifications include, but are not limited to, conjugation to an effector molecule. Additionally, modifications include, but are not limited to, conjugation to a detectable reporter moiety. Modifications that increase half-life (e.g., pegylation) are also included. Modifications for deimmunization are also included. Proteins and non-protein agents may be conjugated to the fusion proteins by methods well known in the art. Conjugation methods include direct linkage, linkage via a covalent linker, and specific binding pair members (e.g., avidin-biotin). Such methods include, for example, those described in Greenfield et al., Cancer Research 50, 6600-6607 (1990), which is incorporated herein by reference, and Amon et al., Adv. Exp. Med. Biol. 303, 79-90 (1991) and Kiseleva et al., MoI. Biol. (USSR) 25, 508-514 (1991), both of which are incorporated herein by reference.

[0188] Fusion proteins Described herein are fusion proteins. The fusion proteins comprise nanocage monomers linked to SARS-CoV-2 binding moieties. Multiple fusion proteins self-assemble to form nanocages. In this manner, the SARS-CoV-2 binding moieties can decorate the inner surface of the self-assembled nanocage, the outer surface of the self-assembled nanocage, or both.

[0189] The SARS-CoV-2 binding moiety is typically an antibody or fragment thereof and can target any part of the SARS-CoV-2 virus, but typically targets the SARS-CoV-2 S glycoprotein. It will be understood that the SARS-CoV-2 binding moiety need not be an antibody or fragment thereof, but can be, for example, a protein that binds to and blocks the virus, or a molecule such as the S glycoprotein or RBD domain within the virus.

[0190] The antibody or fragment thereof may comprise, for example, a heavy and / or light chain of a Fab fragment. The antibody or fragment thereof may comprise, for example, an scFab fragment, an scFv fragment, an sdAb fragment, and / or a VHH region. It will be understood that any antibody or fragment thereof may be used in the fusion proteins described herein.

[0191] Generally, the fusion proteins described herein are associated with a Fab light chain and / or heavy chain, which may be produced separately or sequentially as a fusion protein.

[0192] For example, the SARS-CoV-2 binding moiety may comprise the single chain variable domains VHH-72, BD23 and / or 4A8. Alternatively, or in addition, the SARS-CoV-2 binding moiety may be selected from any one or combination of mAbs listed in Table 4 herein. For example, the SARS-CoV-2 binding moiety may be selected from any one or combination of mAbs 298, 324, 46, 80, 52, 82, and 236 in Table 4.

[0193] In certain embodiments, the nanocage monomers described herein are split into subunits, allowing for more SARS-CoV-2 binding moieties or other moieties to be attached in various ratios. For example, in some embodiments, the nanocage monomer comprises a first nanocage monomer subunit linked to a SARS-CoV-2 binding moiety. In use, the first nanocage monomer subunit self-assembles with a second nanocage monomer subunit to form a nanocage monomer. As described above, multiple nanocage monomers self-assemble to form a nanocage. The nanocage monomer subunits may be provided alone or in combination and may be fused with the same or different SARS-CoV-2 binding moieties.

[0194] Nanocages made from the nanocage monomers and / or nanocage monomer subunits described herein can have biologically active moieties included in addition to one or more SARS-CoV-2 binding moieties.

[0195] For example, the biologically active portion may include, for example, one or both chains of an Fc fragment. The Fc fragment may be derived from any type of antibody, as will be understood, but is typically an IgG4 Fc fragment. The Fc fragment may further include one or more mutations that modulate the half-life and / or effector function of the fusion protein and / or the resulting assembled nanocages that contain the fusion protein, such as one or more mutations at positions 228, 234, 235, 237, and 238 according to EU numbering. For example, the half-life may be on the scale of minutes, days, weeks, or even months.

[0196] Additionally, other substitutions in the fusion proteins and nanocages described herein are contemplated, including modification of the Fc sequence and addition of other agents (e.g., human serum albumin peptide sequences) that allow for altered bioavailability and would be understood by one of skill in the art. Additionally, the fusion proteins and nanocages described herein can be modulated by adjusting the sequence or adding other agents to attenuate immunogenicity and anti-drug responses (therapeutic, e.g., matching the sequence to the host, or adding immunosuppressive therapy (e.g., methotrexate when administering infliximab to treat rheumatoid arthritis, or induction of neonatal tolerance, which is a major strategy to reduce the development of inhibitors to FVIII (reviewed in DiMichele DM, Hoots WK, Pipe SW, Rivard GE, Santagostino E. International workshop on immune tolerance induction: consensus recommendations. Haemophilia. 2007;13:1-22, the entire text of which is incorporated herein by reference)).

[0197] For example, fusion proteins comprising nanocage monomers or subunits thereof linked to Fc polypeptides are described herein. In some embodiments, the Fc polypeptide is an Fc polypeptide comprising one or more human IgG4 Fc chains, i.e., an Fc chain substantially similar to that of the Fc chain in wild-type human IgG4, except for the mutations described herein.

[0198] In some embodiments, the wild-type IgG4 Fc is a human IgG4 Fc and each Fc chain has the amino acid sequence of SEQ ID NO:66.

[0199] For example, an Fc polypeptide may comprise an Fc chain having an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of an Fc chain in a wild-type IgG4 Fc. In some embodiments, an Fc polypeptide comprises an Fc chain having an amino acid sequence that includes a particular residue(s) at a particular position(s) specifically described for that Fc polypeptide, but is otherwise 100% identical to the corresponding Fc chain in a wild-type Fc chain, e.g., a wild-type IgG4 Fc chain. In some embodiments, an Fc polypeptide comprises an Fc chain having an amino acid sequence that differs from the sequence of SEQ ID NO:66 by at least one, at least two, at least three, or at least four amino acid residues. In some embodiments, the Fc polypeptide comprises an Fc chain having an amino acid sequence that differs by no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, or no more than 4 amino acid residues from the sequence of SEQ ID NO: 66. In some embodiments, the Fc polypeptide comprises an Fc chain that differs from the sequence of SEQ ID NO: 66 by at least 3, at least 4, or at least 5 amino acid residues.

[0200] In some embodiments, the Fc polypeptide is a single chain Fc (scFc) that comprises two Fc chains linked together by a covalent linker, e.g., via an amino acid linker.

[0201] In certain embodiments, the fragment crystallizable (Fc) region, such as an IgG4 Fc chain, comprises a mutation at one or more of positions 228, 234, 235, 237, and 238 according to EU numbering. In some embodiments, the IgG4 Fc chain comprises a mutation at positions 234 and 235. In some embodiments, the IgG4 Fc chain comprises a F234A mutation and a L235A mutation. In some embodiments, the IgG4 Fc chain comprises a mutation at position 228. In some embodiments, the IgG4 Fc chain comprises a S228P mutation. In some embodiments, the IgG4 Fc chain comprises a mutation at positions 237 and 238. In some embodiments, the IgG4 Fc chain comprises a G237A mutation and a P238S mutation. In some embodiments, the IgG4 Fc chain does not comprise a mutation at G237 or P238. In some embodiments, the IgG4 Fc chain comprises a S228P mutation, a F234A mutation, and a L235A mutation. In some embodiments, the IgG4 Fc chain comprises a S228P mutation, a F234A mutation, a L235A mutation, a G237A mutation and a P238S mutation. In some embodiments, the IgG4 Fc chain comprises a F234A mutation, a L235A mutation, a G237A mutation and a P238S mutation. In some embodiments, the IgG4 Fc chain does not comprise a mutation at S228. Unless otherwise stated, numbering of mutations throughout this disclosure is according to the EU index.

[0202] In some embodiments, the Fc region is an IgG4 Fc region (e.g., a human IgG4 Fc region), i.e., except for the mutations described herein, the Fc region comprises Fc chains each having an amino acid sequence substantially similar to the sequence of a chain in a wild-type IgG4 Fc. In some embodiments, the wild-type reference IgG4 Fc is a human IgG4 Fc, and each Fc chain has the amino acid sequence of SEQ ID NO:66.

[0203] For example, the IgG4 Fc region may comprise an Fc chain having an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the Fc chain in wild-type IgG4 Fc. In some embodiments, the IgG4 Fc region comprises an Fc chain having an amino acid sequence that includes the Fc mutations specifically described for that IgG4 Fc region, but that is otherwise 100% identical to the Fc chain in wild-type IgG4 Fc.

[0204] In some embodiments, the Fc region is a single chain Fc (scFc), which comprises two Fc chains linked together by a covalent linker, e.g., via an amino acid linker. In some embodiments, the Fc region is an Fc monomer, which comprises a single Fc chain.

[0205] When the antibody or fragment thereof comprises two chains, such as a first and second chain in the case of an Fc fragment, or a heavy and light chain, the two chains are optionally separated by a linker. The linker can be flexible or rigid, but is typically flexible to allow the chains to fold properly. The linker is generally long enough to provide some flexibility to the fusion protein, although it will be understood that the length of the linker will vary depending on the sequence of the nanocage monomer and the biologically active moiety, as well as the three-dimensional structure of the fusion protein. Thus, the linker is typically about 1 to about 130 amino acid residues, e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125 to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 amino acid residues, for example, about 50 to about 90 amino acid residues, for example, 70 amino acid residues.

[0206] The linker can be any amino acid sequence, and in a typical example, the linker comprises GGS repeats, more typically the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In certain embodiments, the linker comprises or consists of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following sequence: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

[0207] In certain embodiments, linkers are used in fusion polypeptides and / or single chain molecules such as scFc. In some embodiments, the linker is an amino acid linker. For example, a linker as used herein can contain about 1 to about 100 amino acid residues, e.g., about 1 to about 70, about 2 to about 70, about 1 to about 30, or about 2 to about 30 amino acid residues. In some embodiments, the linker contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid residues.

[0208] In certain embodiments, the linker comprises a glycine serine sequence, e.g., (G n S) m The sequence includes (e.g., GGS, GGGS, or GGGGS).

[0209] In an exemplary embodiment, the antibody or fragment thereof specifically binds to an antigen associated with SARS-CoV-2. Typically, the antigen is associated with SARS-CoV-2, and the antibody or fragment thereof comprises a binding domain in Table 4, such as, for example, binding domains 298, 52, 46, 80, 82, 236, 324, or a combination thereof.

[0210] In certain embodiments, the SARS-CoV-2 binding antibody fragment can bind to the receptor binding domain (RBD) of SARS-CoV-2. In certain embodiments, the SARS-CoV-2 binding antibody fragment can bind to the spike protein (S protein) of SARS-CoV-2. In some embodiments, the SARS-CoV-2 binding antibody fragment can bind to the N-terminal domain (NTD) of the S protein of SARS-CoV-2.

[0211] In some embodiments, the SARS-CoV-2 binding antibody fragment comprises a heavy chain variable region (e.g., V H or V H In certain embodiments, the SARS-CoV-2 binding antibody fragment comprises a heavy chain variable domain (e.g., V H ) and a light chain variable domain (e.g., V L or V K In certain embodiments, the SARS-CoV-2 binding antibody fragment comprises a heavy chain variable domain (e.g., V H ) and a light chain variable domain (e.g., V L or V K ) containing Fab.

[0212] In some embodiments, the SARS-CoV-2 binding antibody fragment is H Heavy chain variable domain and V K In some embodiments, the SARS-CoV-2 binding antibody fragment comprises a V H Heavy chain variable domain and V K Contains a Fab, which contains a light chain variable domain.

[0213] In certain examples, the antibody or fragment thereof comprises or consists of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to one or more of the following sequences: Fc chain 1: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK; Fc chain 2: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK; Fc chain 3-T10.G PPCPSCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Fc chain 4-T10.A PPCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Fc chain 5-T10.B PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 298 light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 298 Fab heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGTFSTYGISWVRQAPGQGLEWMGWISPNSGGTDLAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASDPRDDIAGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 52 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQGISNNLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNGFPLTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 52 light chain N92T mutant DIQMTQSPSSLSASVGDRVTITCRASQGISNNLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGTGFPLTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 52Fab heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGGIIPMFGTTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDRGDTIDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 46 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 46Fab heavy chain EVQLLESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDSRDAFDIWGQG TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 80 Light Chain DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAPLTFGGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 80Fab heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGTFNRYAFSWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTRELPEVVDWYFDL WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 82 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQVISNYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPPPTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 82Fab heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGSFSTSAFYWVRQAPGQGLEWWMGWINPYTGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRALYGSGSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 236 light chain DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 236Fab heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGTFTSYGINWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASRGIQLLPRGMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC 324 light chain DIQMTQSPSSLSASVGDRVTITCRASQSITTYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 324Fab heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGTFNNYGISWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVGDYGDYIVSPFDLW GRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Or a combination thereof.

[0214] In further embodiments, the antibody or fragment thereof may be conjugated or associated with a further moiety, such as a detectable moiety (e.g., a small molecule, a fluorescent molecule, a radioisotope, or a magnetic particle), a pharmaceutical agent, a diagnostic agent, or a combination thereof, e.g., constituting an antibody drug conjugate.

[0215] In embodiments where the biologically active moiety is a detectable moiety, the detectable moiety may include fluorescent proteins such as GFP, EGFP, ametrine, and / or flavin-based fluorescent proteins such as LOV proteins, such as iLOV.

[0216] In aspects in which the biologically active moiety is a pharmaceutical agent, the pharmaceutical agent can include, for example, a small molecule, a peptide, a lipid, a carbohydrate, or a toxin.

[0217] In typical embodiments, nanocages assembled from the fusion proteins described herein comprise about 3 to about 100 nanocage monomers, e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 96, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 157, 158, 15 4, 96, or 98 to about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 nanocage monomers, for example, 24, 32, or 60 monomers. The nanocage monomer can be any known nanocage monomer, natural, synthetic, or partially synthetic, and in some embodiments is selected from monomers of ferritin, apoferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and mutants thereof. Typically, the nanocage monomer is ferritin or apoferritin.

[0218] When apoferritin is selected as the nanocage monomer, typically the first and second nanocage monomer subunits comprise alternating "N" and "C" regions of apoferritin. It will be appreciated that other nanocage monomers can be split into bipartite subunits similar to the apoferritin described herein, with the subunits self-assembling and each susceptible to fusion with a bioactive moiety.

[0219] In some embodiments, the nanocage monomer is a ferritin monomer. The term "ferritin monomer" is used herein to refer to a single chain of ferritin that can self-assemble into a polypeptide complex containing multiple ferritin chains in the presence of other ferritin chains. In some embodiments, the ferritin chain self-assembles into a polypeptide complex containing 24 or more ferritin chains. In some embodiments, the ferritin monomer is a ferritin light chain. In some embodiments, the ferritin monomer does not contain a ferritin heavy chain or other ferritin components that can bind iron.

[0220] In some embodiments, each fusion polypeptide in the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of a ferritin light chain. In these embodiments, the self-assembled polypeptide complex does not comprise a ferritin heavy chain or a subunit of a ferritin heavy chain.

[0221] In some embodiments, the ferritin monomer is a human ferritin chain, e.g., a human ferritin light chain, e.g., a human ferritin light chain having a sequence of at least residues 2-175 of SEQ ID NO: 1. In some embodiments, the ferritin monomer is a mouse ferritin chain.

[0222] A "subunit" of a ferritin monomer refers to a portion of a ferritin monomer that can spontaneously associate with another, separate subunit of a ferritin monomer such that the subunits together form a ferritin monomer, which can then self-assemble with other ferritin monomers to form a polypeptide complex.

[0223] In some embodiments, the ferritin monomer subunit comprises about half of a ferritin monomer. As used herein, the term "N-half ferritin" refers to about half of a ferritin chain that includes the N-terminus of the ferritin chain. As used herein, the term "C-half ferritin" refers to about half of a ferritin chain that includes the C-terminus of the ferritin chain. The exact point at which a ferritin chain may be split to form N-half ferritin and C-half ferritin may vary depending on the embodiment. For example, in the context of a ferritin monomer subunit based on a human ferritin light chain, the half is split at a point corresponding to positions from about 75 to about 100 of SEQ ID NO:1. For example, in some embodiments, an N-half ferritin based on a human ferritin light chain has an amino acid sequence corresponding to residues 1-95 (or a substantial portion thereof) of SEQ ID NO:1, and a C-half ferritin based on a human ferritin light chain has an amino acid sequence corresponding to residues 96-175 (or a substantial portion thereof) of SEQ ID NO:1.

[0224] In some embodiments, the halves are split at a point corresponding to about positions 85 to about 92 of SEQ ID NO: 1. For example, in some embodiments, an N-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 1-90 (or a substantial portion) of SEQ ID NO: 1, and a C-half ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 91-175 (or a substantial portion) of SEQ ID NO: 1.

[0225] Typically, the "N" region of apoferritin comprises or consists of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following sequence: MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW.

[0226] Typically, the "C" region of apoferritin comprises or consists of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following sequence: GKTPDAMKAAMALEKKLNQALLLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD or GKTPDAMKAAMALEKKLNQALLLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD.

[0227] In some aspects, the fusion proteins described herein further comprise a linker between the nanocage monomer subunit and the biologically active moiety, similar to the linkers described above. Additionally, the linker can be flexible or rigid, but is typically flexible, allowing the biologically active moiety to retain activity and the pair of nanocage monomer subunits to retain self-assembly properties. The linker is generally long enough to provide some flexibility to the fusion protein, although it will be understood that the length of the linker will vary depending on the sequence of the nanocage monomer and biologically active moiety, as well as the three-dimensional structure of the fusion protein. Thus, the linker is typically from about 1 to about 30 amino acid residues, e.g., from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, e.g., from about 8 to about 16 amino acid residues, e.g., 8, 10, or 12 amino acid residues.

[0228] The linker can be any amino acid sequence, and in a typical example, the linker comprises GGS repeats, more typically the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats. In certain embodiments, the linker comprises or consists of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following sequence: GGGGSGGGGSGGGGSGGGGSGGGGSGG.

[0229] Similarly, the fusion protein may further comprise a C-terminal linker to improve one or more attributes of the fusion protein. In some embodiments, the linker comprises a GGS repeat, more typically the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, for example about 4 GGS repeats. In certain embodiments, the C-terminal linker comprises or consists of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following sequence: GGSGGSGGSGGSGGGSGGSGGSGGSG.

[0230] Also described herein are pairs of the above fusion proteins that self-assemble to form a nanocage monomer, where the first and second nanocage monomer subunits are fused to different SARS-CoV-2 binding moieties, thereby providing multivalency and / or multispecificity to a single nanocage monomer assembled from the paired subunits.

[0231] A substantially identical sequence may include one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may produce a variant peptide without any substantial change in physiological, chemical, or functional properties compared to the reference sequence, and in such cases, the reference and variant sequences will be considered as "substantially identical" polypeptides. Conservative amino acid mutations may include addition, deletion, or substitution of amino acids, and conservative amino acid substitutions are defined herein as the replacement of an amino acid residue with another amino acid residue having similar chemical properties (e.g., size, charge, or polarity).

[0232] In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may replace a basic, neutral, hydrophobic, or acidic amino acid with another amino acid of the same group. The term "basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7 that is typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). The term "neutral amino acid" (also "polar amino acid") refers to a hydrophilic amino acid with a side chain that is uncharged at physiological pH but has at least one bond in which the electron pair shared by two atoms is held in close proximity by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine ​​(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term "hydrophobic amino acid" (also "nonpolar amino acid") is intended to include amino acids that exhibit a hydrophobicity greater than zero according to the standardized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).

[0233] "Acidic amino acid" refers to a hydrophilic amino acid that is typically negatively charged at physiological pH and has a side chain pK value of less than 7. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0234] Sequence identity is used to evaluate the similarity of two sequences, and it is determined by calculating the percentage of residues that are identical when the two sequences are aligned to maximize the match between residue positions.Any known method can be used to calculate sequence identity, for example, computer software is available to calculate sequence identity.Without being limited thereto, sequence identity can be calculated by software such as NCBI BLAST2 service (can be found at ca.expasy.org / tools / blast / ) managed by Swiss Institute of Bioinformatics, BLAST-P, Blast-N, FASTA-N, or other suitable software known in the art.

[0235] Substantially identical sequences of the invention may be at least 85% identical, and in another example, substantially identical sequences may be at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% (or any percentage therebetween) identical at the amino acid level to the sequences described herein. In certain aspects, substantially identical sequences retain the activity and specificity of the reference sequence. In non-limiting embodiments, differences in sequence identity may be due to conservative amino acid mutation(s).

[0236] The polypeptide or fusion protein of the present invention may also include additional sequences to aid in expression, detection, or purification. Any such sequence or tag known to those skilled in the art may be used. For example, but not limited to, the fusion protein may include a target sequence or signal sequence (e.g., but not limited to, ompA), a detection tag, an exemplary tag cassette includes a Strep tag or any variant thereof (see, e.g., U.S. Pat. No. 7,981,632), a His tag, a Flag tag with sequence motif DYKDDDDK, an Xpress tag, an Avi tag, a calmodulin tag, a polyglutamic acid tag, an HA tag, a Myc tag, a Nus tag, an S tag, an SBP tag, a Softag1, a Softag3, a V5 tag, a CREB binding protein (CBP), a glutathione S-transferase (GST), a maltose binding protein (MBP), a green fluorescent protein (GFP), a thioredoxin tag, or any combination thereof, a purification tag (e.g., but not limited to, His5 or His6), or any combination thereof.

[0237] In another example, the additional sequence may be a biotin recognition site, for example as described in Cronan et al., WO95 / 04069 or Voges et al., WO / 2004 / 076670. As known to those skilled in the art, linker sequences may be used in combination with additional sequences or tags.

[0238] More specifically, the tag cassette may comprise an extracellular moiety that can specifically bind to an antibody with high affinity or avidity. Within the single-chain fusion protein structure, the tag cassette may be located (a) immediately amino-terminal to the connector region, (b) between and connecting the linker modules, (c) immediately carboxy-terminal to the binding domain, (d) between and connecting the binding domain (e.g., scFv or scFab) and the effector domain, (e) between and connecting the subunits of the binding domain, or (f) at the amino-terminus of the single-chain fusion protein. In certain embodiments, one or more junction amino acids may be located between the tag cassette and the hydrophobic portion, connecting the tag cassette and the hydrophobic portion, or between the tag cassette and the connector region, connecting the tag cassette and the connector region, or between the tag cassette and the linker module, connecting the tag cassette and the linker module, or between the tag cassette and the binding domain, connecting the tag cassette and the binding domain.

[0239] Also included herein are isolated or purified fusion proteins, polypeptides, or fragments thereof, immobilized to a surface using various methodologies, for example, but not limited to, a polypeptide may be linked or attached to a surface via His-tag binding, biotin binding, covalent binding, adsorption, etc. The solid surface may be any suitable surface, such as, but not limited to, the well surface of a microtiter plate, a channel of a surface plasmon resonance (SPR) sensor chip, a membrane, a bead (such as magnetic-based or sepharose-based beads, or other chromatographic resins), glass, a film, or other useful surface.

[0240] In another aspect, the fusion protein can be linked to a cargo molecule, which can deliver the cargo molecule to a desired site and can be linked to the cargo molecule using any method known in the art (recombinant techniques, chemical conjugation, chelation, etc.) The cargo molecule can be any type of molecule, such as a therapeutic or diagnostic agent.

[0241] In some embodiments, the cargo molecule is a protein and is fused to a fusion protein such that the cargo molecule is contained internally within the nanocage. In other embodiments, the cargo molecule is not fused to a fusion protein and is contained within the interior of the nanocage. The cargo molecule is typically a protein, a small molecule, a radioisotope, or a magnetic particle.

[0242] The fusion proteins described herein specifically bind to their targets. The antibody specificity (which refers to the selective recognition of an antibody for a specific epitope of an antigen) of the antibodies or fragments described herein can be determined based on affinity and / or avidity. Affinity is determined by the equilibrium constant for the dissociation of an antigen with an antibody (K D Avidity is a measure of the strength of binding between an antigenic determinant (epitope) and an antibody binding site. Avidity is a measure of the strength of binding between an antibody and its antigen. Antibodies are typically -5 ~10 -11 K of M D Combine with 10 -4 K exceeds M D is generally considered to represent non-specific binding. D The smaller the value of , the stronger the binding strength between the antigenic determinant and the antibody binding site. In some embodiments, the antibodies described herein have a 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, or 10 -15 K under MD has.

[0243] Also described herein is a nanocage comprising at least one fusion protein described herein and at least one second nanocage monomer subunit that self-assembles with the fusion protein to form a nanocage monomer. Further described herein are pairs of fusion proteins that self-assemble to form a nanocage monomer, where the first and second nanocage monomer subunits are fused to different biologically active moieties.

[0244] It will be appreciated that nanocages can self-assemble from multiple identical fusion proteins, multiple different fusion proteins (thus being multivalent and / or multispecific), combinations of fusion proteins and wild-type proteins, and any combination thereof. For example, nanocages can be decorated internally and / or externally with at least one fusion protein described herein in combination with at least one anti-SARS-CoV-2 antibody. In typical embodiments, about 20% to about 80% of the nanocage monomers constitute the fusion proteins described herein. Given the modular solution described herein, each nanocage monomer can be split into two subunits, each of which can be independently bound to a different bioactive moiety, so that in theory a nanocage could contain up to twice as many bioactive moieties as monomers within the nanocage. It will be appreciated that this modularity can be exploited to achieve any desired ratio of bioactive moieties, up to a 4:2:1:1 ratio of four different bioactive moieties, as described herein in certain examples. For example, the nanocages described herein can include at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different biologically active moieties. In this manner, the nanocages can be multivalent and / or multispecific, the degree of which can be relatively easily controlled.

[0245] In some embodiments, the nanocages described herein may further comprise at least one entire nanocage monomer, optionally fused to a bioactive moiety, which may be the same as or different from the bioactive moieties described herein, as linked to a nanocage monomer subunit.

[0246] In an exemplary embodiment, the nanocages described herein comprise first, second, and third fusion proteins to subunits or monomers, and optionally at least one entire nanocage monomer, optionally fused to a biologically active moiety, wherein the biologically active moieties of the first, second, and third fusion proteins and the biologically active moiety of the entire nanocage monomer are all different from one another.

[0247] More typically, the first, second and third fusion proteins comprise an antibody or Fc fragment thereof fused to N-half ferritin or C-half ferritin, respectively, with at least one of the first, second and third fusion proteins being fused to N-half ferritin and at least one of the first, second and third fusion proteins being fused to C-half ferritin. For example, the antibody or fragment thereof of the first fusion protein is typically an Fc fragment, the second and third fusion proteins typically comprise an antibody or fragment thereof specific for a different antigen of a virus, such as SARS-CoV-2, respectively, and the entire nanocage monomer is fused to another biologically active moiety specific for a different, optionally, antigen of the same virus, such as SARS-CoV-2.

[0248] In some embodiments, the antibody or fragment thereof of the second fusion protein is 46 or 52, and the antibody or fragment thereof of the third fusion protein is 324 or 80. In an exemplary embodiment, the nanocages described herein comprise the following four fusion proteins, optionally in a ratio of 4:2:1:1: a.298 (optionally sc298) fused to full-length ferritin, b Fc (optionally scFc) fused to N-ferritin or C-ferritin; 46 or 52 fused to cN-ferritin or C-ferritin (optionally sc46 or sc52); 324 or 80 (optionally sc324 or sc80) fused to N-ferritin or C-ferritin.

[0249] In some embodiments, the nanocages described herein comprise or consist of a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to one or more of the following sequences, where ferritin subunits are shown in bold, linkers are underlined, light chains are italics, and heavy chains are lowercase: [ka] b.Fc-N-ferr (PAAAS mutation) (included in T10.A) [ka] or Fc-N-ferr (AAAS mutation) (contained within T10.G) [ka] or Fc-N-ferr (PAA mutation) (included in T10.B) [ka] or Fc-N-ferr (PAAAS mutation) (included in T10.A) [ka] or Fc-N-ferr (AAAS mutation) (contained within T10.G) [ka] or Fc-C-ferr (PAA mutation) (included in T10.B) [ka] [ka] [ka]

[0250] In one aspect, a self-assembling polypeptide complex is provided that includes a plurality of fusion polypeptides as disclosed herein. In many embodiments, the self-assembling polypeptide complex includes (1) a plurality of first fusion polypeptides, each of which includes an Fc region linked to a nanocage monomer (e.g., a ferritin monomer, e.g., a human ferritin monomer, or a subunit thereof) as disclosed herein, and (2) a plurality of second fusion polypeptides, each of which includes a SARS-CoV-2-binding antibody fragment (e.g., a Fab fragment of an antibody capable of binding to a SARS-CoV-2 protein (e.g., a spike protein, or a receptor binding domain (RBD))), wherein the SARS-CoV-2-binding antibody fragment is linked to a nanocage monomer (e.g., a ferritin monomer, e.g., a human ferritin monomer) or a subunit thereof. In some embodiments, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each of which is separate from the second fusion polypeptide and each of which is comprised of (1) a nanocage monomer (e.g., a ferritin monomer, e.g., a human ferritin monomer) bound to (2) a SARS-CoV-2-binding antibody fragment (e.g., a Fab fragment of an antibody capable of binding to a SARS-CoV-2 protein).

[0251] In some embodiments, one of the fusion polypeptides (e.g., the first fusion polypeptide or the second fusion polypeptide) comprises an N-half nanocage monomer (e.g., N-half ferritin) (but not a full-length nanocage (e.g., ferritin) monomer) and the other of the fusion polypeptides comprises a C-half nanocage monomer (e.g., C-half ferritin) (but not a full-length nanocage (e.g., ferritin) monomer). In many of these embodiments, the ratio of fusion polypeptides comprising N-half nanocage monomers (e.g., N-half ferritin) to fusion polypeptides comprising C-half nanocage monomers (e.g., C-half ferritin) within the self-assembled polypeptide complex is about 1:1.

[0252] In some embodiments, the self-assembling polypeptide complex comprises 24 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises more than 24 fusion polypeptides, for example, at least 26, at least 28, at least 30, at least 32 fusion polypeptides, at least 34 fusion polypeptides, at least 36 fusion polypeptides, at least 38 fusion polypeptides, at least 40 fusion polypeptides, at least 42 fusion polypeptides, at least 44 fusion polypeptides, at least 46 fusion polypeptides, or at least 48 fusion polypeptides. In some embodiments, the self-assembling polypeptide complex comprises 32 fusion polypeptides.

[0253] In some embodiments, the self-assembled polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 first fusion polypeptides.

[0254] In some embodiments, the self-assembled polypeptide complex comprises at least 4, at least 5, at least 6, at least 7, or at least 8 second fusion polypeptides.

[0255] In some embodiments, the self-assembled polypeptide complex further comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 third fusion polypeptides.

[0256] In some embodiments, the self-assembled polypeptide complex comprises about a 1:1, 1:2, 1:3, or 1:4 ratio of a first fusion polypeptide to every other fusion polypeptide.

[0257] In some embodiments, each fusion polypeptide in the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of a ferritin light chain, in these embodiments, the self-assembled polypeptide complex does not comprise a ferritin heavy chain, a subunit of a ferritin heavy chain, or any other ferritin component capable of binding iron.

[0258] Also described herein are compositions comprising the nanocages, such as therapeutic or prophylactic compositions. Related methods and uses for treating and / or preventing COVID-19 are also described, which methods or uses comprise administering the nanocages or compositions described herein to a subject in need thereof.

[0259] Also described herein are nucleic acid molecules encoding the fusion proteins and polypeptides described herein, as well as vectors containing the nucleic acid molecules and host cells containing the vectors.

[0260] Polynucleotides encoding fusion proteins described herein include polynucleotides having substantially the same nucleic acid sequence as the nucleic acid sequence of a polynucleotide of the invention. A "substantially the same" nucleic acid sequence is defined herein as a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity with another nucleic acid sequence when the two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine the exact nucleotide matches between the two sequences.

[0261] Suitable sources of polynucleotides encoding antibody fragments include any cell, such as hybridomas and spleen cells, that express the full-length antibody. Fragments can be used as antibody equivalents by themselves or can be recombined into equivalents as described above. The DNA deletions and recombinations described in this section can be carried out by known methods, such as those described in the published patent applications listed in the "Functional Equivalents of Antibodies" section above, and / or other standard recombinant DNA techniques as described below. Another source of DNA is single chain antibodies generated from phage display libraries, well known in the art.

[0262] Further provided is an expression vector comprising the aforementioned polynucleotide sequence operably linked to an expression sequence, a promoter, and an enhancer sequence.Various expression vectors have been developed for efficient synthesis of antibody polypeptides in prokaryotes such as bacteria, and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems.The vectors of the present invention can include segments of chromosomal, non-chromosomal, and synthetic DNA sequences.

[0263] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids derived from E. coli, such as colEl, pCRl, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include phage DNA derivatives, such as M13 and other filamentous single-stranded DNA phages. Examples of vectors useful in yeast include the 2μ plasmid. Vectors suitable for expression in mammalian cells include well-known derivatives of DNA sequences derived from SV-40, adenovirus, retrovirus, and shuttle vectors derived from combinations of functional mammalian vectors, as described above, with functional plasmids and phage DNA.

[0264] Additional eukaryotic expression vectors are known in the art (e.g., P. J. Southern & P. ​​Berg, J. Mol. Appl. Genet, 1:327-341 (1982); Subramani et al, Mol. Cell. Biol, 1:854-864 (1981); Kaufhiann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol, 159:601-621 (1982); Kaufhiann & Sharp, Mol. Cell. Biol, 159:601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,'Proc. Nat'l Acad. Sci USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980), all of which are incorporated herein by reference).

[0265] An expression vector typically contains at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. The control sequence is inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences include the lac system, the trp system, the tac system, the trc system, the phage lambda major operator and promoter region, the fd coat protein control region, yeast glycolysis promoters (e.g., the promoter of 3-phosphoglycerate kinase), the promoter of yeast acid phosphatase (e.g., Pho5), the promoter of yeast alpha mating factor, and promoters from polyoma, adenovirus, retrovirus, and simian virus (e.g., the early and late promoters or SV40), as well as other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and their viruses or combinations thereof.

[0266] Also described herein are recombinant host cells comprising the expression vectors described above. The fusion proteins described herein can also be expressed in cell lines other than hybridomas. Nucleic acids comprising sequences encoding the polypeptides according to the invention can be used to transform suitable mammalian host cells.

[0267] Particularly preferred cell lines are selected based on high expression levels, constitutive expression of the protein of interest, and minimal contamination from host proteins. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines, such as HEK293 cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, etc. Additional suitable eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, E. coli, such as E. coli SG-936, E. coli HB101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis, Streptomyces.

[0268] These recombinant host cells of the invention can be used to produce fusion proteins by culturing the cells under conditions that allow expression of the polypeptide and purifying the polypeptide from the host cells or the medium surrounding the host cells. Targeting the expressed polypeptide for secretion in recombinant host cells is facilitated by inserting a sequence encoding a signal or secretory leader peptide at the 5' end of the gene encoding the antibody of interest (Shokri et al, (2003) Appl Microbiol Biotechnol. 60(6):654-664; Nielsen et al, Prot. Eng., 10:1-6(1997); von Heinje et al., Nucl. Acids Res., 14:4683-4690(1986), all of which are incorporated herein by reference). These secretory leader peptide elements can be derived from prokaryotic or eukaryotic sequences. Thus, suitably, a secretory leader peptide is used that is an amino acid attached to the N-terminus of the polypeptide and causes the polypeptide to move out of the cytoplasm of the host cell and be secreted into the medium.

[0269] The fusion proteins described herein can be fused with additional amino acid residues. Such amino acid residues can be, for example, peptide tags to facilitate isolation. Other amino acid residues for homing the antibody to a specific organ or tissue are also contemplated.

[0270] It will be appreciated that Fab nanocages can be generated by co-transfection of HC ferritin and LC. Alternatively, single chain Fab ferritin nanocages can be used, which require only transfection of one plasmid. This can be done using linkers of different lengths, for example 60 or 70 amino acids, between the LC and HC. Using a single chain Fab ensures that the heavy and light chains are paired. As mentioned before, tags (Flag, HA, myc, His6x, Strep, etc.) can also be added to the N-terminus of the construct or within the linker to facilitate purification. Furthermore, using a tag system, if different Fab nanoparticle plasmids are co-transfected, successive / additional affinity chromatography steps can be used to ensure that many different Fabs are present on the same nanoparticle. This confers multispecificity to the nanoparticles. If desired, a protease site (e.g., TEV, 3C, etc.) can be inserted to cleave the linker and tag after expression and / or purification.

[0271] Any suitable method or route can be used to administer the fusion proteins described herein, including, for example, oral, intravenous, intraperitoneal, subcutaneous, and intramuscular injection.

[0272] It is understood that when the fusion protein described herein is used in a mammal for prophylactic or therapeutic purposes, it is administered in the form of a composition further comprising a pharma- ceutically acceptable carrier. Suitable pharma- ceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. The pharma- ceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the binding protein. The composition of the injection may be formulated to provide quick, sustained, or delayed release of the active ingredient after administration to a mammal, as is well known in the art.

[0273] Although human antibodies are particularly useful for administration to humans, they can also be administered to other mammals. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, pets, farm animals, and the like.

[0274] In one aspect, methods are provided that may be useful for treating, alleviating, or preventing a SARS-CoV-2 associated condition, generally comprising administering to a subject a composition comprising a self-assembling polypeptide complex of the present disclosure.

[0275] "SARS-CoV-2 associated condition" refers to a condition (e.g., a symptom or sign) associated with SARS-CoV-2 infection. In some embodiments, the condition is a level of SARS-CoV-2 RNA, protein, or viral particles in a sample from a subject (e.g., a subject administered a self-assembling polypeptide complex disclosed herein), which is indicative of SARS-CoV-2 infection (e.g., because the level meets a threshold or exceeds a reference level indicative of SARS-CoV-2 infection). In some embodiments, the condition is a symptom associated with COVID-19 disease, such as fever, cough, fatigue, shortness of breath or difficulty breathing, muscle pain, chills, sore throat, runny nose, headache, chest pain, conjunctivitis, nausea, vomiting, diarrhea, loss of smell, loss of taste, or stroke. In some embodiments, the condition is associated with downstream sequelae of COVID-19 disease and / or is a symptom of long-term COVID-19 disease.

[0276] In some embodiments, the subject is a mammal, such as a human.

[0277] Compositions for administration to a subject generally comprise the self-assembling polypeptide complexes disclosed herein. In some embodiments, such compositions further comprise a pharma- ceutically acceptable excipient.

[0278] The compositions can be formulated for administration by any of a variety of routes, including systemic routes (eg, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration).

[0279] The above disclosure generally describes the present invention. A more complete understanding can be obtained by referring to the following specific examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention is not limited to the following examples, but should be construed to encompass any variations that become evident as a result of the teachings provided herein.

[0280] The following examples do not include detailed descriptions of conventional methods used to construct vectors or plasmids, insert genes encoding polypeptides into such vectors or plasmids, or introduce plasmids into host cells. Such methods are well known to those of skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, EF and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0281] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following examples specifically point out exemplary aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. EXAMPLES

[0282] Example 1: Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers This example describes the design, expression, purification, and characterization of a fusion protein with apoferritin. The apoferritin protomer self-assembles into an octahedral symmetric structure with a hydrodynamic radius (Rh) of approximately 6 nm, composed of 24 identical polypeptides. The N-terminus of each apoferritin subunit faces the outside of the spherical nanocage and is therefore accessible for genetic fusion of proteins of interest. The fusion protein is designed such that, when folded, the apoferritin protomer serves as a building block to promote multimerization of the 24 proteins fused to the apoferritin termini.

[0283] summary SARS-CoV-2, the causative virus of COVID-19, has caused a global pandemic. Antibodies can be powerful biological therapeutics to fight viral infections. Here, we use human apoferritin protomers as modular subunits to promote oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, we have achieved up to 10,000-fold increased potency compared to the corresponding IgG, resulting in a 9 × 10 -14 The half-maximal inhibitory concentration (IC 50 ) values ​​are achieved. Combining three different antibody specificities and fragment crystallizable (Fc) domains into a single multivalent molecule conferred the ability to overcome viral sequence variability along with superior potency and IgG-like bioavailability. Thus, the MULTi specific, multi-affinity antibody (Multabody or MB) platform uniquely combines binding avidity and multispecificity to provide ultra-potent and broad neutralizers against SARS-CoV-2. The modular nature of this platform also lends itself to rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are promising therapeutics against SARS-CoV-2.

[0284] Introduction The ongoing threat to public health from respiratory viruses, such as the novel SARS-CoV-2, highlights the urgent need to rapidly develop and deploy preventive and therapeutic interventions to combat pandemics. Monoclonal antibodies (mAbs) have been effectively used to treat infectious diseases, for example in high-risk infants. 1 Palivizumab for prophylaxis of respiratory syncytial virus in 2 Zmapp, mAb114, and REGN-EB3 for the treatment of SARS-CoV-2. As a result, mAbs targeting the spike (S) protein of SARS-CoV-2 have become a focus of the development of biomedical countermeasures against COVID-19. To date, several antibodies targeting the S protein have been identified. 3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19Bamlanivimab was the first antibody approved by the U.S. Food and Drug Administration (FDA) in November 2020 as an emergency treatment for SARS-CoV-2. 20 Receptor-binding domain (RBD)-directed mAbs that inhibit binding to angiotensin-converting enzyme 2 (ACE2), a marker of immune-mediated immune response, are typically associated with the highest neutralizing potency. 6,18,19 .

[0285] mAbs can be isolated by B cell sorting from infected donors, immunized animals, or by identifying binders in pre-organized libraries. Although these methodologies are robust and reliable for discovering virus-specific mAbs, identifying the best antibody clones usually comes with a time-cost penalty. In addition, RNA viruses have a higher mutation rate than DNA viruses, and such mutations can significantly alter the potency of neutralizing antibodies. Indeed, several studies have demonstrated a reduced neutralization potency by convalescent sera and the recent B.1.1.7 mutation in SARS-CoV-2. 24 , B.1.351 25 , and B.1.1.28 26、27 Specific mAbs against mutants 21、22、23 Resistance to HIV-1 has already been demonstrated. Thus, there is an unmet need for the development of platforms that bridge antibody discovery with the rapid identification and deployment of potent neutralizing agents that are less susceptible to viral sequence variation.

[0286] The potency of an antibody is largely influenced by its ability to interact with its epitope multiple times simultaneously. 28、29、30 This apparent increase in affinity is known as avidity and is the mechanism by which nanobodies against SARS-CoV-2 are 31、32 and Fab 8、10、16It has been previously reported that Multabody Fabs enhance the neutralization potency of IgG over IgG. To take full advantage of binding avidity, we developed an antibody scaffolding technology using human apoferritin protomers as modular subunits to multimerize antibody fragments and turn mAbs into ultra-potent neutralizers against SARS-CoV-2. Indeed, the resulting Multabody molecules can enhance potency by up to four orders of magnitude over the corresponding IgG. Furthermore, we demonstrated that this technology allows combining three different Fab specificities to more effectively overcome spike point mutations. Multabodies provide a versatile IgG-like "plug-and-play" platform to enhance the antiviral properties of mAbs against SARS-CoV-2, demonstrating the power of avidity as a mechanism to be exploited against viral pathogens.

[0287] Materials and Methods Protein expression and purification Genes encoding VHH-human apoferritin fusions, Fc fusions, Fab, IgG and RBD mutations were synthesized and cloned into the pcDNA3.4 expression vector by GeneArt (Life Technologies). All constructs were transfected at 0.8 × 10 cells / 200 mL of cells using FectoPRO (Polyplus Transfections) unless otherwise specified. 6Transient expression was performed at a density of 1000 cells / mL, in a 1:1 ratio. After 6–7 days of incubation in a Multitron Proshaker (Infors HT) at 37°C, 8% CO2, 70% humidity, and 125 rpm shaking, the cell suspension was harvested by centrifugation at 5000 × g for 15 min, and the supernatant was filtered through a 0.22 μm Steritop filter (EMD Millipore). Fab and IgG were transiently expressed by co-transfecting 90 μg of LC and HC in a 1:2 ratio, respectively, and purified using Kappa Select affinity columns (GE Healthcare) and HiTrapProteinAHP columns (GE Healthcare), using 100 mM glycine pH 2.2 as elution buffer. The eluted fractions were immediately neutralized with 1 M Tris-HCl, pH 9.0, and further purified using a Superdex200Increase size-exclusion column (GE Healthcare). Fc fusions of ACE2 and VHH-72 were purified in the same way as IgG. VHH-72 apoferritin fusions were purified by hydrophobic interaction chromatography using a HiTrapPhenylHP column and the eluted fraction was loaded onto a Superose 610 / 300GL size exclusion column (GE Heathcare) in 20 mM sodium phosphate pH 8.0, 150 mM NaCl. Wild-type (BEI NR52309) and mutant RBD, pre-fusion S extracellular domain (BEI NR52394) and Fc receptors (FcRn and FcγRI) from mouse and human were purified using a HisTrapNi-NTA column (GE Healthcare). After Ni-NTA purification, Superose6 for S trimers and Superdex200Increase size exclusion columns (GE Heathcare) for RBD and Fc receptors were used, in all cases in 20 mM phosphate pH 8.0, 150 mM NaCl buffer.

[0288] Multibody design, expression, and purification All molecules referred to herein as multibodies contain scFab and scFc fragments. The scFab and scFc polypeptide constructs are linked together via a flexible linker of 70 amino acids [(GGGGS) x14

[0043] were used to generate heterodimeric and homodimeric fragments, respectively. Specifically, the C-terminus of the Fab light chain was fused to the N-terminus of the Fab heavy chain via a linker. In the case of scFc, two single Fc chains were fused in tandem to form a functional homodimeric Fc. The individual domains were separated by a 25 amino acid linker (GGGGS) x5 The scFab and scFc fragments were fused to apoferritin monomers with 100% Fab fragments. Genes encoding scFab and scFc fragments linked to half apoferritin were generated by deletion of residues 1-90 (C-ferritin) and 91-175 (N-ferritin) of the light chain of human apoferritin. Transient transfection of Multabody in HEK293F cells was obtained by mixing 66 μg of plasmids scFab-human apoferritin:scFc-human N-ferritin:scFab-C-ferritin in a 2:1:1 ratio. The addition of scFab-human apoferritin allowed efficient Multabody assembly, increasing the number of Fabs compared to Fc in the final molecule and thus favoring Fab avidity over Fc avidity. For multispecific Multabodies, a 4:2:1:1 ratio of scFab1-human apoferritin:scFc-human N-ferritin:scFab2-C-ferritin:scFab3-C-ferritin was used. The DNA mixture was filtered and incubated in 66 μl of FectoPRO at room temperature (RT) before being added to the cell culture. Split Multabodies were purified by affinity chromatography using a HiTrap Protein A HP column (GE Healthcare) with 20 mM Tris pH 8.0, 3 M MgCl2, 10% glycerol elution buffer. Protein-containing fractions were concentrated and further purified by gel filtration on a Superose 6 10 / 300GL column (GE Healthcare).

[0289] Negative staining electron microscopy Three microliters of Multabody at a concentration of approximately 0.02 mg / mL was placed on the surface of a carbon-coated copper grid that had previously been glow-discharged in air for 15 seconds and allowed to adsorb for 30 seconds, then stained with 3 μL of 2% uranyl formate. Excess stain was immediately removed from the grid using Whatman No. 1 filter paper, and an additional 3 μL of 2% uranyl formate was added for 20 seconds. The grid was imaged using an FEI Tecnai T20 electron microscope operated at 200 kV and equipped with an Orius charge-coupled device (CCD) camera (Gatan Inc).

[0290] Biolayer Interferometry Direct binding kinetics measurements were performed using an Octet RED96 BLI system (SartoriusForteBio) in PBS pH 7.4, 0.01% BSA and 0.002% Tween® at 25 °C. His-tagged RBD, SARS-CoV-2 spike, was loaded onto a Ni-NTA (NTA) biosensor (SartoriusForteBio) to achieve a BLI signal response of 0.8 nm. Association kinetics was measured by transferring the loaded biosensor into wells containing a two-fold dilution series from 250 to 8 nM (Fab), 125 to 4 nM (IgG), 16 to 0.5 nM (MB). Dissociation kinetics was measured by immersing the biosensor into wells containing buffer. The duration of each step was 180 s. The Fc properties of the split Multabody design were evaluated by measuring binding to hFcγRI and hFcRn loaded onto Ni-NTA (NTA) biosensors according to the experimental conditions and concentration ranges described above. To investigate the theoretical ability of the Multabody to be recycled in endosomes, binding to the hFcRnβ2-microglobulin complex was measured at physiological pH (7.4) and endosomal pH (5.6). Similarly, the Fc properties of the mouse surrogate MB were evaluated by measuring binding to mFcγRI and mFcRn pre-immobilized on Ni-NTA (NTA) biosensors. Two-fold dilution series from 100 to 3 nM (IgG) and 10 to 0.3 nM (MB) were used. Analysis of the sensorgrams was performed using Octet software using a 1:1 fitting model. The competitive assay was performed in a two-step binding process. Ni-NTA biosensors pre-loaded with His-tag RBD were first immersed in wells containing 50 μg / mL of primary antibody for 180 s. After a 30 second baseline period, the sensors were immersed in wells containing 50 μg / ml of secondary antibody for an additional 300 seconds. All incubation procedures were carried out at 25° C. in PBS pH 7.4, 0.01% BSA, and 0.002% Tween®. ACE2-Fc was used to map mAb binding to the receptor binding site.

[0291] Dynamic Light Scattering The Rh of the Multabodies was determined by dynamic light scattering (DLS) using a DynaPro plate reader III (WyattTechnology). Approximately 20 μL of Multabodies at a concentration of 1 mg / mL was added to a 384-well black clear bottom plate (Corning) and measured for 5 seconds per reading at a fixed temperature of 25°C. Particle size determination and polydispersity were obtained by accumulating 5 readings using Dynamics software (WyattTechnology).

[0292] Coagulation temperature Aggregation temperature (T agg ) was determined using a UNit instrument (Unchained Labs). Samples were concentrated to 1.0 mg / mL and subjected to a thermal gradient from 25° C. to 95° C. in 1° C. increments. T agg was determined as the temperature at which a 50% increase in static light scattering at a wavelength of 266 nm was observed compared to the baseline (i.e., the maximum of the differential curve). The mean and standard error of two independent measurements were calculated using UNit analysis software.

[0293] Pharmacokinetics and immunogenicity In this study, a surrogate Multabody was used, consisting of scFab and scFc fragments of mouse HD37 (anti-hCD19) IgG2a fused to the N-terminus of the light chain of mouse apoferritin (mFerritin). HD37 scFab-mFerritin:Fc-mFerritin:mFerritin was transfected in a 2:1:1 ratio and purified according to the procedure described above. L234A, L235A, and P329G (LALAP) mutations were introduced into the mouse IgG2a Fc construct, and the Multabody 48The effector function of . In vivo studies were performed using 12-week-old male C57BL / 6 mice (strain code: 027) purchased from Charles River and housed in individually ventilated cages under a 12-h light / dark cycle (7 am / 7 pm) at temperatures of 21-23 °C and humidity of 40-55%. All procedures were approved by the Local Animal Care Committee of the University of Toronto Scarborough. Approximately 5 mg / kg of Multabody or control samples (HD37 single chain IgG-IgG1 or IgG2a subtypes) and a single injection of Helicobacter pylori ferritin (HpFerritin)-PfCSP malaria peptide in 200 μL PBS (pH 7.5) were injected subcutaneously. Blood samples were collected at multiple time points and serum samples were assessed for levels of circulating and anti-drug antibodies by ELISA. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were pre-coated with 0.5 μg / ml His 6x Tagged antigen hCD19 was coated in 50 μL and circulating HD37-specific concentrations were measured using IgG and reagent-specific calibration curves from Multibodies. HRP-Protein A (Invitrogen) was used to detect the levels of bound IgG / MB (dilution 1:10,000). For the measurement of anti-drug antibodies, NuncMaxiSorp plates (Biolegend) were coated with 12-mer HD37 scFab-mFerritin or HpFerritin-PfCSP malaria peptides. 1:100 serum dilutions were incubated for 1 h at room temperature and further developed using HRP-Protein A (Invitrogen) as secondary molecule (dilution 1:10,000). Chemiluminescence signal at 450 nm was quantified using a Synergy Neo2 multimode assay microplate reader (Biotek Instruments).

[0294] Biodistribution Eight-week-old male BALB / c mice were purchased from Jackson Laboratory and housed in individually ventilated cages. Mice were housed in a 14-h light / 10-h dark environment with graded light from dawn to dusk, with a temperature of 20–21°C and humidity of 40–60%, maximizing at noon. All procedures were approved by the Local Animal Care Committee of the University of Toronto. This study used a Multabody composed of scFab and scFc fragments of mouse HD37 IgG2a fused to the N-terminus of mouse apoferritin light chain. HD37 IgG2a Multabody or control sample (HD37 single-chain IgG2a) was fluorescently conjugated with Alexa-647 using the Alexa Fluor™ 647 Antibody Labeling Kit (Invitrogen) according to the manufacturer's instructions. Alexa Fluor™ 647-labeled 15-nm gold nanoparticles were purchased from Creative Diagnostics (GFLV-15). For noninvasive biodistribution experiments, a Perkin Elmer IVIS Spectrum (Perkin Elmer) was used. BALB / c mice were subcutaneously injected with approximately 5 mg / kg of MB, HD37IgG2a, or gold nanoparticles in 200 μL of PBS (pH 7.5) into the loose skin of the shoulders and imaged at 0, 1 h, 6 h, 24 h, 2, 3, 4, 8, and 11 days after injection. Before imaging, mice were placed in an anesthesia induction chamber containing a mixture of isoflurane and oxygen for 1 min. Anesthetized mice were then placed in a prone position in the center of an integrated heated docking system (maintained at 37 °C and supplied with a mixture of isoflurane and oxygen) within the IVIS imaging system. For whole-body 2D imaging, mice were imaged for 1–2 s (excitation 640 nm, emission 680 nm) within the imaging system. Data were analyzed using IVIS software (LivingImageSoftware for IVIS). After confirming the fluorescence signal from the 2D epi-illumination images, 3D transillumination fluorescence imaging tomography (FLIT) was performed on the region of interest using an integral scan field of 3x3 or 3x4 transillumination positions.A series of 2D fluorescence surface brightness images were taken at various transmission positions using 640 nm and 680 nm emission excitation. A series of CT scans were also taken at the corresponding positions. The fluorescence signal and the CT scan were combined to reconstruct a 3D distribution map of the fluorescence signal. The resulting 3D fluorescence images were thresholded based on the 3D images of PBS-injected mice taken at the corresponding body positions. The images were mapped into a rainbow LUT in the IVIS software, and the upper limit of the color scale was set to 50 pmol M for mice injected with gold nanoparticles. -1 cm -1 1000 pmol M -1 cm -1 to allow for better visualization of biodistribution over time. The Mouse Organ Registration feature in the IVIS software was used as a general guideline to assess the body position of the samples from the 3D images.

[0295] Panning of phage libraries against the RBD of SARS-CoV-2 A commercially available SuperHuman2.0 phage library (DistributedBio / CharlesRiverLaboratories) was used to identify monoclonal antibody binders against the SARS-CoV-2 RBD. For this purpose, the RBD-Fc-Avi tagged construct of SARS-CoV-2 was expressed in the EXPi-293 mammalian expression system. The protein was then purified by Protein G Dynabeads, biotinylated, and quality controlled for biotinylation and binding to ACE2 recombinant protein (SinoBiologicsInc). The SuperHuman2.0 phage library (5x10 12) were heated to 72°C for 10 min and deselected against ProteinG Dynabeads™ (Invitrogen), M-280 Streptavidin Dynabeads™ (Invitrogen), calf thymus histones (Sigma), human IgG (Sigma), ssDNA-biotin NNK from Integrated DNA Technologies, and DNA-biotin NNK from Integrated DNA Technologies. The library was then panned against the RBD captured on M-280 Streptavidin Dynabeads™ using an automated protocol on KingfisherFLEX (Thermofisher). Selected phages were acid eluted from the beads and neutralized using Tris-HCl pH 7.9 (Teknova). ER2738 cells were cultured at OD 600 After incubating at 100 rpm at 37°C for 40 min, the phage pool was centrifuged and incubated overnight at 30°C on antibiotic selection agar. Rescued phages were precipitated with PEG and subjected to three further rounds of soluble phase automated panning. Deselection, washing, and selection rounds used PBST / 1% BSA buffer and / or PBS / 1% BSA.

[0296] Screening of anti-SARS-CoV-2 scFv in bacterial PPE using SARS-CoV-2 RBD Anti-SARS-CoV-2 RBD scFvs selected from phage display were expressed and screened using high-throughput surface plasmon resonance (SPR) on a Carterra LSA array SPR instrument (Carterra) equipped with a HC200M sensor chip (Carterra) at 25 °C. A V5 epitope tag was added to the scFv to enable capture via immobilized anti-V5 antibody (Abcam, Cambridge, MA) that was pre-immobilized to the chip surface by standard amine coupling. Briefly, the chip surface was first activated by injecting a 1:1:1 (v / v / v) mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 0.1 M N-hydroxysulfosuccinimide (sNHS), and 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5 for 10 min. Next, 50 μg / ml of anti-V5 tag antibody prepared in 10 mM sodium acetate pH 4.3 was allowed to bind for 14 min and excess reactive esters were blocked with 1 M ethanolamine HCl pH 8.5 during a 10 min injection. For screening, a 384-ligand array was prepared consisting of crude bacterial periplasmic extracts (PPE) containing scFvs (one spot per scFv). Each extract was prepared at 2-fold dilutions in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.01% (v / v) Tween®-20 (HBSTE)) and printed onto the anti-V5 surface for 15 min. SARS-CoV-2 RBDAviTevHis-tagged was then prepared at 0, 3.7, 11.1, 33.3, 100, 37, and 300 nM in 10 mM HEPES pH 7.4, 150 mM NaCl, and 0.01% (v / v) Tween®-20 (HBST) supplemented with 0.5 mg / ml BSA and injected as analyte for 5 min with a dissociation time of 15 min. Samples were injected at increasing concentrations without a regeneration step. Binding data from a local reference spot was used to subtract the signal from the active spot, and data were double-referenced by subtracting the nearest buffer blank analyte response.The double-referencing data were fitted to a simple 1:1 Langmuir binding model in Carterra's KineticInspectionTool (October 2019 version). In this study, 20 medium affinity binders were selected from a phage display screen.

[0297] Pseudovirus production and neutralization A SARS-CoV-2 pseudovirus (PsV) was developed using an HIV-based lentivirus system with some modifications. 49 Briefly, 293T cells were co-transfected with a lentiviral backbone encoding a luciferase reporter gene (BEI NR52516), a plasmid expressing spike (BEI NR52310), and plasmids encoding the HIV structural and regulatory proteins Tat (BEI NR52518), Gag-pol (BEI NR52517), and Rev (BEI NR52519) using BioT transfection reagent (Bioland Scientific) according to the manufacturer's instructions. After 24 h of transfection at 37 °C, 5 mM sodium butyrate was added to the medium and the cells were incubated at 30 °C for an additional 24–30 h. The SARS-CoV-2 spike mutant D614G was provided by D.R. Burton (The Scripps Research Institute), the SARS-CoV-2 PsV mutant B.1.351 was provided by D.D. Ho (Columbia University), and the remaining PsV mutants were generated by the KOD-Plus mutagenicity kit (Toyobo, Osaka, Japan) using the primers listed in Table 1. PsV particles were harvested, passed through a 0.45 μm pore sterile filter, and finally concentrated using a 100K Amicon (Merck Millipore Amicon-Ultra 2.0 centrifugal filter unit). [Table 1]

[0298] Neutralization was determined in a single cycle neutralization assay using 293T-ACE2 cells (BEI NR52511) and HeLa-ACE2 cells (DR Burton; provided by The Scripps Research Institute). Cells were seeded at a density of 10,000 cells / well in a volume of 100 μl the day before the experiment. For 293T cells, plates were pre-coated with poly-L-lysine (Sigma-Aldrich). On the day of the experiment, 50 μl of serially diluted IgG and MB samples were incubated with 50 μl of PsV for 1 h at 37°C. After 1 h of incubation, the incubation volume was added to the cells and incubated for 48 h. PsV neutralization was monitored by adding 50 μl of Britelite plus reagent (PerkinElmer) to 50 μl of cells and incubating for 2 min before transferring the volume to a 96-well white plate (Sigma-Aldrich) and measuring luminescence in relative light units (RLU) using a Synergy Neo2 multimode assay microplate reader (Biotek Instruments). Two to three biological replicates and two technical replicates each were performed. IC 50 The fold increase was calculated as follows: IgG IC 50 (μg / mL) / MB IC 50 (μg / mL).

[0299] Genuine virus neutralization VeroE6 cells were seeded in 96F plates at a concentration of 30,000 / well in DMEM supplemented with 100U penicillin, 100U streptomycin, and 10% FBS. Cells were allowed to attach to the plates and left overnight. After 24 hours, 5-fold serial dilutions of IgG and MB samples were prepared in quadruplicate (25 μL / well) in 96R plates in DMEM supplemented with 100U penicillin and 100U streptomycin. Approximately 25 μL of SARS-CoV-2 / SB2-P4-PB 50Clone 1 was added at 100 TCID / well to each well and incubated at 37°C for 1 h with shaking every 15 min. After co-culture, medium was removed from the VeroE6 plates and 50 μL of antibody-virus sample was used to inoculate VeroE6 cells in quadruplicate for 1 h at 37°C, 5% CO2 with shaking every 15 min. One hour after inoculation, the inoculum was removed and 200 μL of fresh DMEM supplemented with 100 U penicillin, 100 U streptomycin, and 2% FBS was added to each well. Plates were incubated for an additional 5 days. Cytopathic effect (CPE) was monitored and IC was determined using PRISM. 50 Values ​​were calculated. Three biological replicates and four technical replicates each were performed.

[0300] Cross-linking of spike protein with Fab80, 298, and 324 Approximately 100 μg of spike trimer was mixed with a 2-fold molar excess of Fab80, 298, or 324 in 20 mM HEPES pH 7.0 and 150 mM NaCl. Proteins were cross-linked by adding 0.075% (v / v) glutaraldehyde (SigmaAldrich) and incubated for 120 min at room temperature. Complexes were purified by size-exclusion chromatography (Superose6Increase10 / 300GL, GE Healthcare), concentrated to 0.5 mg / mL, and used directly for the preparation of Cryo-EM grids.

[0301] Crosslinking of Fab46-RBD complex Approximately 100 μg of Fab46 was mixed with a 2-fold molar excess of RBD in 20 mM HEPES pH 7.0 and 150 mM NaCl. The complex was cross-linked by adding 0.05% (v / v) glutaraldehyde (SigmaAldrich) and incubated at room temperature for 45 min. The cross-linked complex was purified by size-exclusion chromatography (Superdex200Increase10 / 300GL, GE Healthcare), concentrated to 2.0 mg / ml, and used directly for the preparation of Cryo-EM grids.

[0302] Cryo-EM data collection and image processing Three microliters of sample was placed on a holey gold grid prepared in-house. 51 The samples were glow discharged in air for 15 seconds using a PELCO easeGlow (Ted Pella) before use. Samples were blotted for 6 seconds in a modified FEI Mark III Vitrobot (maintained at 4°C and 100% humidity) using an offset of -5, and then plunge frozen in a mixture of liquid ethane and propane. Data were acquired at 300 kV at 250 frames / s in electron counting mode using a Thermo Fisher Scientific Titan Krios G3 electron microscope and a prototype Falcon 4 camera. Movies were recorded in 29 exposure splits, with a camera exposure speed of approximately 5e - / pix / sec, and the total exposure of the sample is approximately 44e - / Å 2 The images were collected for 9.6 seconds at 100 nm. No objective aperture was used. The pixel size was adjusted to 1.03 Å / pixel from a gold diffraction standard. The microscope was automated with the EPU software package and data collection was performed using the cryoSPARC Live 52 was monitored.

[0303] Gradient data collection was employed to overcome the preferred orientation that occurred in some samples. 5 3. For the spike-Fab80 complex, 820 0° tilt videos and 2790 40° tilt videos were collected. For the spike-Fab298 complex, 4259 0° tilt videos and 3513 40° tilt videos were collected. For the spike-Fab324 complex, 1098 0° tilt videos and 3380 40° tilt videos were collected. For the RBD-Fab46 complex, 4722 0° tilt videos were collected. For the 0° tilt videos, cryoSPARC patch motion correction was performed. For the 40° tilt videos, RelionMotionCorr 54,55Micrographs were then imported into cryoSPARC and patch CTF estimation was performed. Templates generated from 2D classification during a cryoSPARC Live session were used for template selection of particles. Unnecessary particle images were removed using 2D classification, resulting in a dataset of 80,951 particle images of spike-Fab80 complexes, 203,138 particle images of spike-Fab298 complexes, 64,365 particle images of spike-Fab324 complexes, and 2,143,629 particle images of RBD-Fab46 complexes. Particle image stacks were cleaned using multiple rounds of multiclass abinitio refinement, and a consensus structure was obtained using homogeneous refinement. For tilted particles, particle polishing was performed within Relion at this stage and re-imported into cryoSPARC. Extensive flexibility was observed for the spike-Fab complexes. 3D fluctuation analysis was performed. 56 ,In combination with heterogeneous refinement, it was used to classify the various states present.,Then, non-uniform refinement was performed on the final set of particle,images. 57 For the RBD-Fab46 complex, three-class cryoSPARC ab initio refinement was used iteratively to clean up the particle image stack. The Euler angle refined particle image stack was then brought into the cisTEM for reconstruction. 58 A 4.0-Å resolution map was generated. Data transfer between Relion and cryoSPARC was performed using pyem. 59 .

[0304] Crystallization and structure determination Ternary complexes of 52Fab-298Fab-RBD were obtained by mixing 200 μg of RBD with a 2-fold molar excess of each Fab in 20 mM Tris pH 8.0, 150 mM NaCl, followed by purification by size exclusion chromatography (Superdex200 Increase10 / 300GL, GE Healthcare). The fractions containing the complex were concentrated to 7.3 mg / ml and mixed in a 1:1 ratio with 20% (w / v) 2-propanol, 20% (w / v) PEG4000, and 0.1 M sodium citrate pH 5.6. Crystals appeared after about 1 day and were cryoprotected with 10% (v / v) ethylene glycol, followed by flash freezing in liquid nitrogen.

[0305] The data were collected at the 23-ID-D beamline of the Argonne National Laboratory Advanced Synchrotron Radiation Facility. The data set is available under the XDS 60 and XPREP. Phase analysis was performed using Phaser 61 The structures were determined by molecular replacement using phenix.refine. CNTO88Fab was used as a model for 52Fab (PDBID:4DN3), 20358Fab was used as a model for 298Fab (PDBID:5CZX), and PDBID:6XDG was used as a search model for RBD. 62 and Coot 63 The structure was analyzed and the diagrams were rendered using PyMOL. 64 All software access is through SBGrid 65 Representative electron densities for the two Fab-RBD interfaces are shown in Fig. 2e,f.

[0306] Obtaining materials Electron microscopy maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-22738, EMD-22739, EMD-22740, and EMD-22741 (Table 2). The crystal structure of the 298-52-RBD complex (Table 3) is available in the Protein Data Bank under accession number PDBID:7K9Z. The sequences of the monoclonal antibodies used are provided herewith (Table 4). Additional PDB / EMDB entries were used throughout the manuscript to perform a comparative analysis of the various epitope bins targeted by the mAbs. The entries used in this analysis were REGN10933 (PDBID: 6XDG), CV30 (PDBID: 6XE1), C105 (PDBID: 6XCM), COVA2-04 (PDBID: 7JMO), COVA2-39 (PDBID: 7JMP), CC12.1 (PDBID: 6XC2), BD23 (PDBID: 7BYR), B38 (PDBID: 7BZ5), P2C-1F11 (PDBID: 7BWJ), 2-4 (PDBID: 6XEY), CB6 (PDBID: 7C01), REGN10987 (PDBID: 6XDG), S309 (PDBID: 6WPS, 6WPT), EY6A (PDBID: 6ZCZ), CR3022 (PDBID: 6YLA), H014 (PDBID: 7CAH), 4-8 (EMDBID: 22159, 4A8 (PDBID: 7C2L), and 2-43 (EMDBID: 22275). [Table 2] [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

[0307] result Avidity enhances neutralizing efficacy We exploited the self-assembly of the light chain of human apoferritin to multimerize the antigen-binding moiety that targets the SARS-CoV-2 S glycoprotein. The apoferritin protomer self-assembles into an octahedral symmetric structure with a hydrodynamic radius (Rh) of approximately 6 nm, composed of 24 identical polypeptides. 33 The N-terminus of each apoferritin subunit faces the outside of the spherical nanocage and is therefore accessible for genetic fusion of proteins of interest. Once folded, the apoferritin protomer serves as a building block to promote multimerization of 24 proteins fused to its N-terminus (Figure 1a).

[0308] First, we investigated the impact of multivalency on the ability of the single variable domain VHH-72 to block viral infection, which has previously been reported to neutralize SARS-CoV-2 when fused to an Fc domain, but not in a monovalent format. 31 The light chain of human apoferritin displaying 24 copies of VHH-72 organized into monodisperse, well-ordered spherical particles (Fig. 1b, c) and showed enhanced binding avidity to the S glycoprotein compared to bivalent VHH-72-Fc (Fig. 1d). Strikingly, displaying VHH-72 on the light chain of human apoferritin achieved a ∼10,000-fold increased neutralizing potency against SARS-CoV-2 pseudovirus (PsV) compared to a conventional Fc fusion (Fig. 1e), demonstrating the power of avidity that transforms a binding moiety into a potent neutralizer.

[0309] Multibodies have IgG-like properties Fc confers in vivo half-life and effector functions to IgG through interactions with the neonatal Fc receptor (FcRn) and Fc gamma receptor (FcγR). To confer these IgG-like properties to the multimeric scaffold, we next explored incorporating both binding moieties and Fc domains. Because Fab is a heterodimer consisting of a light and heavy chain, and Fc is a homodimer, we created single-chain Fab (scFab) and single-chain Fc (scFc) polypeptide structures. The scFab and scFc domains were fused directly to the N-terminus of an apoferritin protomer. For in vivo proof-of-principle experiments, we generated species-matched surrogate molecules composed of mouse light chain apoferritin fusions to mouse scFab and mouse scFc (IgG2a subtype). Binding kinetics showed that the resulting MB molecules, similar to the parent IgG, bound to mouse FcRn in a pH-dependent manner—at endosomal pH (5.6) but not at physiological pH (7.4) (Figure 3a). As expected, binding to the high affinity mouse FcγR1 was enhanced by an avidity effect compared to the parental IgG. Therefore, to reduce Fc binding in a multimeric context, we generated a modified mouse scFc version containing the FcγR-silencing mutation LALAP (Figure 3a). Subcutaneous administration of MB to C57BL / 6 or BALB / c mice was well tolerated, without weight loss or visible adverse events. MB showed a good IgG-like serum half-life (Figure 3b), with prolonged detectable titers in serum for the low FcγR-binding MB (LALAPFc sequence) compared to WTMB, indicating that the Fc plays a role in determining bioavailability in vivo. Live 2D and 3D imaging revealed that fluorescently labeled MB biodistributed throughout the body and did not accumulate in specific tissues, similar to the corresponding IgG (Figure 3c and Figure 4). In contrast, 15 nm gold nanoparticles (GNPs), with an Rh similar to that of MB, rapidly diffused away from the injection site (Figure 3c and Figure 4). Presumably because all sequences are host-derived, the surrogate mouse MB did not elicit an anti-drug antibody response in mice (Figure 3d), thus further highlighting the IgG-like properties of the MB platform.

[0310] Protein engineering to achieve higher valency Given these favorable results with mouse MB surrogates, we have developed a previously reported IgGBD23 that targets the SARS-CoV-2 spike RBD and N-terminal domain (NTD), respectively. 12 and IgG4A8 13 We aimed to generate a fully human MB derived from . Addition of scFc to MB reduces the number of scFabs that can multimerize. To confer Fc to the MB platform without compromising Fab avidity and therefore neutralization potency, the apoferritin protomer was modified to accommodate more than 24 components per particle. Based on the folding of a four-helical bundle, the human apoferritin protomer was split into two halves, namely two N-terminal α-helices (N-ferritin) and two C-terminal α-helices (C-ferritin). In this configuration, an scFc fragment of human IgG1 and an scFab of anti-SARS-CoV-2 IgG were genetically fused at the N-terminus of each apoferritin half, respectively. Complementation of the split apoferritin led to heterodimerization of the two halves, resulting in a highly efficient heterodimerization process of the fusion protein. Excessive co-expression of the scFab-C-ferritin and scFc-N-ferritin genes with the scFab-ferritin gene resulted in complete apoferritin self-assembly, displaying a high number of scFabs and a low number of scFcs at the periphery of the nanocage (Figure 5a and Materials and Methods). Conveniently, this design allows for easy purification of MBs using protein A, similar to IgG purification.

[0311] This split MB design forms 16 nm Rh spherical particles with a continuous ring of density and regularly spaced protruding scFab and scFc (Figure 5b, c). Thus, the MBs are capable of binding to natural IgM. 34Although in the smaller size range of , they pack more weight in a similar size, achieving high multivalency. Binding kinetics experiments demonstrated that the high binding avidity of MBs to the spike was maintained upon addition of Fc fragments (Fig. 5d and Table 5). Binding to human FcγRI and FcRn at both pH 5.6 and 7.4 confirmed that the scFc was properly folded in the split-MB design (Tables 6 and 7). Furthermore, the LALAP mutation in the scFc reduced the binding affinity to human FcγRI (Fig. 5e), as previously observed with surrogate mouse MBs (Fig. 3a). SARS-CoV-2 PsV neutralization assays with split-design MBs showed that the improved binding affinity to the spike translated into improved neutralization potency compared to the IgG counterpart, with an increase of approximately 1600-fold and over 2000-fold for BD23 and 4A8, respectively (Fig. 5f). Combined, these data support further investigation that MB is an IgG platform that confers superior binding avidity and PsV neutralization across a range of epitopes on the spike domain. [Table 5] [Table 6] [Table 7]

[0312] Fc mutations in the IgG1 backbone that have been evaluated in multibodies include LALAP (L234A, L235A, P329G) and I235A, as well as combinations thereof that reduce antibody binding to FcγRs. (Numbering follows the EU numbering scheme.)

[0313] k on , k off , and the equilibrium dissociation constant of the obtained multibody (K DThe determined values ​​for β-terminal ... [Table 8] [Table 9] [Table 10]

[0314] From antibody discovery to super-powerful neutralizers We next assessed the ability of the MB platform to convert mAb binders identified from the initial phage display screen into potent neutralizing agents against SARS-CoV-2 (Figure 6a). -6 M~10 -8 Twenty human mAb binders with moderate affinities in the range of M were selected (Table 4, Table 11). These mAbs were produced as full-length IgG and MB and compared for their ability to block viral infection in a neutralization assay against SARS-CoV-2 PsV (Figure 6b and Figure 7a). Notably, the expression yield, homogeneity, and thermostability of MB were similar to those of the parental IgG (Figure 8 and Table 12), and MB improved the potency of 18 out of 20 (90%) IgGs by up to four orders of magnitude (Table 13). The largest increase was observed for mAb298, with a mean IC 50The IC values ​​ranged from approximately 0.3 μg / mL as IgG to 0.0001 μg / mL as MB. Surprisingly, in the concentration range tested, 11 mAbs were converted from non-neutralizing IgG to neutralizing MB. Seven MBs showed IC values ​​of 0.2–2 ng / mL against SARS-CoV-2 PsV using two different target cells (293T-ACE2 cells and HeLa-ACE2 cells, Figures 6b and 7b). 50 PsV neutralization assays using the benchmark recombinant mAbs REGN10933 and REGN10987 showed IC values ​​similar to those previously reported. 50 values ​​(0.0044 and 0.030 μg / mL, respectively). 8 This confirmed the exceptional potency of MB observed in the assay. The enhanced neutralization potency of MB was further confirmed with the most potent mAbs using authentic SARS-CoV-2 virus (Figures 6c and 7c) and also benchmarked with two recombinant REGN mAbs. The less sensitive neutralization phenotype observed against authentic virus compared to PsV is also consistent with previous reports. 5、6、9、12 It matches. [Table 11] [Table 12] [Table 13-1] [Table 13-2]

[0315] Retrospectively, all IgGs and MBs were tested for their ability to bind to the spike glycoprotein and RBD of SARS-CoV-2 (Figure 9). No detectable off-rates for the spike glycoprotein were observed due to increased avidity, and the apparent binding affinity was increased, likely due to the cross-linking between spikes resulting in higher neutralization potency (Figures 6b-d and Figure 9). Overall, the data demonstrate the compatibility of the MB platform to rapidly deliver ultrapotent IgG-like molecules, even starting with mAbs with moderate neutralizing properties.

[0316] Epitope mapping Based on neutralization potency, seven mAbs were selected for further characterization: 298 (IGHV1-46 / IGKV4-1), 82 (IGHV1-46 / IGKV1-39), 46 (IGHV3-23 / IGKV1-39), 324 (IGHV1-69 / IGKV1-39), 236 (IGHV1-69 / IGKV2-28), 52 (IGHV1-69 / IGKV1-39), and 80 (IGHV1-69 / IGKV4-1) (Figure 6b and Table 4). Epitope binning experiments showed that these mAbs targeted two major sites on the RBD, one of which overlapped with the ACE2 binding site (Figure 10a and Figure 11). Cryo-EM structures of Fab-SARS-CoV-2S complexes at approximately 6-7 Å global resolution confirmed that mAbs 324, 298, and 80 bind overlapping epitopes (Figure 10b, Figures 12a-c, and Table 2). To gain insight into the binding of mAbs targeting other bins, the cryo-EM structure of Fab 46 in complex with the RBD was obtained at 4.0 Å global resolution (Figure 10c, Figure 12d, and Table 2), and crystal structures of Fabs 298 and 52 in ternary complexes with the RBD were obtained at 2.95 Å resolution (Figure 10d, Figure 2, and Table 3).

[0317] The crystal structure shows that Fab298 binds almost exclusively to the ACE2 receptor-binding motif (RBM) of the RBD (residues 438-506). Indeed, 12 of the 16 RBD residues involved in Fab298 binding are also involved in ACE2-RBD binding (Figure 2a-c and Table 14). The RBM is stabilized by 11 hydrogen bonds from heavy and light chain residues of Fab298. Furthermore, RBMPhe486 is located approximately 170 Å away from the RBM. 2 It contacts 11 buried Fab298 residues (24% of the total buried surface area on the RBD) and is therefore central to antibody-antigen interactions (Figure 2a and Table 14).

[0318] Detailed analysis of the RBD-52Fab interface revealed that the epitope of mAb52 is shifted towards the core of the RBD, including 20 residues of the RBM and 7 residues of the core domain (Fig. 10c, Fig. 2b, and Table 14). Consistent with the competition data, antibody 52 and antibody 46 share a similar binding site but approach the RBD at slightly different angles (Fig. 10c, d, and Fig. 2d). Examination of previously reported structures of RBD-antibody complexes revealed that antibodies 46 and 52 target previously undescribed vulnerable sites on the SARS-CoV-2 spike (Fig. 10e). The epitopes targeted by these antibodies are partially occluded by the NTD in the “closed” conformation of the S form, suggesting that the mechanism of action of this class of antibodies may involve spike destabilization. Collectively, these data demonstrate that the avidity-driven enhanced neutralization potency observed with the MB platform is associated with mAbs that can target distinct epitope bins on the RBD. [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] vdW: van der Waals interactions (5.0 Å cutoff) HB: Hydrogen bond (3.8 Å cutoff) SB: salt bridge (4.0 Å cutoff)

[0319] Multibodies overcome spike sequence variability To examine whether MB can increase binding avidity and resist viral escape, we assessed four naturally occurring RBD mutations on the binding and neutralization of the seven most potent human mAbs. 35 The seven human mAbs tested the effects of L452R (bin 1), which is located within the epitopes of antibodies 46 and 52, A475V and V483A (bin 2), which are located within the ACE2 binding site, and the circulating RBD mutant N439K. 36 In addition, we also evaluated the effect of mutating the N-linked glycosylation site Asn234 to Gln, which is consistent with the lack of glycosylation at this site in the RBD. 35 This is because it has previously been reported that the more infectious PsV variant D614G is less susceptible to neutralizing antibodies targeting the 37were also included in the panel. As expected, mutant L452R significantly reduced binding and potency of mAbs 52 and 46, whereas antibody 298 was sensitive to mutant A475V (Fig. 13b, c). Deletion of the N-linked glycan at position Asn234 increased viral resistance to most antibodies, especially mAbs 46, 80, and 324, highlighting the importance of glycans in viral antigenicity (Fig. 13c). Surprisingly, the following antibody specificities in the MB format were minimally affected by the S mutations in their exceptional neutralizing potency: 298, 80, 324, and 236 (Fig. 13d). Mutant L452R reduced the sensitivity of 46-MB and 52-MB, but maintained neutralizing activity against this PsV mutant in contrast to their parental IgG (Fig. 13d). The more infectious SARS-CoV-2 PsV mutant D614G was neutralized with similar potency as WT PsV by both IgG and MB (Figures ​(Figure13c and14a).

[0320] The MB cocktail consisting of three monospecific MBs resulted in pan-neutralization across all PsV variants without a significant loss of potency, thus achieving 100-1000-fold higher potency compared to the corresponding IgG cocktail (Fig. 13e and Fig. 14c, d). To achieve breadth within a single molecule, a trispecific MB was generated by combining multimerized subunits displaying three different Fabs within the same MB assembly (Fig. 14b). Remarkably, the resulting trispecific MB showed pan-neutralization while maintaining the exceptional neutralization potency of the monospecific version, including the B.1.351 PsV variant (Fig. 13e, f and Fig. 14c, d). The highest potency was observed with the 298-324-46 combination (Fig. 14c, e), with the trispecific MB achieving exceptional potency, exceeding that observed with some of the most potent IgGs reported so far, and those recombinantly generated from available sequences (Fig. 13g). Furthermore, the MB format was able to further enhance the previously reported potency of these highly potent IgGs against PsV and live-replicating SARS-CoV-2 virus by an additional 1–2 orders of magnitude (Figure ​(Figure13h), thus highlighting the plug-and-play nature of MBs and their ability for multivalency to enhance the neutralizing capacity of mAbs across a range of potencies.

[0321] Median Neutralization IC 50 The values ​​determined are summarized in Tables 13 and 15. [Table 15]

[0322] Consideration In this study, we reveal how binding avidity can be exploited as an effective mechanism to promote antibody neutralization potency and resistance to viral mutations. To this end, we have developed a plug-and-play antibody multimerization platform that utilizes protein engineering to increase the avidity of mAbs targeting SARS-CoV-2. The seven most potent MBs ranged in concentration from 0.2 to 2 ng / mL (9 × 10 -14 ~9×10 -13IC of M against SARS-CoV-2 PsV 50 value and are therefore, to the best of our knowledge, within the range of the most potent antibody-like molecules reported so far against SARS-CoV-2.

[0323] The MB platform is designed to contain important advantageous attributes from a developability perspective. First, the ability to enhance antibody potency is independent of the antibody sequence, format, or epitope targeted. The modularity and flexibility of the platform was demonstrated by enhancing the potency of VHH and multiple Fabs targeting non-overlapping regions on the two SARS-CoV-2S subdomains (RBD and NTD). Using MBs to enhance the potency of VHH domains may bring special value to this class of molecules, as their small size allows for very efficient multimerization. Second, the ability to enhance the potency of single-chain variable fragments is independent of the antibody sequence, format, or epitope targeted. 38、39 In contrast to other approaches that increase avidity through the tandem fusion of streptavidin, MBs do not suffer from low stability and, in fact, self-assemble into highly stable particles with an aggregation temperature similar to that of the parent IgG. 40 , Verotoxin B subunit scaffold 41 , or virus-like nanoparticles 42Alternative multimerization strategies such as , face immunogenicity challenges and / or reduced bioavailability due to the absence of Fc fragments and therefore the inability to undergo FcRn-mediated recycling. The light chain of apoferritin is fully human, biologically inactive, and engineered to contain an Fc domain, with an Rh similar to IgM, despite the multimerization of more than 24 Fab / Fc fragments. Therefore, surrogate mouse MBs did not elicit anti-drug antibodies in mice and were detected in serum for more than a week, similar to parental mouse IgG. However, the in vivo bioavailability of MBs is dependent on their binding affinity to FcγR, suggesting that Fc avidity must be carefully fine-tuned to efficiently translate MBs into clinical practice. Furthermore, further studies will be required to assess how MBs are distributed in anatomical sites of interest, such as the lungs in the case of SARS-CoV-2 infection. The plug-and-play nature of multibodies also lends itself to exploring alternative half-life extending moieties other than the Fc (e.g., human serum albumin) when bioavailability is the only desired property without effector function. 43 , or human serum albumin 44、45 (the binding moiety that binds to the

[0324] Differential increases in neutralization potency were observed with different mAb sequences tested with MB against SARS-CoV-2. This suggests that the ability of MB to increase potency may depend on the location of the epitope on the spike, or the shape of how the Fab binds the antigen to achieve neutralization. The fact that neutralization of two of the 20 SARS-CoV-2 RBD binders was not rescued by the MB platform suggests limitations based on mAb sequence and binding properties alone. Nevertheless, the ability of MB to translate avidity into neutralization potency across a range of epitope specificities on the SARS-CoV-2 spike highlights the potential for broad use of this technology. HIV-1 46It will be interesting to explore the potency-enhancing capabilities of the MB platform against viruses with low surface spike density, such as HIV-1, or other targets, such as the tumor necrosis factor receptor superfamily, where the bivalency of conventional antibodies limits efficient activation. 47 .

[0325] Viral escape can occur in response to therapeutic selective pressure or natural selection. The conventional approach to combat escape mutants is to use antibody cocktails targeting different epitopes. MB showed reduced sensitivity to S mutations compared to the parent IgG, presumably because the reduced affinity was compensated for by enhanced binding affinity. Thus, when used in a cocktail, MB overcame viral sequence variability with exceptional potency. Furthermore, the split-MB design allows combining multiple antibody specificities within a single multimeric molecule, achieving similar potency and breadth as the MB cocktail. Importantly, several mAbs 21、22、23 There is a B.1.351 mutant of interest that is able to escape neutralization by the trispecific Multabody, which is neutralized with high potency by the trispecific Multabody, thus further highlighting the ability of these molecules to resist viral escape. Multispecificity within the same particle may bring additional benefits, such as intra-S avidity and synergistic effects of appropriate combinations of mAbs, providing a basis for further investigation of different combinations of mAb specificity on MBs. It is also possible to exploit avidity and multispecificity to deliver a single molecule that potently neutralizes an entire viral genus.

[0326] Overall, the MB platform provides tools to overcome the limitations of antibody affinity and generate broadly potent neutralizing molecules while avoiding extensive antibody discovery and engineering efforts. This platform is an example of how binding avidity can be leveraged to accelerate time to discovery of the most potent biologics against infectious diseases of global health importance.

[0327] Example 2 summary SARS-CoV-2, the causative agent of COVID-19, has caused a global pandemic. Monoclonal antibodies have been used as antiviral therapeutics, but efficacy has been limited by the diversity of viral sequences in emerging variants of concern (VOCs), and deployment has been limited by the need for high doses. In this study, we leverage a MULTI-specificity, multi-affinity antibody (Multabody, MB) platform derived from human apoferritin protomers to promote multimerization of antibody fragments to generate exceptionally potent and broad-spectrum SARS-CoV-2 neutralizers. CryoEM revealed a high degree of homogeneity in the core of these engineered antibody-like molecules at 2.1 Å resolution. We demonstrated that the improved neutralization potency of MBs relative to their corresponding IgGs translated into superior in vivo protection. In a SARS-CoV-2 mouse challenge model, we achieved comparable in vivo protection with MBs administered at a 30-fold lower dose compared to their corresponding IgGs. Furthermore, we show how MBs leverage enhanced avidity to potently neutralize SARS-CoV-2 VOCs even when the corresponding IgG has lost its potent neutralizing capacity. Our study demonstrates how combining avidity and multispecificity can provide protection and resilience against viral diversity that exceeds traditional monoclonal antibody therapy.

[0328] Introduction Emerging infectious pathogens, including viruses such as SARS-CoV-2, pose a major challenge to global public health through the lack of preexisting immunity in people. Despite the availability of vaccines against SARS-CoV-2 infection (COVID-19), global vaccination coverage remains low, with only 19.9% ​​of people in low-income countries having received at least one dose. The relatively short-term protective effect of vaccines, as well as the emergence of new viral variants, further highlight the need for effective prevention and treatment options. Monoclonal antibodies (mAbs) are effective and promising options for treating infectious diseases such as respiratory syncytial virus (RSV) and Ebola virus. Several mAbs, such as the combination of bamlanivimab and etesevimab and the REGEN-COV cocktail of casirivimab and imdevimab, have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of COVID-19, but they have struggled to overcome the diversity of the virus and are limited by the need for high doses and intravenous administration. Both combinations were de-approved following the emergence of omicron BA.1VOC, which has 37 mutations in the spike domain, the target of most clinical antibodies against SARS-CoV-2, and 15 mutations in the receptor binding domain (RBD). To date, the only mAb that retains sufficient in vitro activity against circulating omicron subvariants and is FDA-approved for use against SARS-CoV-2 is bebuterovimab. Approval was also updated to allow for dose escalation of the cocktail of tixagevimab and silgavimab, which is expected to maintain activity against subvariants despite losing potency at the original dose. Despite these limited approvals, a number of additional antibodies targeting the SARS-CoV-2 spike epitope have been identified. However, such broadening of mAb is often accompanied by reduced potency, highlighting the need to identify therapies that combine potency and breadth.

[0329] Increasing the valency of antibodies is a promising approach to increase apparent binding affinity, potentially lowering therapeutic doses, improving breadth, and allowing administration by alternative routes such as subcutaneous or intramuscular administration. A wide range of antibody engineering strategies have been described with the aim of exploiting avidity to enhance the functional response of antibodies. Among them, biologics assembled based on IgM, synthetic nanocage, and minibinder formats have demonstrated superior neutralizing properties against SARS-CoV-2 compared to traditional mAb formats. Furthermore, the potent molecules GEN3009, INBRX-106 (Inhibrx), and IGM-8444 have been tested in phase I / II clinical trials aimed at treating hematological and solid tumors, highlighting the clinical advantages of multivalent antibody presentation formats. Following a similar principle, but using human light chain apoferritin protomers to promote oligomerization of antibody fragments, we developed a platform called Multabody (MB) to enhance the neutralizing potency of antibodies targeting SARS-CoV-2 and HIV-1. With this platform, enhanced affinity can be combined with multispecificity (the incorporation of multiple antibody fragments recognizing different epitopes) to provide antigen recognition that is more resistant to viral mutations, which is particularly important given the immune pressure that is causing the continued emergence of new variants of SARS-CoV-2, including some that reduce the efficacy of existing vaccines and drugs. Here, we investigated whether the improved in vitro SARS-CoV-2 neutralization by multibodies translates into in vivo protection at lower doses. Furthermore, we assessed whether MBs can restore the reduced neutralization potency against various variants of interest (VOCs) observed with conventional mAbs. Our data provide proof of concept that MBs are a tractable platform that exploits avidity to improve both the in vitro and in vivo potency and breadth of antibody-based molecules against SARS-CoV-2.

[0330] method Biolayer Interferometry Direct binding kinetics measurements were performed at 25 °C in PBS pH 7.4, 0.01% BSA, 0.002% Tween® using an OctetRED96 BLI system (SartoriusForteBio). His-tagged RBD or SARS-CoV-2 spike protein were loaded onto Ni-NTA (NTA) biosensors (SartoriusForteBio) to achieve a BLI signal response of 0.8 nm. Association kinetics were measured by transferring the loaded biosensor into wells containing a two-fold dilution series from 250 to 16 nM (Fab), 125 to 4 nM (IgG), and 16 to 0.5 nM (MB). Dissociation kinetics were measured by immersing the biosensor into wells containing buffer. The duration of each of these two steps was 180 s. Fc properties in the split Multabody design were assessed by measuring binding to hFcγRI and hFcRn. To investigate the theoretical ability of Multabody to be recycled in endosomes, binding to hFcRnb2-microglobulin complex was measured at physiological pH (7.5) and endosomal pH (5.6). In some cases, association of Multabody with hFcRnb2-microglobulin complex was performed at pH 5.6 and dissociation was performed at pH 7.4. Competitive assays were performed with a two-step binding process. Ni-NTA biosensors preloaded with His-tagged RBD were first immersed in wells containing 50 μg / mL primary antibody for 180 s. After a 30 s baseline period, the sensors were immersed in wells containing 50 μg / mL secondary antibody for an additional 300 s.

[0331] Virus production and pseudovirus neutralization assays SARS-CoV-2 pseudoviruses (PsVs) were generated using an HIV-based lentiviral system with some modifications as previously described. Briefly, 293T cells were co-transfected with a lentiviral backbone encoding a luciferase reporter gene (BEI NR52516), a plasmid expressing spike (BEI NR52310), and plasmids encoding the HIV structural and regulatory proteins Tat (BEI NR52518), Gag-pol (BEI NR52517), and Rev (BEI NR52519). After 24 h of transfection at 37 °C, 5 mM sodium butyrate was added to the medium, and cells were incubated at 30 °C for an additional 24–30 h. The SARS-CoV-2 spike mutant D614G was provided by D.R. Burton (The Scripps Research Institute), and the remaining PsV mutants were generated by the KOD-Plus mutagenicity kit (Toyobo, Osaka, Japan). The SARS-CoV-2 spike variants of interest, B.1.117, B.1.351, P.1, and B.1.617.2, were kindly provided by David Ho (Columbia). Neutralization was determined in a single cycle neutralization assay using 293T-ACE2 cells (BEI NR52511) and HeLa-ACE2 cells (DR Burton; kindly provided by The Scripps Research Institute). PsV neutralization was monitored by adding Britelite Plus reagent (PerkinElmer) to cells and measuring luminescence in relative light units (RLU) using a Synergy Neo2 multimode assay microplate reader (Biotek Instruments). IC 50 Fold increase is IgG IC50 (μg / mL) / MB IC50 Calculated as (μg / mL). Two to three biological replicates and two technical replicates each were performed.

[0332] Authentic virus neutralization assay VeroE6 cells were seeded in 96F plates at a concentration of 30,000 / well in DMEM supplemented with 100U penicillin, 100U streptomycin, and 10% FBS. Cells were allowed to adhere to the plates and left overnight. After 24 hours, 5-fold serial dilutions of IgG and MB samples were prepared in quadruplicate (25uL / well) in 96R plates in DMEM supplemented with 100U penicillin and 100U streptomycin. 25μL of SARS-CoV-2 / SB2-P4-PB50 clone 1 was added to each well at 100TCID / well and incubated at 37°C for 1 hour with shaking every 15 minutes. After co-culture, the medium was removed from the VeroE6 plates and 50μL of antibody-virus samples were used to inoculate VeroE6 cells in quadruplicate and incubated at 37°C, 5% CO2 for 1 hour with shaking every 15 minutes. One hour after inoculation, the inoculum was removed and 200 μL of fresh DMEM supplemented with 100 U penicillin, 100 U streptomycin, and 2% FBS was added to each well. The plates were incubated for an additional 3 days. Cytopathic effect (CPE) was monitored and IC was determined using PRISM. 50 Values ​​were calculated. Three biological replicates and four technical replicates each were performed.

[0333] Antibody-dependent cell-mediated phagocytosis assay Immune complexes were formed by incubating SARS-CoV-2 spike-coated fluorescent beads with diluted MB or IgG preparations for 2 h at 37 °C + 5% CO (10 μL of beads and 10 μL of 1 mg / mL antibody sample). THP-1 cells (ATCC, TIB-202) were incubated at 5 × 10 5 Maintain less than 5 x 10 cells / mL in 200 µL 4Cells / well were added to immune complexes for 1 h at 37°C + 5% CO2. Cells were washed according to the provided protocol and stained with Live Dead Fixable Violet stain (Invitrogen, L34995), followed by washing with 1% PFA and fixing for 20 min at room temperature. Fixed cells were washed with FACS buffer (PBS + 10% FBS, 0.5 mM EDTA) and collected on an LSRII flow cytometer (BD Biosciences). Data were analyzed with FlowJo (BD Biosciences, Ashland, OR) and phagocytosis was quantified as the percentage of live THP-1 cells that phagocytosed red fluorescent SARS-CoV-2 spiked beads.

[0334] SARS-CoV-2 Challenge Study Six- to eight-week-old female hFcRn / hACE2 double transgenic mice were purchased from Jackson Laboratories (stock number 034902). All procedures were approved by the Local Animal Care Committee of the University of Toronto. Trispecific 298-80-52 (T10) MB or PGDM1400 negative control IgG were administered by intraperitoneal (i.p.) injection one day prior to infection, for a total of 6–60 μg MB, 60–180 μg IgG, depending on the experiment. Twenty-four hours later, mice were immunized with 1 × 10 4 ~1×10 5 Mice were infected with SARS-CoV-2 / SB2-P4-PB clone 1 at a dose of 100 PFU / mouse. Mice were monitored daily for weight until day 12 post-infection. At endpoint, described as when mice were sacrificed due to weight loss >20% or survived until day 12, lungs were harvested and analyzed for lung viral titers and MB / IgG quantification. Data shown in Figure 19 are cumulative results from n=2-6 independent experiments, while data shown in Figures 21 and 22 represent n=1 experiment.

[0335] Quantification of viral titers and MB / IgG in the lungs at endpoint Lungs were harvested from mice at endpoint, weighed, and then homogenized in 1 mL of incomplete DMEM. Samples were centrifuged and the supernatants were collected and frozen until sample analysis. For quantification of lung viral titers, samples were added to VeroE6 cells in 1:10 serial dilutions and allowed to infect for 1 h at 37 °C. After infection, the supernatant was removed and cells were replenished with 100 μL of fresh medium and allowed to incubate for 5 days. Cytopathic effect (CPE) was monitored and ID was determined using PRISM. 50 Values ​​were calculated. Three technical replicates were performed for each. For quantification of MB / IgG levels in the lungs, 96-well Pierce nickel-coated plates (ThermoFisher) were filled with 50 μL of His 6x Tagged RBD antigen (0.5 μg / ml) or His 6x Tagged BG505 was coated and T10 MB and PGDM1400IgG levels were measured, respectively. HRP-ProteinA (Invitrogen) was used as the secondary molecule, and the chemiluminescence signal was quantified using a SynergyNeo2 multimode assay microplate reader (Biotek Instruments).

[0336] Quantification of T10 MB in the lungs 2 days after challenge SARS-CoV-2 challenge studies performed to quantify T10 MB levels in the lungs on day 2 post-infection were performed with n = 3 mice / group as previously described. Briefly, 96-well Pierce nickel-coated plates (Thermo Fisher) were filled with 50 μL of His 6x The plates were coated with tagged RBD antigen (0.5 μg / ml).Anti-human FabIgG (Jackson Immuno Research) was used as the secondary molecule, and the optical signal was quantified using a SynergyNeo2 multimode assay microplate reader (Biotek Instruments).

[0337] CryoEM data collection and image processing Trispecific MB(298-52-80) samples were concentrated to 2.0 mg / mL and 3.0 μl of sample was placed on a homemade holey gold grid that was glow discharged in air for 15 s before use. Samples were blotted for 3.0 s and then plunge frozen in liquid ethane using a Leica EMGP2 automated plunge freezer (maintained at 4 °C and 100% humidity). Data collection was performed on a Thermo Fisher Scientific Titan KriosG3 operating at 300 kV with a Falcon4i camera automated with EPU software. A nominal magnification of 75,000× and a defocus range of 0.5–2.0 μm were used for data collection. Exposures were recorded with a camera exposure speed of approximately 6.3 e per pixel per second. - , total exposure is 49.6 electrons / Å 2 The images were collected as 8.3 second long, 30 frame videos, resulting in a total of 4,385 raw videos.

[0338] Image processing was performed in cryoSPARCv3. Initial specimen motion correction, exposure weighting, and CTF parameter estimation were performed using a patch-based algorithm. Micrographs were classified based on their CTF-fit resolution, and only micrographs with a fit better than 5.0 Å were accepted for further processing. Manual picking was performed to create templates for template-based picking, and 955,995 particle images were selected. Particle images were classified by multiple rounds of 2D classification, resulting in the selection of 358,036 particle images. A preliminary 3D model was obtained ab initio, without applying symmetry. To further select the best quality particle images, 151,443 particle images with a CTF-fit resolution better than 3.0 Å were re-extracted from the micrographs and subjected to non-uniform refinement without applying symmetry. 75The resulting map was a 2.4 Å resolution map of the trispecific MB. 65,478 particle images with a CTF fit better than 2.7 Å were extracted from the micrograph and subjected to non-uniform refinement applying octahedral symmetry, resulting in a 2.1 Å resolution map. Non-uniform refinement was performed by defocus refinement and group-wise CTF parameter optimization. The pixel size was calibrated to 1.04 Å per pixel by fitting the structure of human apoferritin light chain (PDBID: 2FFX).

[0339] To obtain 3D reconstructions of Fab and Fc molecules on the surface of the trispecific MB, manual picking was performed and templates were created for template-based picking, resulting in the selection of 6,692,141 particle images. The particle images were classified by multiple rounds of 2D classification, resulting in the selection of 668,214 particle images. Preliminary 3D maps were acquired ab initio without applying symmetry. Further cleaning of the dataset was performed by multiple rounds of non-uniform refinement, resulting in 73,163 Fab and 13,328 Fc particle images. Final cryoEM maps with a resolution of 6.7 Å for Fab and 7.1 Å for Fc were acquired using a local refinement job with a custom soft mask.

[0340] To assess the quality of the maps obtained, human apoferritin light chain (PDBID: 6WX6), human IgG1Fc (PDBID: 6CJX) 78 The models of Fab298 (PDBID: 7K9Z) were generated using UCSF Chimera 79 The structures were manually docked into the cryoEM map using Pymol, UCSF Chimera, and UCSF ChimeraX.

[0341] Crystallization and structure determination Purified 80Fab-RBD binary complex was obtained by mixing Fab:RBD at a molar ratio of 2:1. After incubation at 4°C for 30 min, the complex was purified by size-exclusion chromatography (Superdex200Increase size-exclusion column, GE Healthcare, Chicago, IL) in 20 mM Tris pH 8.0, 150 mM NaCl buffer. The fraction of interest was then concentrated to 10 mg / mL and crystallization trials were performed using the sitting drop vapor diffusion method with a JCSGTop96 screen at a protein:reservoir ratio of 1:1. Crystals appeared at day 70 in conditions containing 0.2 M diammonium tartrate and 20% (w / v) PEG3350. Crystals were cryoprotected with 10% (v / v) ethylene glycol and flash frozen in liquid nitrogen. X-ray diffraction data were collected at the 23-ID-D beamline at the Argonne National Laboratory Advanced Synchrotron Radiation Facility. Data sets were processed using XDS and XPREP. Phases were determined using Phaser with 80Fab predicted by ABodyBuilder and SARS-CoV-2 RBD (PDBID: 7LM8) as search models. Iterative refinement was performed using PhenixRefine and manual building was done in Coot. All software was accessed through SBGrid.

[0342] result Identifying sequence vulnerabilities in mAb52 In silico analysis of the lead VH / VL sequences identified a deamidation site at position N92 in CDRL3 of mAb52. Deamidation sites in mAbs can contribute to both altered binding kinetics and drug product heterogeneity. To circumvent this potential effect, a mutant was generated in which the asparagine residue was mutated to threonine (N92T). Figure 15 shows that this mutation had no effect on potency as an IgG or monospecific MB in the WT pseudovirus neutralization assay. The 298-80-52 trispecific MB, containing the N92T mutation in the VL of mAb52, was subsequently screened in the P.1PsV neutralization assay, confirming that no reduction in potency was observed compared to the parent trispecific MB (Figure 16).

[0343] Neutralization of T10 MB against mutants of interest The potency of T10 MB against WT SARS-CoV-2 and variants of interest (VOCs) was evaluated in both pseudovirus and authentic virus neutralization assays. As shown in Table 16 and Figure 17, T10 MB showed >1000-fold improved potency against WT SARS-CoV-2 compared to the corresponding IgG cocktail and maintained activity across alpha, beta, gamma, delta and omicron (BA.1) PsVs. The breadth and potency of T10 MB was further evaluated in authentic virus neutralization assays, confirming the highly potent nature of T10 MB across the VOCs tested (Figure 18). Taken together, the PsV and authentic virus neutralization assays highlight the ability of the trispecific T10 MB to overcome viral escape of SARS-CoV-2 with exceptional potency. [Table 16]

[0344] In vivo protection by T10 MB in SARS-CoV-2 challenge studies Following the identification of the ultrapotent T10 MB in PsV and authentic virus neutralization assays, we investigated the ability of this MB to exert a protective effect in a SARS-CoV-2 challenge study using hFcRn / hACE2 double transgenic mice. The nomenclature of the T10 MBs described below is shown in Table 17. [Table 17]

[0345] To specifically evaluate the effect of neutralization potency on in vivo protection from lethal SARS-CoV-2 challenge, we generated T10 MB and corresponding IgG cocktails using IgG4 Fc containing mutations (S228P, F234A, L235A, G237A, P238S) that abolish binding to Fcγ receptors, hereafter referred to as MB* (or T10.AMB) and IgG4*, respectively. As expected, replacing the Fc subtype from IgG1 to IgG4* did not affect the neutralization potency of IgG or MB, as previously reported. The trispecific MB* showed a >1000-fold increase in potency compared to the corresponding cocktail IgG (Figure 19a). Binding kinetics studies revealed that the trispecific MB* and IgG4* antibody cocktails both bound to mouse and human FcRn in a pH-dependent manner, but not to human and mouse Fcγ receptors (Figure 19b-c). This was in contrast to the FcγR binding observed with the corresponding IgG1 antibody cocktail control (Figure 19c). Antibody-dependent cell-mediated phagocytosis (ADCP) experiments using fluorescently labeled beads coated with SARS-CoV-2 spike protein further confirmed the inability of the trispecific MB* and IgG4* cocktail to bind Fc receptors, whereas the IgG1 antibody cocktail was shown to significantly internalize SARS-CoV-2 spike-coated beads (Figure 19d).

[0346] To assess whether the increased neutralization potency achieved by MB translates into improved in vivo protection against SARS-CoV-2, hACE2 and hFcRn double transgenic mice were treated with 30 μg (1.5 mg / kg) of FcγR-binding deficient IgG4* and MB* molecules 1 day prior to infection, followed by a high dose (1 × 10 5The animals were challenged intranasally with 100 TCID50 of SARS-CoV-2. The trispecific MB* showed significantly better protection (60% survival) compared to the IgG4* cocktail, with all animals receiving the cocktail succumbing to the challenge at D67 (Figure 19e). The improved protection was associated with a significantly lower viral titer in the lungs, especially in animals that survived the challenge (open circles, Figure 19f). Subsequent studies found that comparable in vivo protection was achieved when the trispecific MB* was delivered at 3 μg (0.15 mg / kg) and the IgG4* cocktail at 90 mg (4.5 mg / kg) (Figure 19g). The difference in dosage can be observed in the circulating serum concentrations of the administered molecules 2 days after challenge (Figure 19h). This data not only provides the first evidence of in vivo protection from a lethal challenge via MB, but also indicates that the improved neutralization potency provided by the trispecific MB* format provides enhanced protection against SARS-CoV-2 challenge compared to the corresponding IgG mixture. In one particular experiment, MB and IgG titers in the lung were also evaluated, and at the endpoint, trispecific MB* was detected in the lung, highlighting the ability of MB to enter the lung (Figure 19i).

[0347] Generation of IgG4MB mutants To confer different putative effector function properties in vivo to MBs, three sets of mutations were used to generate IgG4-based mutants with different binding profiles to human FcγR. Set #1 used S228P, F234A, and L235A mutations (T10.BMB); set #2 used F234A, L235A, G237A, and P238S mutations (T10.G MB); set #3 used S228P, F234A, L235A, G237A, and P238S mutations (T10.AMB). Figure 20a shows the binding profiles of these MBs to hFcRn, hFcγRI, hFcγRIIa, and hFcγRIIb. The binding of these MB mutants to hFcRn was not significantly altered, but striking differences were observed in the binding to FcγR. T10.A showed no detectable binding to any FcγR tested, and T10.G showed little binding even at the highest concentration tested. Interestingly, removal of the G237A and P238S mutations in T10.AMB to generate T10.BMB restored binding to all three hFcγRs tested. Similar trends in the binding of T10.A, T10.B, and T10.G to human FcRn and FcγRI were also observed for CynoFcRn and FcγRI (Figure 20b). However, in contrast to the binding patterns observed for human and cynomolgus monkey FcγRI, none of T10.A, T10.B, or T10.G showed detectable binding to mouse FcγRI (Figure 20c).

[0348] In vivo protection by T10.B and T10.G MB in a SARS-CoV-2 challenge study Following generation of T10.B and T10.G MBs, we investigated the ability of these MBs to exert a protective effect in a SARS-CoV-2 challenge study. hACE2 / hFcRn transgenic mice (n = 8 mice / group) were treated with a total of 60 μg of either T10.B MB, T10.G MB, or negative control IgG, and 5 × 10 4PFU / mouse were challenged intranasally with SARS-CoV-2. The results of this study show that both T10.B and T10.G MB were able to achieve 75% survival at day 12 compared to the negative IgG control, where all mice died by day 7 (Figure 21a). In vivo protection was accompanied by both reduced weight loss throughout the experiment in surviving mice (Figure 21b) and reduced viral titers to the limit of detection in the lungs of surviving mice at day 12 (Figure 21c).

[0349] T10.G MB was then tested for protective effects in hACE2 / hFcRn double transgenic mice homozygous for the hFcRn transgene (JAX#037043). In this study, mice (n=4 / group) were treated with 30 μg (1.5 mg / kg) of T10.G MB or negative control IgG, followed by 1×10 4 PFU / mouse were challenged intranasally with SARS-CoV-2. The results of this study show that T10.G MB was able to confer 75% protection compared to the negative IgG control group, where all mice succumbed to infection by day 8 (Figure 22a). In vivo protection was accompanied by reduced weight loss in mice that survived the duration of the experiment (Figure 22b).

[0350] Pharmacokinetic evaluation of T10.BMB in non-human primates (NHPs) To examine MB exposure in NHPs, T10.BMB was administered subcutaneously at 1.5 mg / kg to male and female cynomolgus monkeys (n=3 per group). Figure 23 shows that T10.BMB achieved the expected maximum serum concentration (Cmax) and was detectable in the circulation for several weeks after administration.

[0351] Assembly of the trispecific 298-52-80 multibody defined at atomic resolution We previously reported the generation of trispecific MB molecules using a modified apoferritin split design (Figure 24A). In this design, human apoferritin protomers were split into two N-terminal α-helices (N-Ferr) and two C-terminal α-helices (C-Ferr) based on the folding of a four-helix bundle. Genetic fusion of single-chain (sc) Fab or scFc at the N-terminus of each apoferritin half and full apoferritin and transfection into mammalian cell expression systems resulted in the secretion of self-assembled oligomeric molecules with ultrapotent neutralizing capacity. Specifically, a trispecific MB incorporating antibody specificities 298, 52, and 80 exhibited an approximately 1000-fold increase in neutralizing potency compared to the corresponding IgG cocktail. This trispecific MB is described as possessing antibody-like biochemical properties as assessed by biophysical characterization after purification and under accelerated heat stress. To gain molecular insight into the assembly of the multibody design, this trispecific MB was next characterized by cryo-electron microscopy (cryoEM) (Figure 25).

[0352] Analysis of cryoEM micrographs revealed the formation of highly decorated, homogenous nanocage-like particles (Figure 24B). Flexible (GGS) nanoparticles connect the scFab and scFc components to the apoferritin scaffold. x Consistent with the presence of linkers, the density of these antibody fragments is not well resolved in the 2D classes (Figure 24C) and in the 3D reconstruction of the trispecific MB (Figure 24D). However, manual selection of scFab and scFc particles, followed by template-based particle selection and subsequent purification of these molecules confirmed proper assembly of the Fab and Fc components on the MB at ∼7 Å resolution (Figures 24B-D, Figures 25 and 26).

[0353] 3D reconstructions of the apoferritin scaffold of the MB reached 2.4 Å and 2.1 Å resolution when applying no symmetry (C1; Fig. 24D, Fig. 27A-D) or octahedral symmetry (O; Fig. 27E-H), respectively. The apoferritin scaffold of the trispecific MB is virtually identical to that of the human apoferritin light chain (PDBID: 6WX6), with cross-correlation (cc) coefficients between the maps measured to be 0.97 (C1) and 0.92 (O). The N- and C-termini of the core MB scaffold are arranged on 3- and 4-fold symmetry axes similar to the native human apoferritin light chain (Fig. 24D), indicating minimal impact on scFab and scFc gene fusions. Furthermore, the cryoEM map showed no evidence that structural elements at the split-design site (between residues Trp93 and Gly94, Figure 24D, bottom right panel) deviated from the apoferritin fold. In summary, our cryoEM analysis of the trispecific 298-52-80 MB provided atomic level details indicating that the MB constructed on the apoferritin split-design scaffold adopted the intended structural arrangement.

[0354] Molecular basis of Fab80 binding to the SARS-CoV-2 RBD Next, we sought to understand the molecular basis of mAb80 binding, as the structure of mAb80 has yet to be solved. We solved the crystal structure of 80Fab in complex with the RBD at 3.1 Å resolution (Figure 28). Epitope recognition is mediated by 20 residues that form an interface with the RBD, 14 of which are involved in ACE2 binding (Table 18). This shows how mAb80 inhibits SARS-CoV-2 infection via receptor blockade, preventing the interaction of ACE2 with the receptor-binding motif (Figure 29A). The heavy chain of mAb80 is primarily responsible for the interaction with the RBD, accounting for 10 of the 11 hydrogen bonds at the binding interface (Figure 30A-B, Table 18). Furthermore, the interaction of F54 of the antibody heavy chain with Y489 of the RBD leads to the formation of a novel triple pi-stacking between residues Y473, F456, and Y421 in the RBD structure (Figure 30C). [Table 18]

[0355] Detailed analysis of the RBD-80Fab interface revealed that residues S477 and T478 of the RBD correspond to Y92 and D93 of the antibody. 100D forms hydrogen bonds with 124Å of its surface area 2 It was found that 80 mAb is buried and accounts for 15% of the total buried surface area (BSA) of the RBD (Figure 29B-C, Table 18). These residues have been mutated in several VOCs, including Omicron (BA.1, BA.2), which significantly reduces the binding affinity of the antibody to the BA.1 RBD (Figure 29D-E), however, the increased avidity achieved in the MB format compensates for this weak binding. As a result, the interaction of 80 mAb with the mutated Omicron BA.1 RBD exhibits high apparent binding affinity with no detectable off-rate (Figure 29D-E), which likely contributes to its strong neutralization potency against Omicron BA.1 (Figure 29G). The potency of 80 mAb against Omicron BA.2 was further confirmed using replication-competent virus, and as expected, a significantly reduced potency against Omicron BA.2 live virus was observed for 80 mAb, whereas in the MB format, the high neutralization potency is retained (Figure 29F).

[0356] Consideration The rapid emergence of new SARS-CoV-2 VOCs has hampered mAb therapy and driven antibody discovery efforts focused on expanding the breadth of viral sequences recognized by a single antibody. Evidence that several FDA-approved mAb therapies have lost efficacy against Omicron VOCs supports the urgent need for new therapeutic interventions with improved breadth. Furthermore, strategies to increase the potency of such mAbs may reduce the therapeutic dose and enable more practical routes of administration, which has the potential to reduce production costs and make them available worldwide. We previously described a Multabody platform capable of delivering highly potent and broadly acting molecules in vitro. Indeed, 2 years after the identification of the trispecific 298-52-80MB, derived from three mAbs with moderate potency, to the best of our knowledge, the in vitro neutralization potency of this molecule against WT SARS-CoV-2 (IC of 0.0002 μg / mL) has not yet been demonstrated. 50 ) has not yet been described. Here, we demonstrate that potent in vitro neutralization translates into in vivo protection against SARS-CoV-2 at low doses, and that the combination of avidity and multispecificity results in a molecule with a broad neutralization spectrum against SARS-CoV-2.

[0357] The MB platform offers various advantages as next-generation polyvalent biologics, including high stability, efficient assembly, ease of production and purification, and plug-and-play genetic fusion of selected antibodies. Here, we further confirmed the proper assembly of trispecific MBs by cryoEM. This structural technique has been useful for the characterization of large and complex biological designs such as subunit vaccines, including self-assembled protein nanoparticles displaying ectodomains of influenza and respiratory syncytial virus glycoprotein trimers, bicomponent protein nanoparticles exhibiting stabilized HIV-1 Env trimers, or COVID-19 vaccine candidate nanoparticles utilizing SpyCatcher multimerization of the SARS-CoV-2 spike protein RBD. Although the flexibility of the connecting linkers makes it difficult to simultaneously visualize both the nanoparticle scaffold and the molecules displayed at its periphery, recent advances in cryoEM data processing have allowed the analysis of different nanoparticle components individually. By employing this strategy, we were able to confirm the proper folding of scFab and scFc at the periphery of the MB. Furthermore, we confirm the proper assembly of the human light chain apoferritin scaffold of MB at 2.1 Å resolution in the context of an engineered partitioning design from which an assembly of multispecific antibody components is constructed. These analyses further support the central role of structure-guided protein engineering in the rational design of novel biologics and are critical for validating MB as a homogeneous biologic.

[0358] We next investigated the ability of MB to protect against lethal challenge in vivo. The specific role of enhanced neutralization potency in mediating in vivo protection was assessed using trispecific MB* molecules expressing a mutant IgG4 Fc defective in Fcg receptor binding. To maintain IgG-like bioavailability as previously described, we maintained the ability of MBFc to interact with FcRn, a receptor associated with antibody recycling and extended half-life. In vivo, the dramatic increase in neutralization potency of trispecific MB compared to the IgG4* cocktail significantly improved protection against lethal SARS-CoV-2 challenge and facilitated a reduction in the dose required for protection. Previous studies have shown that some mAbs targeting SARS-CoV-2 require Fc-mediated effector functions for optimal efficacy. Our data illustrate that enhanced neutralization potency is sufficient to protect against lethal challenge even in the absence of effector functions, supporting the use of avidity-based increased potency to facilitate dose sparing of antibody-based therapeutics. We have previously shown that the MB format is able to induce ADCP in vitro, indicating that the format itself does not preclude the incorporation of effector functions into the molecules.

[0359] Monospecific MBs show high tolerance to viral sequence variability through enhanced apparent binding affinity compared to IgG, allowing these molecules to retain neutralizing capacity even when mAbs lose potency. In the case of mAb80, mutations within the RBD epitope found in VOCs cause the mAb to lose potency. In contrast, given the specificity of 80 on MBs, mutations present within VOCs minimally alter the high binding affinity and potent neutralization profile of this molecule. The ability of MBs to better tolerate sequence variability is likely driven by increased avidity through reduced off-rates and a higher spike density on the viral particle surface. The potential for potent antibody technology to tolerate sequence variability could benefit antibody discovery timelines by extending the lifespan of early identified mAbs with the ability to neutralize emerging VOCs.

[0360] Identifying potent bnAbs can take years or even decades of antibody discovery and engineering efforts, as seen in the examples of HIV-1 and influenza. Although ongoing monitoring and screening of emerging variants will be required to confirm the durability of neutralization, the larger footprint of RBD covered by trispecific MBs compared to traditional mAbs offers the MBs a unique advantage of remaining resistant to future VOCs compared to mAbs alone. Furthermore, the ability to combine multiple specificities into a single molecule may provide an additional potential advantage of ensuring bioavailability of all components throughout the course of treatment, which is a limiting feature of mAb cocktail combinations.

[0361] Sequence Listing SEQ ID NO:1 hFerritin LC MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD SEQ ID NO:2 Linker 1 GGGGSGGGGSGGGGSGGGGSGGGGSGG SEQ ID NO:3 VHH-hFerr (The underlined parts indicate the linker sequences, and the bolded parts indicate the hFerritinLC.) [ka] SEQ ID NO: 4 VHH-Fc (The underlined parts indicate the linker sequences.) [ka] SEQ ID NO: 5 N-hFerritinLC MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW SEQ ID NO:6 C-hFerritinLC GKTPDAMKAAMALEKKLNQALLLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD SEQ ID NO: 7 Signal sequence MGILPSPGMPALLSLVSLLSVLLMGCVAE SEQ ID NO:8 Linker 1 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:9 Linker 2 GGGGSGGGGSGGGGSGGGGSGGGGSGG SEQ ID NO: 10 BD23-scFab-hFerritinLC (The underlined parts indicate the linker sequences, and the bolded parts indicate the hFerritinLC.) [ka] SEQ ID NO: 11 BD23 V K DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPYTFGQGTKLEIK SEQ ID NO: 12 BD23 V H QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARPQGGSSWYRDYYYGMDVWGQGTTVTVSS SEQ ID NO: 13 BD23-scFab-C_hFerritinLC (The underlined sequence indicates the linker sequence, and the bold text indicates C_hFerritinLC.) [ka] SEQ ID NO: 14 scFc-N_hFerritinLC (The underlined parts indicate the linker sequences, and the bolded parts indicate the hFerritinLC.) [ka] SEQ ID NO: 15 scFc(LALAP) (The residue(s) mutated relative to wild-type Fc are boxed.) [ka] SEQ ID NO: 164A8-scFab-hFerritinLC (The underlined parts indicate the linker sequences, and the bolded parts indicate the hFerritinLC.) [ka] SEQ ID NO: 17 4A8 V K EIVMTQSPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCTQATQFPYTFGQGTKVDIK SEQ ID NO: 18 4A8 V H EVQLVESGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETMYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATSTAVAGTPDLFDYYYGMDVWGQGTTVTVSS SEQ ID NO: 19 4A8-scFabC_hFerritinLC (The underlined sequence indicates the linker sequence, and the bold text indicates C_hFerritinLC.) [ka] Sequence number 20: mFerritin MTSQIRQNYSTEVEAAVNRLVNLHLRASYTYLSLGFFFDRDDVALEGVGHFFRELAEEKREGAERLLEFQNDRGGRALFQDVQKPSQDEWGKTQEAMEAALAMEKNLNQALLLDLHALGSARTDPHLCDFLESHYLDKEVKLIKKMGNHLTNLRRVAGPQPAQTGAPQGSLGEYLFERLTLKHD SEQ ID NO: 21 HD37-scIgG (The underlined parts indicate the linker sequences.) [ka] SEQ ID NO: 22 IgG2aFc_mFerr (The underlined parts indicate the linker sequences, and the bolded parts indicate mFerritin.) [ka] SEQ ID NO: 23 scFc-N-hFerrLALAPI253A (The underlined sequence is the linker sequence, the bold text is hFerritinLC, and the boxes indicate the residues mutated compared to the wild-type IgG1Fc.) [ka] SEQ ID NO: 24 Wild type human IgG1Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 25 Antibody 56 light chain DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPSTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:26 Antibody 56 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDIGPIDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 27 Antibody 349 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDTSNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPWTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:28 Antibody 349 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSGISSAGSITNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGNHAGTTVTSEYFQHW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 29 Antibody 178 light chain EIVMTQSPATLSVSPGERATLSCKASQSVSGTYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQTHSYPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 30 Antibody 178 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYHMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDISSWYEITKFDPW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 31 Antibody 108 light chain DIQMTQSPSSLSASVGDRVTITCRASQVITNNLAWYQQKPGKAPKLLIYDASTLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTFPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 Antibody 108 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYIFSRYAIHWVRQAPGQGLEWMGWMNPISGNTDYAPNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKDGSQLAYLVEYFQHW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 33 Antibody 128 light chain DIQMTQSPSSLSASVGDRVTITCRASQNISRYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPPTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 34 Antibody 128 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYYMHWVRQAPGQGLEWMGIINPSSSSASYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDGRYGSGSYPFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 35 Antibody 160 light chain DIQMTQSPSSLSASVGDRVTITCRASQSVSSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTTPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 36 Antibody 160 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHDMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARANSLRYYYGMDVWG QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 37 Antibody 368 light chain DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPATFGPGTKV DIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 38 Antibody 368 heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGAIMPMFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGSSGYYYGWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 39 Antibody 192 light chain DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYAASSLQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 40 Antibody 192 heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWINPNSGGANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCSTYYYDSSGYSTDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 41 Antibody 158 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYDASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 42 Antibody 158 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPLNGGTNFAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDPGGSYSNDAFDIW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 43 Antibody 180 light chain DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYAASSLQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYSSPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 44 Antibody 180 heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAMHWVRQAPGQGLEWMGRISPRSGGTKYAQRFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREAVAGTHPQAGDFDL WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 45 Antibody 254 light chain DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASSLQIGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQSYSTPPWTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 46 Antibody 254 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGTFSSSAMHWVRQAPGKGLEWVSAIGTGGDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGDGYNFYFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 47 Antibody 120 light chain EIVMTQSPATLSVSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYYTTPRTFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 48 Antibody 120 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGMIDPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKDFGGGTRYDYWYFDL WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 49 Antibody 64 light chain DIQMTQSPSSLSASVGDRVTITCRASQGISSHLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:50 Antibody 64 heavy chain EVQLLESGGGLVQPGGSLRLSCAASGFPFSQHGMHWVRQAPGKGLEWVSAIDRSGSYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTYGGKVTYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:51 Antibody 298 light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPPTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:52 Antibody 298 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGTFSTYGISWVRQAPGQGLEWMGWISPNSGGTDLAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASDPRDDIAGGYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:53 Antibody 82 light chain DIQMTQSPSSLSASVGDRVTITCRASQVISNYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPPPTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:54 Antibody 82 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGSFSTSAFYWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRALYGSGSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:55 Antibody 46 light chain DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:56 Antibody 46 heavy chain EVQLLESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDSRDAFDIWGQG TMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:57 Antibody 324 light chain DIQMTQSPSSLSASVGDRVTITCRASQSITTYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:58 Antibody 324 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGTFNNYGISWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVGDYGDYIVSPFDLW GRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO:59 Antibody 236 light chain DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPTFGQGTRL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 60 Antibody 236 heavy chain QVQLVQSGAEVKKPGASVKVSCKASGGTFTSYGINWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASRGIQLLPRGMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 61 Antibody 52 light chain DIQMTQSPSSLSASVGDRVTITCRASQGISNNLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNGFPLTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 62 Antibody 52 light chain N92T [ka] SEQ ID NO: 63 Antibody 52 heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGGIIPMFGTTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDRGDTIDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 64 Antibody 80 light chain DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAPLTFGGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 65 Antibody 80 heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGGTFNRYAFSWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTRELPEVVDWYFDL WGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 66 Wild type human IgG4 Fc PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 67 Fc chain 3-T10.G PPCPSCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 68 Fc chain 4-T10.A PPCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 69 Fc chain 5-T10.B PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 7052 Light chain N92T mutant DIQMTQSPSSLSASVGDRVTITCRASQGISNNLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGTGFPLTFGPGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 71 Nferr-Fc (PAAAS mutation) (contained within T10.A) PPCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGPPCPSCPAPEAAGASSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGSGGSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW SEQ ID NO: 72 Nferr-Fc (AAAS mutation) (contained within T10.G) PPCPSCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGPPCPSCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW Sequence number 73 Nferr-Fc (in PAA fart) (contained within T10.B) PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW Sequence number 74 Cferr-Fc (PAAAS mutation) (contained within T10.A) PPCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD SEQ ID NO: 75 Cferr-Fc (AAAS mutation) (contained within T10.G) PPCPSCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD SEQ ID NO: 76 Cferr-Fc (PAA mutation) (contained within T10.B) PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGPPCPPCPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

[0362] Equivalents / Alternative Embodiments While the invention has been described in relation to specific embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover generally any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including such departures from the present disclosure that are within known or customary practice in the art to which this invention pertains, and may be applied to the essential features of the invention as described hereinabove.

Claims

1. 1. A self-assembling polypeptide complex comprising: (a) one or more fusion proteins comprising a nanocage monomer or subunit thereof linked to an Fc polypeptide; (b) one or more fusion proteins comprising a nanocage monomer or subunit thereof linked to a SARS-CoV-2 binding moiety; The self-assembled polypeptide complex, wherein a plurality of the fusion proteins self-assemble to form a nanocage.

2. The self-assembling polypeptide complex of claim 1 , wherein the Fc polypeptide does not bind to an Fcγ receptor.

3. 3. The self-assembling polypeptide complex of claim 2, wherein the Fc polypeptide comprises an IgG4 Fc chain having a mutation at one or more of positions 228, 234, 235, 237, and 238 according to EU numbering.

4. The self-assembling polypeptide complex of claim 3 , wherein the IgG4 Fc chain comprises mutations at positions 234 and 235.

5. The self-assembling polypeptide complex of claim 4 , wherein the IgG4 Fc chain comprises an F234A mutation and an L235A mutation.

6. The self-assembling polypeptide complex of claim 3 , wherein the IgG4 Fc chain comprises a mutation at position 228.

7. The self-assembling polypeptide complex of claim 6 , wherein the IgG4 Fc chain comprises an S228P mutation.

8. The self-assembling polypeptide complex of claim 3 , wherein the IgG4 Fc chain comprises mutations at positions 237 and 238.

9. The self-assembling polypeptide complex of claim 8 , wherein the IgG4 Fc chain comprises a G237A mutation and a P238S mutation.

10. The self-assembling polypeptide complex of claim 3 , wherein the IgG4 Fc chain does not contain mutations at G237 and P238.

11. The self-assembling polypeptide complex of claim 3 , wherein the IgG4 Fc chain comprises an S228P mutation, an F234A mutation, and an L235A mutation.

12. The self-assembled polypeptide complex of claim 3 , wherein the IgG4 Fc chain comprises an S228P mutation, an F234A mutation, an L235A mutation, a G237A mutation, and a P238S mutation.

13. The self-assembling polypeptide complex of claim 3 , wherein the IgG4 Fc chain comprises a F234A mutation, a L235A mutation, a G237A mutation, and a P238S mutation.

14. The self-assembling polypeptide complex of claim 13 , wherein the IgG4 Fc chain does not contain a mutation at S228.

15. The self-assembled polypeptide complex of claim 1 , wherein the nanocage monomer or subunit thereof is a ferritin monomer or subunit thereof.

16. The self-assembled polypeptide complex of claim 15, wherein the ferritin monomer or subunit thereof is a ferritin light chain or subunit thereof.

17. The self-assembled polypeptide complex of claim 15, wherein the ferritin monomer or subunit thereof is a ferritin monomer subunit.

18. The self-assembled polypeptide complex of claim 17, wherein the ferritin monomer subunit is C-half ferritin.

19. 19. The self-assembling polypeptide complex of claim 18, wherein the Fc polypeptide comprises a single-chain Fc (scFc) comprising two Fc chains, the two Fc chains being linked via an amino acid linker.

20. The self-assembling polypeptide complex of claim 1 , wherein the SARS-CoV-2 binding moiety comprises an antibody or a fragment thereof.

21. The self-assembling polypeptide complex of claim 20 , wherein the antibody or fragment thereof comprises a Fab fragment.

22. The self-assembled polypeptide complex of claim 1, which exhibits binding to hFcRn.

23. The self-assembling polypeptide complex of claim 1, which does not exhibit binding to at least one human Fcγ receptor as determined by an in vitro assay.

24. The self-assembling polypeptide complex of claim 23, which does not exhibit binding to one or more human Fcγ receptors selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof, as determined by an in vitro assay.

25. The self-assembling polypeptide complex of claim 24, which does not exhibit binding to hFcγRI, hFcγRIIa, and hFcγRIIb as determined by an in vitro assay.

26. 24. The self-assembling polypeptide complex of claim 23, which exhibits substantially no IgG4 effector function.

27. 2. The self-assembling polypeptide complex of claim 1, which exhibits binding to at least one human Fcγ receptor as determined by an in vitro assay.

28. 28. The self-assembling polypeptide complex of claim 27, which exhibits binding to one or more human Fcγ receptors selected from the group consisting of hFcγRI, hFcγRIIa, hFcγRIIb, hFcγRIIIa, hFcγRIIIb, and combinations thereof, as determined by an in vitro assay.

29. 29. The self-assembling polypeptide complex of claim 28, which exhibits binding to hFcγRI, hFcγRIIa, and hFcγRIIb as determined by an in vitro assay.

30. 28. The self-assembling polypeptide complex of claim 27, which exhibits an antibody effector function, such as an IgG effector function.

31. 31. The self-assembling polypeptide complex of claim 30, which exhibits IgG4 effector function.

32. A composition comprising a plurality of self-assembled polypeptide complexes according to any one of claims 1 to 31.

33. A composition comprising the self-assembled polypeptide complex of any one of claims 1 to 31 for use in the treatment and / or prevention of SARS-CoV-2.

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