CN116615255A

CN116615255A - Polypeptides targeting SARS-CoV-2 and related compositions and methods

Info

Publication number: CN116615255A
Application number: CN202180082176.4A
Authority: CN
Inventors: J-P·朱利恩; E·鲁哈斯迪兹
Original assignee: Hospital for Sick Children HSC
Current assignee: Hospital for Sick Children HSC
Priority date: 2020-10-09
Filing date: 2021-10-08
Publication date: 2023-08-18

Abstract

Provided herein is a fusion protein comprising a nanocage monomer linked to a SARS-CoV-2 binding moiety, wherein a plurality of fusion proteins self-assemble to form a nanocage. A trispecific antibody construct targeting SARS-CoV-2 is also provided. Also provided are fusion polypeptides comprising (1) a fragment crystallizable (Fc) region linked to (2) a nanocage monomer or subunit thereof, wherein the Fc region comprises an I253A mutation, wherein numbering is according to the EU index.

Description

Polypeptides targeting SARS-CoV-2 and related compositions and methods

Technical Field

The present invention relates to polypeptides. In particular, the invention relates to SARS-CoV-2 specific polypeptides and related constructs, compositions and methods.

Background

Nanoparticles contribute to the progress of various disciplines. Their use has engineered, sustained release and caged microenvironments that confer targeted delivery and allow for ordered microarrays for catalytic processes.

Protein self-assembly is an attractive approach to the manufacture of nanoparticles containing sensitive and metastable proteins. In fact, self-assembled nanoparticles are formed by non-covalent interactions under physiological conditions and reliably produce uniform and generally symmetrical nanocapsules or nanocages.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a strain of coronavirus responsible for the 2019 coronavirus disease (COVID-19), a respiratory disease that results in a pandemic of COVID-19.

There is a need for improved compositions and methods for treating and/or preventing SARS-CoV-2.

Summary of The Invention

According to one aspect, there is provided a fusion protein comprising a nanocage monomer linked to a SARS-CoV-2 binding moiety, wherein a plurality of fusion proteins self-assemble to form a nanocage.

In one aspect, the SARS-CoV-2 binding moiety targets a SARS-CoV-2S glycoprotein.

In one aspect, the SARS-CoV-2 binding moiety decorates the inner and/or outer surfaces, preferably the outer surfaces, of the assembled nanocage.

In one aspect, the SARS-CoV-2 binding moiety comprises an antibody or fragment thereof.

In one aspect, the antibody or fragment thereof comprises a Fab fragment.

In one aspect, the antibody or fragment thereof comprises a scFab fragment, scFv fragment, sdAb fragment, VHH domain, or combination thereof.

In one aspect, the antibody or fragment thereof comprises the heavy and/or light chain of a Fab fragment.

In one aspect, the SARS-CoV-2 binding moiety comprises the single chain variable domain VHH-72, BD23 and/or 4A8.

In one aspect, the SARS-CoV-2 binding moiety comprises the mAbs listed in Table 4.

In one aspect, the SARS-CoV-2 binding moiety comprises mAbs 298, 324, 46, 80, 52, 82 or 236 from Table 4.

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

In one aspect, the nanocage monomer comprises a first nanocage monomer subunit linked to a SARS-CoV-2 binding moiety; wherein the first nanocage monomer subunit self-assembles with the second nanocage monomer subunit to form a nanocage monomer.

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

In one aspect, the fusion protein is provided in combination with a second nanocage monomeric subunit.

In one aspect, the second nanocage monomeric subunit is linked to a biologically active moiety.

In one aspect, the biologically active portion comprises an Fc fragment.

In one aspect, the Fc fragment is an IgG1 Fc fragment.

In one aspect, the Fc fragment comprises one or more mutations, such as LS, YTE, LALA, I253A and/or LALAP, which modulate the half-life of the fusion protein from, for example, minutes or hours to days, weeks or months.

In one aspect, the Fc fragment is a scFc fragment.

In one aspect, about 3 to about 100 nanocage monomers, e.g., 24, 32, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or more, optionally in combination with one or more intact nanocage monomers, self-assemble to form nanocages.

In one aspect, the nanocage monomers are selected from the group consisting of ferritin, apoferritin, encapsulin, SOR, tetrahydropteridine dioxygenase (lumazine synthase), pyruvate dehydrogenase, carboxylase, vault protein, groEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

In one aspect, the nanocage monomer is apoferritin, optionally human apoferritin.

In one aspect, the first and second nanocage monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

In one aspect, the "N" region of the 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 sequence:

MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW。

in one aspect, the "C" region of the 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 sequence:

GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

or (b)

GKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD。

In one aspect, the fusion protein further comprises a linker between the nanocage monomer subunit and the biologically active moiety.

In one aspect, the linker is flexible or rigid and comprises from about 1 to about 30 amino acid residues, for example from about 8 to about 16 amino acid residues.

In one aspect, the linker comprises a GGS repeat sequence, e.g., 1, 2, 3, 4 or more GGS repeats.

In one aspect, 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:

GGGGSGGGGSGGGGSGGGGSGGGGSGG。

In one aspect, the fusion protein further comprises a C-terminal linker.

In one aspect, 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 sequence:

GGSGGSGGSGGSGGGSGGSGGSGGSG。

according to one aspect, there is provided 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.

In one aspect, each nanocage monomer comprises a fusion protein described herein.

In one aspect, about 20% to about 80% of the nanocage monomers comprise the fusion proteins described herein.

In one aspect, the nanocage comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different SARS-CoV-2 binding moieties, e.g., 3 different SARS-CoV-2 binding moieties.

In one aspect, the nanocages are multivalent and/or multispecific.

In one aspect, the nanocage comprises one or more mabs from table 4.

In one aspect, the nanocage comprises 3 mabs from table 4.

In one aspect, the nanocages comprise mabs 298, 324, 46, 52, 80, 82, and/or 236 from table 4.

In one aspect, the nanocage comprises scFab1 human apoferritin to scFc human N-ferritin to scFab 2-C-ferritin to scFab 3-C-ferritin in a ratio of 4:2:1:1.

In one aspect, the nanocages carry cargo molecules, such as pharmaceutical agents, diagnostic agents, and/or imaging agents.

In one aspect, the cargo molecule is not fused to a fusion protein, but is contained internally within a nanocage.

In one aspect, 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 one aspect, the cargo molecule is a fluorescent protein, e.g., GFP, EGFP, ametrine, and/or a flavin-based fluorescent protein, e.g., a LOV protein, e.g., iLOV.

According to one aspect, a trispecific antibody construct is provided that targets SARS-CoV-2.

According to one aspect, there is provided a SARS-CoV-2 therapeutic or prophylactic composition comprising a nanocage or antibody as described herein.

According to one aspect, there is provided a nucleic acid molecule encoding a fusion protein described herein.

According to one aspect, there is provided a vector comprising a nucleic acid molecule as described herein.

According to one aspect, there is provided a host cell comprising a vector as described herein and producing a fusion protein as described herein.

According to one aspect, there is provided a method of treating and/or preventing SARS-CoV-2, the method comprising administering a nanocage or an antibody or composition described herein.

According to one aspect, there is provided the use of a nanocage or antibody or composition as described herein for the treatment and/or prophylaxis of SARS-CoV-2.

According to one aspect, there is provided a nanocage or antibody or composition as described herein for use in the treatment and/or prophylaxis of SARS-CoV-2.

According to one aspect, there is provided a polypeptide or functional fragment thereof comprising an amino acid sequence having at least 70% identity to any of the sequences listed in the following table:

in one aspect, the polypeptide comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the listed sequences.

In one aspect, the polypeptide consists of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the listed sequences.

According to one aspect, there is provided an antibody or fragment thereof comprising a polypeptide described herein.

According to one aspect, there is provided a fusion polypeptide comprising (1) a fragment crystallizable (Fc) region linked to (2) a nanocage monomer or subunit thereof, wherein the Fc region comprises an I253A mutation, wherein numbering is according to the EU index.

In one aspect, the Fc region further comprises a LALAP (L234A/L235A/P329G) mutation, wherein numbering is according to the EU index.

In one aspect, the Fc region is an IgG1 Fc region.

In one aspect, the nanocage monomers are ferritin monomers.

In one aspect, the ferritin monomer is a ferritin light chain.

In one aspect, the ferritin light chain is a human ferritin light chain.

In one aspect, the Fc region is linked to the nanocage monomer or subunit thereof via an amino acid linker.

In one aspect, the Fc region is linked to the N-terminus of the nanocage monomer or subunit thereof.

In one aspect, the Fc region is a single chain Fc (scFc).

In one aspect, the Fc region is an Fc monomer.

According to one aspect, there is provided a self-assembled polypeptide complex comprising:

(a) A plurality of first fusion polypeptides, each first fusion polypeptide comprising (1) an Fc region linked to (2) a nanocage monomer or subunit thereof, and

(b) A plurality of second fusion polypeptides, each comprising (1) a SARS-CoV-2 binding antibody fragment linked to (2) a nanocage monomer or subunit thereof.

In one aspect, the nanocage monomers are ferritin monomers.

In one aspect, the nanocage monomer is a ferritin light chain.

In one aspect, the self-assembled polypeptide complex does not comprise any ferritin heavy chain or subunits of ferritin heavy chain.

In one aspect, the nanocage monomers are human ferritin light chains.

In one aspect, the SARS-CoV-2 binding antibody fragment binds to a receptor binding domain or spike protein of SARS-CoV-2.

In one aspect, the SARS-CoV-2 binding antibody fragment comprises a light chain variable domain and a heavy chain variable domain.

In one aspect, the SARS-CoV-2 binding antibody fragment comprises a Fab of an antibody capable of binding SARS-CoV-2.

In one aspect, the SARS-CoV-2 binding antibody fragment comprises a VK domain and a VH domain.

In one aspect, the self-assembling polypeptide complex is characterized by a ratio of the first fusion polypeptide to the second fusion polypeptide of 1:1.

In one aspect, the Fc region is an IgG1 Fc region.

In one aspect, the self-assembled polypeptide complex comprises a total of at least 24 fusion polypeptides.

In one aspect, the self-assembling polypeptide complex comprises a total of at least 32 fusion polypeptides.

In one aspect, the self-assembling polypeptide complex has a total of about 32 fusion polypeptides.

(a) A plurality of first fusion polypeptides, each first fusion polypeptide comprising (1) an IgG1 Fc region linked to (2) a human ferritin monomer or subunit thereof, wherein said IgG1 Fc region comprises LALAP (L234A/L235A/P329G) and an I253A mutation, wherein numbering is according to the EU index, and

(b) A plurality of second fusion polypeptides, each second fusion polypeptide comprising (1) a Fab fragment of an antibody capable of binding to a SARS-CoV-2 protein, said Fab fragment being linked to (2) a human ferritin monomer or subunit thereof.

In one aspect:

(1) Each first fusion polypeptide comprises a ferritin monomeric subunit that is C-half-ferritin, and each second fusion polypeptide comprises a ferritin monomeric subunit that is N-half-ferritin; or (b)

(2) Each first fusion polypeptide comprises a ferritin monomer subunit that is N-half-ferritin and each second fusion polypeptide comprises a ferritin monomer subunit that is C-half-ferritin.

In one aspect, each first fusion polypeptide comprises a monomeric subunit of ferritin, which is C-half-ferritin.

In one aspect, the IgG1 Fc region is linked to C-half-ferritin via an amino acid linker.

In one aspect, the IgG1 Fc region is linked to C-half-ferritin via the N-terminus of C-half-ferritin.

In one aspect, each second fusion polypeptide comprises a monomeric subunit of ferritin, which is N-half-ferritin.

In one aspect, the Fab fragment is linked to N-half-ferritin via an amino acid linker.

In one aspect, the Fab fragment is linked to N-half-ferritin via the N-terminus of N-half-ferritin.

In one aspect, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each third fusion polypeptide comprising (1) a human ferritin monomer linked to (2) a Fab fragment of an antibody capable of binding to a SARS-CoV-2 protein.

In one aspect, the self-assembling polypeptide complex is characterized by a ratio of the first fusion polypeptide to the second fusion polypeptide to the third fusion polypeptide of 1:1:2.

In one aspect, the self-assembling polypeptide complex has a total of 32 fusion polypeptides.

In one aspect, wherein the Fab fragment comprises a VK domain and a VH domain, wherein

(1) The VK domain has the amino acid sequence of SEQ ID No. 11 and the VH domain has the amino acid sequence of SEQ ID No. 12;

(2) The VK domain has the amino acid sequence of SEQ ID No. 17 and the VH domain has the amino acid sequence of SEQ ID No. 18;

(3) The VK domain has the amino acid sequence of VK within SEQ ID No. 25 and the VH domain has the amino acid sequence of VH within SEQ ID No. 26;

(4) The VK domain has the amino acid sequence of VK within SEQ ID No. 27 and the VH domain has the amino acid sequence of VH within SEQ ID No. 28;

(5) The VK domain has the amino acid sequence of VK within SEQ ID No. 29 and the VH domain has the amino acid sequence of VH within SEQ ID No. 30;

(6) The VK domain has the amino acid sequence of VK within SEQ ID No. 31 and the VH domain has the amino acid sequence of VH within SEQ ID No. 32;

(7) The VK domain has the amino acid sequence of VK within SEQ ID No. 33 and the VH domain has the amino acid sequence of VH within SEQ ID No. 34;

(8) The VK domain has the amino acid sequence of VK within SEQ ID No. 35 and the VH domain has the amino acid sequence of VH within SEQ ID No. 36;

(9) The VK domain has the amino acid sequence of VK within SEQ ID No. 37 and the VH domain has the amino acid sequence of VH within SEQ ID No. 38;

(10) The VK domain has the amino acid sequence of VK within SEQ ID No. 39 and the VH domain has the amino acid sequence of VH within SEQ ID No. 40;

(11) The VK domain has the amino acid sequence of VK within SEQ ID No. 41 and the VH domain has the amino acid sequence of VH within SEQ ID No. 42;

(12) The VK domain has the amino acid sequence of VK within SEQ ID No. 43 and the VH domain has the amino acid sequence of VH within SEQ ID No. 44;

(13) The VK domain has the amino acid sequence of VK within SEQ ID No. 45 and the VH domain has the amino acid sequence of VH within SEQ ID No. 46;

(14) The VK domain has the amino acid sequence of VK within SEQ ID No. 47 and the VH domain has the amino acid sequence of VH within SEQ ID No. 48;

(15) The VK domain has the amino acid sequence of VK within SEQ ID No. 49 and the VH domain has the amino acid sequence of VH within SEQ ID No. 50;

(16) The VK domain has the amino acid sequence of VK within SEQ ID No. 51 and the VH domain has the amino acid sequence of VH within SEQ ID No. 52;

(17) The VK domain has the amino acid sequence of VK within SEQ ID No. 53 and the VH domain has the amino acid sequence of VH within SEQ ID No. 54;

(18) The VK domain has the amino acid sequence of VK within SEQ ID No. 55 and the VH domain has the amino acid sequence of VH within SEQ ID No. 56;

(19) The VK domain has the amino acid sequence of VK within SEQ ID No. 57 and the VH domain has the amino acid sequence of VH within SEQ ID No. 58;

(20) The VK domain has the amino acid sequence of VK within SEQ ID No. 59 and the VH domain has the amino acid sequence of VH within SEQ ID No. 60;

(21) The VK domain has the amino acid sequence of VK within SEQ ID No. 61 or SEQ ID No. 62 and the VH domain has the amino acid sequence of VH within SEQ ID No. 63; or (b)

(22) The VK domain has the amino acid sequence of VK within SEQ ID No. 64 and the VH domain has the amino acid sequence of VH within SEQ ID No. 65.

In one aspect, the human ferritin monomer is a human ferritin light chain.

According to one aspect, there is provided a method of treating, ameliorating or preventing a SARS-CoV-2 related disorder, the method comprising administering to a subject a composition comprising a self-assembled polypeptide complex as described herein.

In one aspect, the subject is a mammal.

In one aspect, the subject is a human.

New features of the invention will become apparent to those skilled in the art upon examination of the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and the specific examples presented are given by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description of the invention and the claims that follow.

Brief Description of Drawings

The invention will be further understood from the following description with reference to the drawings, in which:

fig. 1: affinity enhances binding and neutralization of SARS-CoV-2 by VHH. a. Schematic representation of multimerization of monomeric VHH domains using conventional Fc (dark red) scaffolds or human apoferritin (grey). b. Size exclusion chromatography and SDS-PAGE of individual apoferritin (grey) and VHH-72 apoferritin particles (gold). Negative staining electron microscope of vhh-72 apoferritin particles. (scale 50nm, representing two independent experiments). d. When displayed in the divalent (dark red) or 24-mer (gold) form, the binding affinity of VHH-72 to SARS-CoV-2S protein (apparent K _D ) Is a comparison of (c). Bar graphs represent the average of n=2 biologically independent experiments. Apparent K _D Below 10 ^-12 M (dashed line) exceeds the instrument detection limit. e. Neutralization potency (color coding as shown in (d)) against SARS-CoV-2 PsV. One representative of two biologically independent replicates with similar results is shown. The mean ± SD of two technical replicates is shown. Shows two biologically independent repeated ICs ₅₀ Median value.

FIG. 2 shows the binding interface of Fabs 52 and 298 with RBD. The interactions of Fab 298 (a) and 52 (b) with RBD (light green and dark green for core and RBM, respectively) are mediated by Complementarity Determining Region (CDR) heavy chains (H) 1 (yellow), H2 (orange), H3 (red), kappa light chains (K) 1 (light blue) and K3 (purple). The key binding residues are shown as bars (inset). The H-bonds and salt bridges are indicated by black dashed lines. The L and H chains of Fab are shown in tan and white, respectively. c) ACE2 (left) and Fab 298 (right) in combination with RBD are seen from the bottom and side. The RBD side chains that are part of the ACE2-RBD and Fab 298-RBD complex binding interfaces are shown in pink, while the RBD side chains that are unique to a given interface are shown in yellow. The surface of ACE2, fab 298HC and variable regions of Fab 298KC are shown in white, grey and tan, respectively. The color of the RBD is shown in (a). d) Superposition of Fab 46 (light pink) and 52 (dark pink) when bound to RBD (green) reveals a different approach angle for the two mabs. The stereoscopic image of the composite omits the electron density map drawn at 1.3sigma contour at the interface of e) 298-RBD and f) 52-RBD.

Fig. 3: bioavailability, biodistribution and immunogenicity of mice instead of multhabodies. a. Binding kinetics of WT and Fc modified (LALAP mutant) MBs to mouse fcyri (left) and mouse FcRn at endosomal (middle) and physiologic (right) pH compared to the parental IgG. Two-fold serial dilutions from 100 to 3nM (IgG) and 10 to 0.3nM (MB) were used. The red line represents the original data; the black line represents the global fit. b. 5 male C57BL/6 mice per group were used to assess serum concentrations of surrogate mice MB, fc modified MB (LALAP mutation) and parent mice IgG (IgG 1 and IgG2a subtypes) after subcutaneous administration of 5 mg/kg. c. MB and IgG2a samples were labeled with Alexa-647 to visualize biodistribution after subcutaneous injection into three male BALB/c mice/groups by real-time non-invasive 2D whole body imaging. 15nm fluorescent labelled Gold Nanoparticles (GNPs) with Rh values similar to multhabody as a comparison. d. Any anti-drug antibody response induced by mice in place of multhabody was evaluated using 5 male C57BL/6 mice per group, compared to parent IgG and malaria PfCSP peptide that was species-mismatched to helicobacter pylori (Helicobacter pylori) ferritin (hpfer) fusion. Average ± SD of n=5 mice are shown in (b) and (d).

Fig. 4: the 3D biodistribution of the surrogate mouse multhady was comparable to its parent IgG. 15nm Gold Nanoparticle (GNP) biodistribution of the Alexa-647 labeled MB and IgG samples following subcutaneous injection into BALB/c mice was observed by real-time non-invasive 3D whole body imaging. a) Representative 3D rendered fluoroscopic images overlaid with CT scan from PBS injection control. b) CT scan localization description of overlapping mouse major organs. c) 3D rendered fluoroscopic images overlapping CT scans 1 hour (1H), 2 days (D2), 8 days (D8) and 11 days (D11) after subcutaneous injection of gold nanoparticles (upper panel), MB (middle three panels) or IgG (lower panel). Each 3D image set is shown showing a back view overlapping a CT scan (right), and selected front (top left), middle (middle) and cross-section (bottom left) based on signal localization. Mapping the 3D fluorescence image as a Rainbow look-up table (LUT), setting the color scale minimum to background and the maximum to 50pmol M ^-1 cm ^-1 (GNP) or 1000pmol M ^-1 cm ^-1 (MB and IgG).

Fig. 5: protein engineering of multimeric IgG-like particles against SARS-CoV-2. a. Schematic representation of the human apoferritin partition design. Negative staining electron micrograph of mb. (scale bar 50nm, representing two independent experiments). Hydrodynamic radius of mb (Rh). d. Binding to SARS-CoV-2 spike protein (apparent K) for 4A8 (purple) and BD23 (grey) _D ) Affinity influence of (a) is provided. e. Sensorgrams of BD23-IgG and MB binding to fcγri (up), fcRn at endosomal pH (middle row), and FcRn at physiological pH (down) with different Fc sequence variants. The red line represents the raw data and the black line represents the global fit. f. Neutralization of SARS-CoV-2PsV by 4A8 and BD23 IgG and MB. Representative data for three biologically independent samples are shown. Average ± SD of two technical replicates is shown in each neutralization assay. Three biologically independent repeat ICs are shown ₅₀ Average value.

Fig. 6: multhady enhances the efficacy of human monoclonal antibodies by phage display. a. The workflow of identifying potent anti-SARS-CoV-2 neutralizers using MB technology. Created using a Biorender. b. A comparison of neutralization potency between IgG (cyan) and MB (pink) derived from phage display of the same human Fab sequences is shown. c. IC after multimerization ₅₀ The value is increased by a multiple. d. The apparent affinity (K) of MB (pink) was most effectively neutralized compared to the IgG counterpart (cyan) that binds SARS-CoV-2S protein _D ) Binding ratio (k) _on ) Dissociation rate (k) _off ). Three biological repeats and their ICs are shown in (b) and (c) ₅₀ Average of the values.

Fig. 7: neutralization of Multabody and its parent IgG targeting SARS-CoV-2 RBD. a) Representative neutralization titration curves for 20 anti-SARS-CoV-2 PsV antibodies when displayed as IgG (black) and MB (dark red). Three biological repeats are shown Average IC ₅₀ Values are compared. Average ± SD of two technical replicates is shown in each neutralization plot. b) Neutralization profile of selected IgG and MB against SARS-CoV-2PsV targeting 293T-ACE2 (black) and HeLa-ACE2 (grey) target cells. Average IC of three and two biological repeats of 293T-ACE2 and HeLa-ACE2 cells, respectively, are shown ₅₀ Value and respective IC ₅₀ Values. c) Neutralization titration curves for three biological replicates (different gray levels) of the authentic SARS-CoV-2/SB2-P4-PB strain. Shows the average IC ₅₀ . The neutralizing capacity of recombinant mabs REGN10933 (red) and REGN10987 (blue) is included in (a) and (c) as a benchmark for comparison.

Fig. 8: expression yield and homogeneity of Multabody targeting SARS-CoV-2 RBD. a) Seven most potent IgG (white) and their respective MB (dark red) yields (mg/L). Mean ± standard deviation of two biologically independent samples. b) The polymerization temperature (Tagg, DEG C) is shown in (a). The solid line represents the average Tagg value for two biologically independent samples. c) SEC chromatograms from three independently expressed and purified 298IgG (top row, black) and 298MB (bottom row, dark red). In both cases, the sample was purified using protein a affinity chromatography prior to SEC. The arrow indicates the peak value for each batch of PsV neutralization assays. Note that IC ₅₀ Value (μg/mL). Average ± SD of two technical replicates is shown in each neutralization plot.

Fig. 9: binding profile of IgG and MB. Sensorgrams of IgG and MB bound to RBD (left) and S protein (right) of SARS-CoV-2 immobilized on Ni-NTA biosensor. 2-fold serial dilutions of 125 to 4nM (IgG) and 16 to 0.5nM (MB) were used. The red line represents the raw data and the black line represents the global fit.

Fig. 10: epitope profiling specific for the most potent mabs. RBD (light green core, dark green RBM) and ACE2 ⁶⁶ (light brown) combined surface and cartoon schematic. A heat map of the binding competition experiment is shown. A high signal response (red) indicates low contention, while a low signal response (white) corresponds to high contention. Epitope bins (bins) are highlighted by dashed boxes.Frozen EM reconstitution of filtered spike protein (grey) complexes with Fab 80 (yellow), 298 (orange) and 324 (red). RBD and NTD are shown in green and blue, respectively. Frozen electron microscopy reconstitution of fab 46 (pink) and RBD (green) complexes. RBD is put into ⁶⁶ The secondary structure cartoon fits into the local density (partial density) observed for RBD. d. Crystal structure of ternary complex formed by Fab 52 (purple), fab 298 (orange) and RBD (green). e. Synthetic images depicting uncomplexed (PDB 6XM 4) and antibody-bound SARS-CoV-2 spike with available PDB or EMD entries ^{3,4,9,10,13,15,17,67,68,69,70,71,72} Side and top views of (a). Insert: close-up of antibodies targeting different antigenic sites on the RBD. Selection of IC with lowest report for SARS-CoV-2PsV ₅₀ The mAb of the values are representative antibodies of the bins (highlighted in bold), and those with similar binding epitopes are listed under the same color (color coding of spike, NTD and RBD as shown in (b)). The individual protomers in the non-ligand spike are shown in white, pink and purple.

Fig. 11: epitope binding. Binding competition experiments of mAb with His-tagged RBD as measured by Biological Layer Interferometry (BLI). 50 μg/ml mAb 1 was incubated for 3 min and then 50 μg/ml mAb 2 for 5 min.

Fig. 12: cryo-electron microscopy analysis of Fab spikes and Fab RBD complexes. Representative cryo-electron micrographs (scale 50nm, top left), selected 2D class mean (top right), final 3D non-uniformly refined fourier shell correlation curve (bottom left) and local plots plotted on the cryo-electron micrograph surface of Fab 80-spike protein complex (a), fab 298 spike protein complex (b), fab 324 spike protein complex (c) and Fab 46-RBD complex (D) are shown(lower right).

Fig. 13: multhady overcomes the SARS-CoV-2 sequence diversity. a. Four cartoon schematic representations of naturally occurring mutated RBDs are shown as spheres. Epitopes of mAbs 52 (light pink) and 298 (yellow) are shown as representative tables for each bin Bits. Affinity between WT and mutant RBD and PsV and c IC ₅₀ And (5) comparing multiple changes. d. In contrast to WT PsV, igG (gray bar) neutralizes the SARS-CoV-2PsV variant relative to MB (dark red bar). e. Neutralization potency of two IgG mixtures (three IgG), monospecific MB mixtures (three MB) and trispecific MB against wild-type SARS-CoV-2PsV and variants was compared. Mabs sensitive to one or more PsV variants (d) were selected to produce mixed and trispecific MBs. f. Neutralization potency of trispecific 298-80-52MB against SARS-CoV-2B.1.351PsV variant. g. PsV (y-axis) demonstrating the ability of trispecific MB (Red) to enhance the efficacy of a broad spectrum of mAb properties (blue and black) and an IC with replication-competent SARS-CoV-2 virus (SB 2-P4-PB: x-axis) ₅₀ Values. h.IC ₅₀ The value was increased by a multiple after multimerization. The average of three biological replicates is shown in (b-h).

Fig. 14: MB effectively overcomes SARS-CoV-2 sequence variability. a) Comparison of neutralization potency of selected IgG and MB against WT PsV (dark red) and more infectious D614G PsV (gray). b) Schematic representation of a tri-specific MB generated by combining three Fab-specific and Fc fragments using an MB split design. c) Mixed and trispecific MBs combining the specificities of mabs 298, 80 and 52 or 298, 324 and 46 were generated and tested against WT PsV. Average ± SD of two technical replicates is shown in each representative neutralization plot. The source data is provided in the form of a source data file. d) The neutralizing potency of mixed and trispecific MBs against pseudotyped SARS-CoV-2 variants was altered compared to wild-type PsV. Variants of PsV sensitive to the individual antibodies within the mixed antibodies were selected. The area within the dashed line represents an IC ₅₀ The value varies 3 times. This threshold is determined as a cut-off value for an increase in sensitivity (upper bar) or resistance (lower bar). e) Three biologically repeated neutralization titration curves for the mixed and trispecific MB of the baseline SARS-CoV-2/SB2-P4-PB strain are shown. Shows the average IC of three biologically independent replicates ₅₀ Values.

Fig. 15: in the WT pseudovirus neutralization assay, the N92T mutation had no effect on potency as IgG or monospecific MB.

Fig. 16: 298-80-52 trispecific MBs containing the N92T mutation in the VL of mAb 52 were screened in the p.1psv neutralization assay, confirming that no potency loss compared to the parental trispecific MB was observed.

Fig. 17: in the pseudovirus neutralization assay, the efficacy of trispecific MB 298-80-52 in the relevant Variant (VOC) was evaluated.

Detailed description of certain aspects

Definition of the definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin lewis, genes V, oxford University Press,1994 publications (ISBN 0-19-854287-9); kendrew et al (eds.), the Encyclopedia of Molecular Biology, blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, inc.,1995 publication (ISBN 1-56081-569-8). Although any methods and materials similar or equivalent to those described herein can be used in the testing practice of the present invention, the exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

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 cited herein to aid in the understanding of the described aspects. Each of these references is incorporated by reference herein in its entirety.

In understanding the scope of the present application, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. Furthermore, the term "comprising" and derivatives thereof as used herein is intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and/or steps. The above definitions apply to words of similar import such as "comprising", "having" and derivatives thereof.

It should be understood that any aspect described as "comprising" certain components may also "consist of …" or "consist essentially of …", where "consisting of …" has a closed or limiting meaning, "consisting essentially of …" means including the specified components, but excluding other components except for materials present as impurities, unavoidable materials present as a result of the method used to provide the components, and components added to achieve purposes other than the technical effects of the present application. For example, a composition defined using the phrase "consisting essentially of …" encompasses any known acceptable additive, excipient, diluent, carrier, etc. Typically, a composition consisting essentially of a set of components will comprise 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.

It should be understood that any component defined herein as comprising may be explicitly excluded from the claimed invention by way of a conditional or disclaimer limitation. For example, in some aspects, the nanocages and/or fusion proteins described herein may exclude ferritin heavy chains and/or may exclude iron binding components.

Furthermore, all ranges given herein include the end of the range as well as any intermediate range points, whether or not explicitly stated.

Terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. If a deviation of such degree term would not negate the meaning of the word it modifies, it should be interpreted as comprising, up to and including at least + -5% of the deviation of the modified term. For example, the term "about" may encompass a range of values 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 stated reference value. "

The abbreviation "e.g." originates from latin example gratia, used to represent a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example" or "such as (such as)". The term "or" is intended to include "and" unless the context clearly indicates otherwise.

The term "subject" as used herein 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 animals, and farm animals, as well as zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and the like. Typically, the mammal is a human.

The terms "protein nanoparticle", "nanocage" and "multabody" are used interchangeably herein to refer to a multi-subunit, protein-based polyhedral shaped structure. The subunits or nanocage monomers are each comprised of a protein or polypeptide (e.g., glycosylated polypeptide) and optionally consist of single or multiple of the following features: nucleic acids, prosthetic groups, organic 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), encapulein nanoparticles (see, e.g., sutter et al, nature Struct and mol.biol.,15:939-947,2008, incorporated herein by reference), thiooxidoreductase (SOR) nanoparticles (see, e.g., ui et al, science,311:996-1000,2006, incorporated herein by reference), dioxatetrahydropteridine synthase nanoparticles (see, e.g., zhang et al, j.mol.biol.,306:1099-1114,2001), or pyruvate dehydrogenase nanoparticles (see, e.g., ixd et al, PNAS 96:1240-1245,1999, incorporated herein by reference). Ferritin, apoferritin, encapsulin, SOR, tetrahydropteridine dioxygenase and pyruvate dehydrogenase are monomeric proteins that self-assemble into a globular protein complex, consisting in some cases of 24, 60, 24, 60 and 60 protein subunits, respectively. Ferritin and apoferritin are generally referred to interchangeably herein and are understood to both apply to the fusion proteins, nanocages and methods described herein. Carboxylase, vault proteins, groEL, heat shock proteins, E2P and MS2 coat proteins can also produce nanocages, contemplated for use herein. In addition, fully or partially synthesized self-assembled monomers may also be used herein.

It is understood that each nanocage monomer may be split into two or more subunits that will self-assemble into functional nanocage monomers. For example, ferritin or apoferritin may be split into N-and C-subunits, such as those obtained by substantially bisecting full-length ferritin, such that each subunit may be bound to a different SARS-CoV-2 binding moiety or bioactive moiety, respectively, followed by self-assembly into nanocage monomers and then assembly into nanocages. In various aspects, each subunit can bind to a SARS-CoV-2 binding moiety and/or a biologically active moiety at the same or different ends. By "functional nanocage monomers" is meant nanocage monomers that are capable of self-assembling with other such monomers into nanocages as described herein.

The terms "ferritin" and "apoferritin" are used interchangeably herein to generally refer to a polypeptide (e.g., ferritin chain) capable of assembling into a ferritin complex, which typically includes 24 protein subunits. It should be understood that ferritin may be from any species. Typically, the ferritin is human ferritin. In some embodiments, the ferritin is wild-type ferritin. For example, the ferritin may be wild-type human ferritin. In some embodiments, ferritin light chains are used as nanocage monomers and/or subunits of ferritin light chains are used as nanocage monomer subunits. In some embodiments, the assembled nanocages do not include any ferritin heavy chains or other ferritin components capable of binding to iron.

The term "multispecific" as used herein refers to a feature having at least two binding sites at which at least two different binding partners, such as antigens or receptors (e.g., fc receptors), can bind. For example, a nanocage comprising at least two Fab fragments is "multispecific", wherein each of the two Fab fragments binds to a different antigen. As another example, nanocages comprising an Fc fragment (capable of binding to an Fc receptor) and a Fab fragment (capable of binding to an antigen) are also "multispecific".

As used herein, the term "multivalent" refers to a feature having at least two binding sites at which a binding partner, such as an antigen or receptor (e.g., fc receptor), can bind. The binding partners that can bind to at least two binding sites can be the same or different.

The term "antibody", as used herein, also known in the art as "immunoglobulin" (Ig), refers to a protein constructed from paired heavy and light chain polypeptide chains; there are various Ig isotypes, including IgA, igD, igE, igG, such as IgG ₁ 、IgG ₂ 、IgG ₃ And IgG ₄ And IgM. It will be appreciated that the antibodies may be from any species including human, mouse, rat, monkey, llama or shark. When an antibody is properly folded, each chain folds into multiple distinct globular domains, linked by more linear polypeptide sequences. For example, in the case of IgG, immunoglobulin light chains fold to be variable (V _L ) And constant (C) _L ) Domain, while heavy chain is folded to be variable (V _H ) And three constant (C _H ，C _H2 ，C _H3 ) A domain. Heavy and light chain variable domains (V _H And V _L ) The interaction of (a) results in the formation of an antigen binding region (Fv). Each domain has a well-defined structure familiar to those skilled in the art.

The light and heavy chain variable regions are responsible for binding to the target antigen and thus can show significant sequence diversity between antibodies. The constant region shows less sequence diversity and is responsible for binding many natural proteins to elicit important immune events. The variable region of an antibody contains the antigen binding determinants of the molecule, thereby determining the specificity of the antibody for its target antigen. Most sequence variations occur in six hypervariable regions, three for each variable heavy and light chain; the hypervariable region forms an antigen binding site and facilitates binding and recognition of an epitope. The specificity and affinity of an antibody for its antigen depends on the structure of the hypervariable region and the size, shape, and chemical nature of its surface presented to the antigen.

The "antibody fragment" as described herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally occurring antibody fragment, also Can be obtained by manipulation of naturally occurring antigens or by using recombinant methods. For example, antibody fragments may include, but are not limited to, fv, single chain Fv (scFv; V linked by a peptide linker) _L And V _H Composed molecules), fc, single chain Fc, fab, single chain Fab, F (ab') ₂ Single domain antibodies (sdAb; from a single V) _L Or V _H Composition), and multivalent display of any of these antibodies.

As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques. The term should also be construed to refer to antibodies produced by synthesizing DNA molecules encoding the antibodies, and which express the antibody protein, or specify the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

The term "epitope" refers to an antigenic determinant. An epitope is a specific chemical group or peptide sequence that is antigenic on a molecule, i.e., elicits a specific immune response. Antibodies specifically bind to a particular epitope, e.g., an epitope on a polypeptide. Epitopes can be formed either by contiguous amino acids of a protein or by non-contiguous amino acids juxtaposed by tertiary folding. Epitopes formed by consecutive amino acids are typically retained upon exposure to denaturing solvents, whereas the number of epitopes formed by tertiary folding is typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5, about 9, about 11, or about 8 to about 12 amino acids in a unique spatial conformation. Methods for determining epitope spatial conformation 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).

The term "antigen" as used herein is defined as a molecule that elicits an immune response. Such an immune response may involve the production of antibodies, or the activation of specific immune competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including almost any protein or peptide, may be used as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. Those of skill in the art will understand that any DNA comprising a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response, and thus encodes an "antigen" as used herein. Furthermore, one skilled in the art will appreciate that an antigen need not be encoded solely by the full length nucleotide sequence of a gene. It will be apparent that aspects described herein include, but are not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences may be arranged in various combinations to elicit the desired immune response. Furthermore, one skilled in the art will appreciate that antigens need not be encoded by a "gene" at all. It is apparent that the antigen may be synthesized or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, cells, or biological fluids.

"coding" refers to the inherent properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, for use as a template in the synthesis of other polymers and macromolecules in biological processes having a particular nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a particular amino acid sequence, and the biological properties resulting therefrom. Thus, if transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, typically provided in the sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be referred to as encoding the protein or other product of the gene or cDNA.

As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

"isolated" means altered or detached from its natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely isolated from coexisting materials in its natural state, is "isolated. The isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment such as a host cell.

Unless otherwise specified, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, such that the nucleotide sequence encoding the protein may comprise introns in some versions.

As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a subject's response compared to the level of a subject's response in the absence of a treatment or compound, and/or compared to the level of a response of an otherwise identical but untreated subject. The term encompasses interference and/or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject (typically a human).

The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleotide sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, if necessary, join two protein coding regions in the same reading frame.

"parenteral" administration of a composition includes, for example, subcutaneous injection (s.c.), intravenous injection (i.v.), intramuscular injection (i.m.), or intrasternal injection or infusion techniques. Inhalation and intranasal administration are also included.

As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. As is generally known to those skilled in the art, a nucleic acid is a polynucleotide that can be hydrolyzed to a monomer "nucleotide". Monomeric nucleotides can be hydrolyzed to nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR, etc., as well as synthetically.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids a protein or peptide sequence can contain. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and long chains, which are commonly referred to in the art as proteins, which are of many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, the term "specifically binds" with respect to an antibody refers to 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 an antigen from one species may also bind the antigen from one or more species. However, this cross-species reaction does not itself alter the specific classification of antibodies. In another example, antibodies that specifically bind an antigen may also bind different allelic forms of the antigen. However, this cross-reactivity does not itself alter the specific classification of antibodies. In some cases, the term "specifically bind" or "specifically bind" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on the presence of a particular structure (e.g., an epitope or epitope) on the chemical substance; for example, antibodies recognize and bind to a particular protein structure, rather than recognizing and binding to proteins in general. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction containing labeled "A" and the antibody.

The term "therapeutically effective amount," "effective amount," or "sufficient amount" refers 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 can vary depending on such factors as the molecule, age, sex, species and weight of the subject. As will be appreciated by those skilled in the art, the dosage or treatment regimen may be adjusted to provide the optimal therapeutic response. For example, administration of a therapeutically effective amount of a fusion protein described herein is in some aspects sufficient to treat and/or prevent covd-19.

Furthermore, a treatment regimen for treating a subject with a therapeutically effective amount may comprise a single administration, or a series of applications. The frequency and length of the treatment cycle depends on a variety of factors, such as the molecule, the age of the subject, the concentration of the agent, the patient's responsiveness to the agent, or a combination thereof. It will also be appreciated that the effective dose of the agent for treatment may be increased or decreased during a particular treatment regimen. The variation in dosage may be produced and apparent by standard diagnostic assays known in the art. The fusion proteins described herein may in some aspects be administered prior to, during, or after conventional therapies for treating the disease or disorder. For example, the fusion proteins described herein may be used in combination with conventional therapies for viral infections.

As used herein, the term "transfection" or "transformation" or "transduction" refers to the process whereby an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell refers to a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary subject cells and their progeny.

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

A "vector" is a composition of matter comprising an isolated nucleic acid that can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides conjugated to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

"in combination" with one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

The term "pharmaceutically acceptable" means that the compound or combination of compounds is compatible with the remaining ingredients of the pharmaceutical formulation and is generally safe for administration to the human body according to established government standards, including standards promulgated by the U.S. food and drug administration.

The term "pharmaceutically acceptable carrier" includes, but is not limited to, solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and/or absorption delaying agents, and the like. The use of pharmaceutically acceptable carriers is well known.

By "variant" is meant a fusion protein, antibody or fragment thereof having biological activity, the amino acid sequence of which differs from the control sequence by the insertion, deletion, modification and/or substitution of one or more amino acid residues in the comparison sequence. Variants typically have less than 100% sequence identity to the comparison sequence. Typically, however, a biologically active variant has an amino acid sequence that has at least about 70% amino acid sequence identity to the comparison sequence, e.g., 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 a certain level of biological activity of the comparison sequences. Variants also include polypeptides in which one or more amino acid residues are added at the N-or C-terminus of or within the comparison sequence. Variants also include polypeptides in which many amino acid residues are deleted and/or optionally substituted with one or more amino acid residues. Variants may also be modified covalently, for example by substitution with moieties other than naturally occurring amino acids or by modification of amino acid residues to produce non-naturally occurring amino acids.

"percent amino acid sequence identity" is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to residues in a sequence of interest (e.g., a polypeptide of the invention) after the sequences are aligned and gaps are introduced (if necessary) to achieve maximum sequence identity, and without regard to any conservative substitutions as part of sequence identity. Neither the N-terminal, C-terminal, or internal extension, deletion, or insertion 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".

"active" or "activity" as used herein refers to the biological and/or immunological activity of the fusion proteins described herein, wherein "biological" activity refers to the biological function (inhibition or stimulation) caused by the fusion protein.

The fusion proteins described herein may include modifications. Such modifications include, but are not limited to, conjugation to effector molecules. Modifications further include, but are not limited to, conjugation to a detectable reporter moiety. Modifications that extend half-life (e.g., pegylation) are also included. Modifications that deimmunize are also included. Protein and non-protein formulations may be conjugated to fusion proteins by methods known in the art. Conjugation methods include direct ligation, ligation by covalently linked linkers, and specific binding pair members (e.g., avidin-biotin). For example, these include those described by Greenfield et al, cancer Research 50,6600-6607 (1990), which is incorporated herein by reference, and by reference, those described by 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.

Fusion proteins

Described herein are fusion proteins. The fusion protein comprises a nanocage monomer linked to a SARS-CoV-2 binding moiety. The plurality of fusion proteins self-assemble to form a nanocage. In this way, the SARS-CoV-2 binding moiety can decorate the inner surface of the assembled nanocage, the outer surface of the assembled nanocage, or both.

The SARS-CoV-2 binding moiety is typically an antibody or fragment thereof, and while it can target any portion of the SARS-CoV-2 virus, it is typically targeted to the SARS-CoV-2S glycoprotein. It will be appreciated that the SARS-CoV-2 binding moiety need not be an antibody or fragment thereof, and can be, for example, a molecule, such as a protein, that binds to and blocks the viral or RBD domain of a virus.

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

Generally, the fusion proteins described herein are associated with Fab light and/or heavy chains, which may be produced separately or sequentially from the fusion protein.

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

In certain aspects, nanocage monomers described herein may be partitioned into subunits, allowing more SARS-CoV-2 binding moieties or other moieties to be attached thereto in various proportions. For example, in some aspects, 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 the second nanocage monomer subunit to form within the nanocage monomer. As described above, a plurality of nanocage monomers self-assemble to form nanocages. The nanocage monomer subunits may be provided separately or in combination and may have the same or different SARS-CoV-2 binding moiety fused thereto.

Nanocages made from the nanocage monomers and/or nanocage monomer subunits described herein may have a biologically active moiety in addition to one or more SARS-CoV-2 binding moieties.

For example, the biologically active moiety may comprise one or both chains of, for example, an Fc fragment. The Fc fragment may be derived from any type of antibody, but is typically an IgG1 Fc fragment. The Fc fragment may further comprise one or more mutations, such as LS, YTE, LALA, I253A and/or LALAP, which modulate the half-life and/or effector function of the fusion protein and/or the resulting assembled nanocages comprising the fusion protein. For example, the half-life may be minutes, days, weeks, or even months.

In addition, other substitutions in the fusion proteins and nanocages described herein are also contemplated, including Fc sequence modifications and the addition of other agents (e.g., human serum albumin peptide sequences), which allow for variations in bioavailability, and will be understood by the skilled artisan. In addition, the fusion proteins and nanocages described herein can be modulated sequentially or by addition of other agents to attenuate immunogenicity and drug-resistance responses (therapeutic, such as sequences matched to the host, or to add immunosuppressive therapy [ e.g., methotrexate can be added when infliximab is administered to treat rheumatoid arthritis or induce neonatal tolerance), which is a major strategy to reduce the occurrence of inhibitors against FVIII (reviewed in DiMichele DM, houts WK, pipe SW, rivard GE, santagogstino E. International workshop on immune tolerance induction: contacts recommends. Haemophilia.2007;13:1-22, which is incorporated herein by reference in its entirety).

In certain embodiments, the fragment crystallizable (Fc) region comprises an I253A mutation. In some embodiments, the Fc region further comprises a LALAP (L234A/L235A/P329G) mutation. Mutation numbering in the present disclosure is according to EU numbering unless otherwise indicated.

In some embodiments, the Fc region is an IgG1 Fc region (e.g., a human IgG1 Fc region), i.e., in addition to the mutations described herein, the Fc region comprises Fc chains each having an amino acid sequence substantially similar to the amino acid sequence of a wild-type IgG1 Fc chain. In some embodiments, the wild-type reference IgG1 Fc is a human IgG1 Fc, wherein each Fc chain has the amino acid sequence of SEQ ID NO: 24.

For example, an IgG1 Fc region can 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 amino acid sequence of an Fc chain within a wild-type IgG1 Fc. In some embodiments, the IgG1 Fc region comprises an Fc chain comprising an Fc mutation specifically described for the IgG1 Fc region, but having an amino acid sequence 100% identical to an Fc chain within a wild-type IgG1 Fc.

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

Where the antibody or fragment thereof comprises two chains, e.g. a first and a second chain in the case of an Fc fragment, or a heavy and a light chain, the two chains are optionally separated by a linker. The joints may be flexible or rigid, but are typically flexible to allow the chain to fold properly. The linker is typically long enough to impart some flexibility to the fusion protein, but it is understood that the linker length will vary depending on the nanocage monomer and bioactive moiety sequences as well as the three-dimensional conformation of the fusion protein. Thus, the linker is typically from about 1 to about 130 amino acid residues, such as 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, 100, 105110, 115, 120, 125 or 130 amino acid residues, such as about 50 to about 90 amino acid residues, such as 70 amino acid residues.

The linker can be any amino acid sequence, and in a typical example, the linker comprises GGS repeats, and more typically, the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, e.g., about 4 GGS repeats. In particular aspects, 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:

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS。

In certain embodiments, the linker is used in a fusion polypeptide and/or a single chain molecule such as scFc. In some embodiments, the linker is an amino acid linker. For example, a linker as used herein may comprise from about 1 to about 100 amino acid residues, such as from about 1 to about 70, from about 2 to about 70, from about 1 to about 30, or from about 2 to about 30 amino acid residues. In some embodiments, the linker comprises 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.

In certain embodiments, the linker comprises a glycine-serine sequence, e.g. (G _n S) _m Sequences (e.g., GGS, GGGS, or GGGGS sequences) that are present in at least 1, at least 2, at least 3, 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, or at least 14 copies within the linker.

In a typical aspect, 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, e.g., binding domain 298, 52, 46, 80, 82, 236, 324, or a combination thereof, e.g., in Table 4.

In certain embodiments, the SARS-CoV-2 binding antibody fragment is capable of binding to the Receptor Binding Domain (RBD) of SARS-CoV-2. In certain embodiments, the SARS-CoV-2 binding antibody fragment is capable of binding to the spike protein (S protein) of SARS-CoV-2. In some embodiments, the SARS-CoV-2 binding antibody fragment is capable of binding to the N-terminal domain (NTD) of the SARS-CoV-2S protein.

In some embodiments, the SARS-CoV-2 binding antibody fragment comprises a heavy chain variable region (e.g., V _H Or V _H H) A. The invention relates to a method for producing a fibre-reinforced plastic composite 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 Fab comprising a heavy chain variable domain (e.g., V _H ) And a light chain variable domain (e.gV _L Or V _K )。

In some embodiments, the SARS-CoV-2 binding antibody fragment comprises V _H Heavy chain variable domains and V _K Light chain variable domains. In some embodiments, the SARS-CoV-2 binding antibody fragment comprises a Fab comprising V _H Heavy chain variable domains and V _K Light chain variable domains.

In a specific example, 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:

Fc chain 1:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK；

fc chain 2:

298 light chain

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

298Fab heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGTFSTYGISWVRQAPGQGLEWMGWISPNSGGTDLAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASDPRDDIAGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

52 light chain

DIQMTQSPSSLSASVGDRVTITCRASQGISNNLNWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNGFPLTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

52Fab heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGGIIPMFGTTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDRGDTIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

46 light chain

DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

46Fab heavy chain

EVQLLESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGDSRDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

80 light chain

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

80Fab heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGGTFNRYAFSWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSTRELPEVVDWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

82 light chain

DIQMTQSPSSLSASVGDRVTITCRASQVISNYLAWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPPPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

82Fab heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGSFSTSAFYWVRQAPGQGLEWMGWINPYTGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARSRALYGSGSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

236 light chain

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

236Fab heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGTFTSYGINWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCASRGIQLLPRGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

324 light chain

DIQMTQSPSSLSASVGDRVTITCRASQSITTYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

324Fab heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGTFNNYGISWVRQAPGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVGDYGDYIVSPFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

Or a combination thereof.

In a further aspect, the antibody or fragment thereof is conjugated or bound to another moiety, e.g., a detectable moiety (e.g., a small molecule, fluorescent molecule, radioisotope, or magnetic particle), pharmaceutical formulation, diagnostic agent, or combination thereof, and may comprise, e.g., an antibody-drug conjugate.

In some aspects wherein the biologically active moiety is a detectable moiety, the detectable moiety may comprise a fluorescent protein, e.g., GFP, EGFP, ametrine, and/or a flavin-based fluorescent protein, e.g., a LOV protein, e.g., iLOV.

In aspects where the biologically active moiety is a pharmaceutical formulation, the pharmaceutical formulation may comprise, for example, a small molecule, peptide, lipid, carbohydrate, or toxin.

In typical aspects, nanocages assembled from fusion proteins described herein comprise from about 3 to about 100 nanocage monomers, e.g., from 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, 94, 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 monomers may be any known natural, synthetic or partially synthetic nanocage monomers and are selected in some aspects from ferritin, apoferritin, encapsulin, SOR, tetrahydropteridine dioxygenase, pyruvate dehydrogenase, carboxylase, vault protein, groEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof. Typically, the nanocage monomers are ferritin or apoferritin.

When apoferritin is selected as the nanocage monomer, typically the first and second nanocage monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin. It is understood that other nanocage monomers may be split into subunits in duplicate, much like the apoferrins described herein, such that the subunits self-assemble and each subunit is suitable for fusion with a biologically active moiety.

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 is capable of self-assembling in the presence of other ferritin chains into a polypeptide complex comprising a plurality of ferritin chains. In some embodiments, the ferritin chains self-assemble into a polypeptide complex comprising 24 or more ferritin chains. In some embodiments, the ferritin monomer is a ferritin light chain. In some embodiments, the ferritin monomer does not include a ferritin heavy chain or other ferritin component capable of binding to iron.

In some embodiments, each fusion polypeptide within the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of ferritin light chain. In these embodiments, the self-assembled polypeptide complex does not comprise any ferritin heavy chain or subunits of ferritin heavy chain.

In some embodiments, the ferritin monomer is a human ferritin chain, such as 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.

"subunit" of a ferritin monomer refers to a portion of a ferritin monomer that is capable of spontaneously binding to a different subunit of a ferritin monomer such that these subunits together form a ferritin monomer that is in turn capable of self-assembly with other ferritin monomers to form a polypeptide complex.

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 the ferritin chain, the half comprising the N-terminus of the ferritin chain. As used herein, the term "C-half-ferritin" refers to about half of the ferritin chain, including the C-terminus of the ferritin chain. The precise sites at which the ferritin chains may be separated to form N-half-ferritin and C-half-ferritin may vary depending on the embodiment. For example, in the case of a ferritin monomer subunit based on a human ferritin light chain, the two halves may be separated at a site corresponding to a position between about 75 to about 100 of SEQ ID NO. 1. For example, in some embodiments, the N-half-ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 1-95 (or a major portion thereof) of SEQ ID No. 1, and the C-half-ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 96-175 (or a major portion thereof) of SEQ ID No. 1.

In some embodiments, the two halves are separated at a site corresponding to a position between about position 85 to about position 92 of SEQ ID NO. 1. For example, in some embodiments, the N-half-ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 1-90 (or a major portion thereof) of SEQ ID No. 1, and the C-half-ferritin based on the human ferritin light chain has an amino acid sequence corresponding to residues 91-175 (or a major portion thereof) of SEQ ID No. 1.

Typically, the "N" region of the 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 sequence:

MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEG VSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEW。

typically, the "C" region of the 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 sequence:

or (b)

In some aspects, the fusion proteins described herein further comprise a linker between the nanocage monomer subunit and the biologically active moiety, much like the linker described above. Likewise, the linker may be flexible or rigid, but is typically flexible to allow the bioactive moiety to remain active and the paired nanocage monomer subunits to remain self-assembling. The linker is typically long enough to impart some flexibility to the fusion protein, but it is understood that the length of the linker will vary depending on the nanocage monomer and bioactive moiety sequences as well as the three-dimensional conformation of the fusion protein. Thus, the linker is typically from about 1 to about 30 amino acid residues, e.g., 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, 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.

The linker may be any amino acid sequence, and in a typical example, the linker comprises GGS repeats, and more typically the linker comprises about 2, 3, 4, 5 or 6 GGS repeats, e.g., about 4 GGS repeats. In particular aspects, 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:

GGGGSGGGGSGGGGSGGGGSGGGGSGG。

similarly, the fusion protein may further comprise a C-terminal linker for improving one or more properties of the fusion protein. In some aspects, it comprises GGS repeats, and more typically, the linker comprises about 2, 3, 4, 5, or 6 GGS repeats, e.g., about 4 GGS repeats. In particular aspects, 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:

GGSGGSGGSGGSGGGSGGSGGSGGSG

also described herein are a pair of the above fusion proteins, wherein the pair of fusion proteins self-assemble to form a nanocage monomer, wherein the first and second nanocage monomer subunits are fused to different SARS-CoV-2 binding moieties. This provides multivalent and/or multispecific properties to individual nanocage monomers assembled from paired subunits.

Substantially identical sequences may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations of a reference sequence can produce a mutant peptide that has no substantial change in physiological, chemical, or functional properties as compared to the reference sequence; in this case, the reference sequence and the mutant sequence will be considered as "substantially identical" polypeptides. Conservative amino acid mutations may include additions, deletions or substitutions of amino acids; conservative amino acid substitutions are defined herein as the substitution of one amino acid residue with another amino acid residue having similar chemical properties (e.g., size, charge, or polarity).

In one non-limiting example, the conservative mutation may be an amino acid substitution. Such conservative amino acid substitutions may replace another amino acid in the same group with a basic, neutral, hydrophobic or acidic amino acid. The term "basic amino acid" refers to hydrophilic amino acids having a side chain pK value greater than 7, which are generally 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 referred to as "polar amino acid") refers to a hydrophilic amino acid whose side chains are uncharged at physiological pH, but have at least one bond in which a pair of electrons shared by two atoms is more tightly held 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 referred to as "nonpolar amino acid") refers to a standardized common hydrophobicity scale (normalized consensus hydrophobicity scale) according to Eisenberg (1984), including amino acids exhibiting a hydrophobicity greater than zero. 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).

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

Sequence identity was used to evaluate similarity of two sequences; this is determined by calculating the percentage of residues that are identical when two sequences are aligned to obtain the maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software may be used to calculate sequence identity. Without wishing to be limiting, sequence identity may be calculated by software, such as the NCBI BLAST2 service, BLAST-P, blast-N or FASTA-N maintained by Swiss Institute of Bioinformatics (and found in ca. Expasy. Org/tools/BLAST /), or any other suitable software known in the art.

The substantially identical sequences of the invention may be at least 85% identical; in another example, a substantially identical sequence 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 a sequence described herein. In particular aspects, substantially identical sequences retain the activity and specificity of the reference sequence. In one non-limiting embodiment, the differences in sequence identity may be due to conservative amino acid mutations.

The polypeptides or fusion proteins of the invention may also comprise additional sequences to aid in their expression, detection or purification. Any such sequence or tag known to those skilled in the art may be used. For example, but not intended to be limiting, the fusion protein may comprise a targeting or signal sequence (e.g., without limitation ompA), a detection tag, an exemplary tag cassette comprising a Strep tag or any variant thereof; see, e.g., U.S. Pat. No. 7981632,His tag, flag tag with the sequence motif DYKDDDDK, xpress tag, avi tag, calmodulin tag, polyglutamate tag, HA tag, myc tag, nus tag, S tag, SBP tag, softag 1, softag 3, V5 tag, CREB Binding Protein (CBP), glutathione S-transferase (GST), maltose Binding Protein (MBP), green Fluorescent Protein (GFP), thioredoxin tag, or any combination thereof; purification tags (e.g., without limitation His) ₅ Or His ₆ ) Or a combination thereof.

In another example, the additional sequence may be a biotin recognition site, such as that described by Cronan et al in WO 95/04069 or Voges et al in WO 2004/076670. The linker sequence may be used in combination with additional sequences or tags, as known to those skilled in the art.

More specifically, the tag cassette may comprise an extracellular component that can specifically bind to the antibody with high affinity or avidity. Within the single chain fusion protein structure, the tag cassette may be located (a) directly amino-terminal to the linker region, (b) inserted between and linked to the linker module, (c) directly carboxy-terminal to the binding domain, (d) inserted between and linked to the effector domain (e) inserted between and linked to subunits of the binding domain, or (f) amino-terminal to the single chain fusion protein. In certain embodiments, one or more linking amino acids may be disposed between and linking the tag cassette and the hydrophobic moiety, or between and linking the tag cassette and the linking region, or between and linking the tag cassette and the linker module, or between and linking the tag cassette and the binding domain.

Also encompassed herein are isolated or purified fusion proteins, polypeptides, or fragments thereof immobilized on a surface using various methods; for example, but not intended to be limiting, the polypeptide may be attached or coupled to the surface by His-tag coupling, biotin binding, covalent binding, adsorption, or the like. The solid surface may be any suitable surface, such as, but not limited to, a well surface of a microtiter plate, a channel of a Surface Plasmon Resonance (SPR) sensor chip, a membrane, beads (e.g., magnetic or agarose based beads or other chromatographic resins), glass, a thin film, or any other useful surface.

In other aspects, the fusion protein may be linked to a cargo molecule; the fusion protein may deliver the cargo molecule to the desired site and may be attached to the cargo molecule using any method known in the art (recombinant techniques, chemical conjugation, chelation, etc.). The cargo molecule may be any type of molecule, such as a therapeutic or diagnostic agent.

In some aspects, the cargo molecule is a protein and is fused to the fusion protein such that the cargo molecule is contained internally within the nanocage. In other aspects, the cargo molecule is not fused to the fusion protein, but is contained internally within the nanocage. The cargo molecule is typically a protein, a small molecule, a radioisotope or a magnetic particle.

The fusion proteins described herein specifically bind to their targets. Antibody specificity refers to the selective recognition of a particular epitope by an antibody, and the antibody specificity of an antibody or fragment described herein may be determined based on affinity and/or avidity. Affinity, equilibrium constant (K) for dissociation of antigen from antibody _D ) Indicating that the antigen is measuredBinding strength between the determinant (epitope) and the antibody binding site. Avidity is a measure of the strength of binding between an antibody and its antigen. Antibodies are generally in the form of 10 ^-5 To 10 ^- ¹¹ K of M _D And (5) combining. Any of more than 10 ^-4 K of M _D Are generally considered to represent non-specific binding. K (K) _D The smaller the value, the stronger the binding strength between the epitope and the antibody binding site. In some aspects, the antibodies described herein have a binding capacity of less than 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 of M _D 。

Also described herein are nanocages 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 nanocage monomers. Further, described herein are paired fusion proteins, wherein the paired fusion proteins self-assemble to form a nanocage monomer, and wherein the first and second nanocage monomer subunits are fused to different bioactive moieties.

It will be appreciated that the nanocages may self-assemble from a plurality of the same fusion proteins, from a plurality of different fusion proteins (thus multivalent and/or multispecific), from a combination of fusion proteins and wild-type proteins, and any combination thereof. For example, the nanocages may 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 aspects, about 20% to about 80% of the nanocage monomers comprise the fusion proteins described herein. In view of the modular solution described herein, the nanocages may theoretically contain up to twice the number of bioactive moieties in the nanocages, as each nanocage monomer may be split into two subunits, each of which may independently bind a different bioactive moiety. It will be appreciated that such modularity may be utilized to achieve any desired ratio of bioactive molecules, such as a ratio of 4:2:1:1 for the four different bioactive moieties in the specific embodiments described herein. For example, a nanocage described herein may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different bioactive moieties. In this way, the nanocages may be multivalent and/or multispecific, and the extent thereof may be relatively easily controlled.

In various aspects, the nanocages described herein may further comprise at least one intact nanocage monomer, optionally fused to a biologically active moiety, which may be the same or different from the biologically active moiety attached to the nanocage monomer subunits described herein.

In a typical aspect, the nanocages described herein comprise a subunit or monomer optionally fused to a biologically active moiety and optionally first, second and third fusion proteins of at least one intact nanocage monomer, wherein the biologically active moieties of the first, second and third fusion proteins and the intact nanocage monomer are each different from each other.

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

In some aspects, 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 one typical aspect, the nanocages described herein comprise the following four fusion proteins, optionally in a ratio of 4:2:1:1:

a.298 (optionally sc 298), fused to full-length ferritin;

fc (optionally scFc), fused to N-ferritin;

c.46 or 52 (optionally sc46 or sc 52) fused to c-ferritin; a kind of electronic device with high-pressure air-conditioning system

d.324 or 80 (optionally sc324 or sc 80) fused to C-ferritin.

In various aspects, nanocages described herein comprise or consist of sequences that are 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, wherein ferritin subunits are indicated in bold, linkers are indicated in underlined, light chains are indicated in italics, and heavy chains are indicated in lower case:

a.298-hFerr：

or (b)

/>

b.Fc-N-hFerr LALAP I253A

c1.52-C-hFerr

/>

c2.46-C-hFerr

d1.324-C-hFerr

/>

d2.80-C-hFerr

In one aspect, there is provided a self-assembled polypeptide complex comprising a plurality of fusion polypeptides disclosed herein. In many embodiments, the self-assembled polypeptide complex comprises (1) a plurality of first fusion polypeptides, each first fusion polypeptide comprising an Fc region linked to a nanocage monomer (e.g., a ferritin monomer, such as a human ferritin monomer or subunit thereof), as disclosed herein; and (2) a plurality of second fusion polypeptides, each second fusion polypeptide comprising 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 Receptor Binding Domain (RBD)) linked to a nanocage monomer (e.g., a ferritin monomer, e.g., within a human ferritin monomer) or subunit thereof. In some embodiments, the self-assembled polypeptide complex further comprises a plurality of third fusion polypeptides, each third fusion polypeptide being different from the second fusion polypeptide and each comprising (1) a nanocage monomer (e.g., a ferritin monomer, e.g., a human ferritin monomer) linked 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).

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 one of the other 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 polypeptide comprising N-half-nanocage monomers (e.g., N-half-ferritin) to fusion polypeptide comprising C-half-nanocage monomers (e.g., C-half-ferritin) within the self-assembled polypeptide complex is about 1:1.

In some embodiments, the self-assembled polypeptide complex comprises 24 fusion polypeptides. In some embodiments, the self-assembled polypeptide complex comprises more than 24 fusion polypeptides, e.g., 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-assembled polypeptide complex comprises 32 fusion polypeptides.

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.

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.

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.

In some embodiments, the self-assembled polypeptide complex comprises the first fusion polypeptide in a ratio of about 1:1, 1:2, 1:3, or 1:4 to all other fusion polypeptides.

In some embodiments, each fusion polypeptide within the self-assembled polypeptide complex comprises a ferritin light chain or a subunit of ferritin light chain. In these embodiments, the self-assembled polypeptide complex does not comprise any ferritin heavy chain, subunits of ferritin heavy chain or other ferritin components capable of binding to iron.

Also described herein are compositions, e.g., therapeutic or prophylactic compositions, comprising nanocages. Also described are related methods and uses for treating and/or preventing covd-19, wherein the methods or uses comprise administering to a subject in need thereof a nanocage or composition described herein.

Nucleic acid molecules encoding the fusion proteins and polypeptides described herein are also described herein, as well as vectors comprising the nucleic acid molecules and host cells comprising the vectors.

Polynucleotides encoding the fusion proteins described herein include polynucleotides having a nucleic acid sequence that is substantially identical to the nucleic acid sequence of the polynucleotides of the invention. A nucleic acid sequence that is "substantially identical" is defined herein as having 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 to another nucleic acid sequence when the two sequences are optimally aligned (with the appropriate nucleotide insertions or deletions) and compared to determine an exact match of nucleotides between the two sequences.

Suitable sources of polynucleotides encoding antibody fragments include any cell that expresses full length antibodies, such as hybridomas and spleen cells. As described above, the fragments may themselves be used as antibody equivalents, or may be recombined into equivalents. The DNA deletions and recombinations described in this section may be performed by known methods, such as those described in published patent applications listed in the section entitled "functional equivalents of antibodies" above and/or other standard recombinant DNA techniques, such as those described below. Another source of DNA is single chain antibodies generated from phage display libraries, as is known in the art.

In addition, expression vectors containing the polynucleotide sequences described previously operably linked to expression sequences, promoters and enhancer sequences are provided. A variety of expression vectors have been developed for efficient synthesis of antibody polypeptides in prokaryotic, e.g., bacterial, and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems. Vectors of the invention may comprise segments of chromosomal, nonchromosomal and synthetic DNA sequences.

Any suitable expression vector may 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 derivatives of phage DNA, such as Ml3 and other filamentous single-stranded DNA phages. One example of a vector that can be used in yeast is a 2 mu plasmid. Suitable vectors for expression in mammalian cells include the well known SV-40 derivatives, adenoviruses, retrovirus-derived DNA sequences and shuttle vectors, which are derived from a combination of functional mammalian vectors, such as those described above, as well as functional plasmid and phage DNA.

Other eukaryotic expression vectors are known in the art (e.g., P J. Southern & P.Berg, J.Mol.Appl.Genet,1:327-341 (1982); subramanni et al, mol. Cell. Biol,1:854-864 (1981); kaufmann & 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 AHuman 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).

Expression vectors typically contain at least one expression control sequence operably linked to a DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate expression of the cloned DNA sequences. Examples of expression control sequences that can be used are the lac system, trp system, tac system, trc system, the major operator and promoter regions of phage lambda, the control regions of fd coat proteins, yeast glycolytic promoters, such as 3-phosphoglycerate kinase promoters, yeast acid phosphatase promoters, such as Pho5, yeast alpha mating factor promoters, yeast acid phosphatase promoters, and promoters derived from polyoma, adenovirus, retrovirus and simian viruses, such as early and late promoters or SV40, and other sequences known to control gene expression in prokaryotic or eukaryotic cells, and viruses or combinations thereof.

Recombinant host cells containing the expression vectors previously described are also described herein. The fusion proteins described herein may be expressed in cell lines other than hybridomas. Nucleic acids comprising sequences encoding polypeptides of the invention can be used to transform suitable mammalian host cells.

Particularly preferred cell lines are selected based on high levels of expression of the protein of interest, constitutive expression and minimal contamination from the host protein. Mammalian cell lines that can be used as hosts for expression are well known in the art and include many immortalized cell lines such as, but not limited to, HEK 293 cells, chinese Hamster Ovary (CHO) cells, baby Hamster Kidney (BHK) cells, and many others. Suitable other eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, E.coli (E.coli), such as E.coli SG-936, E.coli HB 101, E.coli W3110, E.coli X1776, E.coli X2282, E.coli DHI and E.coli MRC1, pseudomonas, bacillus, such as B.subtilis (Bacillus subtilis) and Streptomyces (Streptomyces).

These existing recombinant host cells 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 cell or from the culture medium surrounding the host cell. The targeting of expressed polypeptides for secretion in recombinant host cells can be facilitated by inserting a signal or secretion leader peptide coding sequence at the 5' end of the antibody-encoding gene of interest (see 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 may be derived from prokaryotic or eukaryotic sequences. Thus, suitably, a secretion leader peptide is used, which is an amino acid linked to the N-terminus of the polypeptide to direct the removal of the polypeptide from the cytoplasm of the host cell and secretion into the culture medium.

The fusion proteins described herein may be fused to additional amino acid residues. For example, such amino acid residues may be peptide tags to facilitate isolation. Other amino acid residues for homing antibodies to specific organs or tissues are also contemplated.

It is understood that Fab nanocages can be generated by co-transfection of HC-ferritin and LC. Alternatively, single chain Fab-ferritin nanocages may be used that require transfection of only one plasmid, as shown in fig. 1C. This can be accomplished with linkers of different lengths between LC and HC, for example 60 or 70 amino acids. When single chain Fab is used, it can be ensured that the heavy and light chains are paired. Tags (e.g., flag, HA, myc, his6x, strep, etc.) may also be added at the N-terminus or within the linker of the construct to facilitate purification, as described above. Furthermore, when co-transfecting different Fab nanoparticle plasmids, a tag system can be used to ensure that there are many different fabs on the same nanoparticle using a series/addition of affinity chromatography steps. This provides multi-specificity for the nanoparticle. Protease sites (e.g., TEV, 3C, etc.) may be inserted after expression and/or purification to cleave linkers and tags, if desired.

Any suitable method or route may be used to administer the fusion proteins described herein. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

It will be appreciated that the fusion proteins described herein, when used for prophylaxis or treatment in a mammal, will be administered in the form of a composition that additionally comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the binding protein. As is well known in the art, injectable compositions may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.

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

In one aspect, methods are provided that can be used to treat, ameliorate or prevent a SARS-CoV-2 associated condition, generally comprising the step of administering to a subject a composition comprising a self-assembled polypeptide complex of the disclosure.

A "SARS-CoV-2 associated disorder" refers to a disorder (e.g., symptom or sign) associated with SARS-CoV-2 infection. In some embodiments, the disorder is a level of SARS-CoV-2RNA, protein, or viral particle in a sample from a subject (e.g., a subject administered the self-assembled polypeptide complex disclosed herein) that is indicative of a SARS-CoV-2 infection (e.g., because the level meets a threshold or exceeds a reference level indicative of a SARS-CoV-2 infection). In some embodiments, the disorder is a symptom associated with a covd-19 disorder, such as fever, cough, tiredness, shortness of breath or dyspnea, muscle pain, aversion to cold, sore throat, runny nose, headache, chest pain, conjunctivitis, nausea, vomiting, diarrhea, loss of sense of smell, loss of taste, or stroke. In some embodiments, the condition is associated with a downstream sequelae of covd-19 disease and/or is a symptom of long-term covd-19 disease.

In some embodiments, the subject is a mammal, e.g., a human.

The composition administered to a subject generally comprises a self-assembled polypeptide complex as disclosed herein. In some embodiments, such compositions further comprise a pharmaceutically acceptable excipient.

The compositions may be formulated for any of a variety of routes of administration, including systemic routes of administration (e.g., oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration).

The foregoing disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Thus, the present invention should in no way be construed as limited to the following examples, but rather should be construed to encompass any and all variations that become evident from the teachings provided herein.

The following examples do not include detailed descriptions of conventional methods, such as those used in constructing vectors and plasmids, inserting genes encoding polypeptides into such vectors and plasmids, or introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, j., fritsch, e.f. and Maniatis, t. (1989), molecular Cloning: A Laboratory Manual,2nd edition,Cold Spring Harbor Laboratory Press, which are incorporated herein by reference.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the following example examples, utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following working examples specifically point out typical aspects of the invention and should not be construed as limiting the remainder of the disclosure in any way.

Examples

Example 1: multivalent conversion of SARS-CoV-2 antibodies to super-neutralizing agents

This example describes the design, expression, purification and identification of fusion proteins with apoferritin. The apoferritin multimers self-assemble into octahedral symmetry structures with hydrodynamic radii (R) of about 6nm _h ) Consists of 24 identical polypeptides. The N-terminus of each apoferritin subunit points to the outside of the spherical nanocage and is therefore useful for genetic fusion of the protein of interest. Fusion proteins were designed whereby, upon folding, the apoferritin multimer served as a building block driving multimerization of 24 proteins fused to the apoferritin terminus.

Abstract

SARS-CoV-2 is a virus that causes COVID-19, which has led to global pandemic. Antibodies can be powerful biological therapeutics against viral infections. Here we used human apoferritin as a modular subunit to drive oligomerization of antibody fragments and convert antibodies targeting SARS-CoV-2 into particularly effective neutralizing agents. Using this platform, as low as 9X 10 can be achieved due to a 10,000-fold increase in potency compared to the corresponding IgG ^-14 Half of MMaximum Inhibitory Concentration (IC) ₅₀ ) Values. The combination of three different antibody specificities and fragment crystallizable (Fc) domains on a single multivalent molecule confers the ability to overcome viral sequence variations, as well as excellent potency and IgG-like bioavailability. Thus, MULTi-specific, MULTi-affinity antibody (multhady or MB) platforms uniquely exploit binding affinity and MULTi-specificity to deliver a super-potent and broad neutralizing agent against SARS-CoV-2. The modularity of the platform also allows it to rapidly assess other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic agent for SARS-CoV-2.

Introduction(s)

The continuing threat of the novel SARS-CoV-2 respiratory viruses to public health has highlighted the urgent need to rapidly formulate and deploy preventive and therapeutic interventions to combat the disease's pandemic. Monoclonal antibodies (mabs) have been effective in the treatment of infectious diseases, such as palivizumab for the prevention of high risk infant respiratory syncytial virus ¹ Or Zmapp, mAb114 and REGN-EB3 for the treatment of ebola virus ² . Thus, mAbs targeting the spike (S) protein of SARS-CoV-2 have been the focus of biomedical countermeasure research against COVID-19. To date, several antibodies to the S protein have been identified ^{3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19} Wherein bamlanivimab is the first antibody approved by the U.S. Food and Drug Administration (FDA) for emergency treatment of SARS-CoV-2 at month 11 of 2020. Receptor Binding Domain (RBD) -directed mabs interfere with binding to angiotensin converting enzyme 2 (ACE 2), a receptor for cell entry ²⁰ Generally with the highest neutralization capacity ^6,18,19 And (5) correlation.

mAb antibodies can be isolated by B cell sorting from infected donors, immunized animals, or by identifying binders in a pre-assembled library. Although these methods are robust and reliable for the discovery of virus-specific mabs, the identification of optimal antibody clones is often associated with a time cost penalty. In addition, RNA viruses have a higher mutation rate than DNA viruses, and such mutations can significantly alter the efficacy of neutralizing antibodies. Indeed, some studies have shown that convalescent serumReduced neutralizing potency and certain mabs ^21, 22 ^,23 For the nearest B.1.1.7 ²⁴ 、B.1.351 ²⁵ And B.1.1.28 ^26,27 The SARS-CoV-2 variant has reduced resistance. Thus, there is an unmet need to develop a platform that links the discovery of antibodies with the rapid identification and deployment of highly potent neutralizing agents that are not susceptible to viral sequence variation.

The efficacy of an antibody is greatly affected by its ability to interact with its epitope multiple times simultaneously ^28, 29 ^,30 . This enhanced apparent affinity, i.e., avidity, has been previously reported to enhance nanobodies ^31,32 And IgG exceeds Fab ^8,10,16 Neutralization potency against SARS-CoV-2. To fully exploit the full capacity of binding affinity, we developed an antibody scaffold technology that uses human apoferritin as a modular subunit, multimerizes antibody fragments, and pushes mabs into a super-potent neutralizing agent against SARS-CoV-2. In fact, the resulting Multabody molecules may be up to four orders of magnitude more potent than the corresponding IgG. Furthermore, we demonstrate the ability of this technique to combine three different Fab specificities to better overcome point mutations in spike proteins. Multhady provides a universal IgG-like "plug and play" platform to enhance the antiviral properties of mabs against SARS-CoV-2 and demonstrate the leveraging ability of avidity as a mechanism against viral pathogens.

Materials and methods

Protein expression and purification

Genes encoding VHH-human apoferritin fusion, fc fusion, fab, igG and RBD mutants were synthesized and cloned into pcdna3.4 expression vectors via GeneArt (Life Technologies). Unless otherwise specified, all constructs were transiently expressed in HEK 293F cells (Thermo Fisher Scientific) at a cell density of 0.8×10 ⁶ Mu.g of DNA per 200mL of cells per mL of cells was used, fectoPRO (Polyplust Transfections) in a 1:1 ratio. Shaking at 125rpm at 37deg.C, 8% CO ₂ And 70% humidity in a Multitron Pro shake flask (Infos HT) for 6-7 days, by incubation inThe cell suspension was harvested by centrifugation at 5000 Xg for 93 minutes and the supernatant was filtered through a 0.22 μm Steritop filter (EMD Millipore). Fab and IgG were transiently expressed by co-transfection of 90 μg LC and HC at a 1:2 ratio and purified using kappa select affinity column (GE Healthcare) and HiTrap Protein a HP column (GE Healthcare), respectively, using 100mM glycine pH 2.2 as elution buffer. The eluted fractions were immediately neutralized with 1M Tris-HCl, pH 9.0, and further purified using Superdex 200 interference size exclusion column (GE Healthcare). The Fc fusion of ACE2 and VHH-72 was purified in the same manner as IgG. VHH-72 apoferritin fusion was purified by hydrophobic interaction chromatography using a HiTrap Phenyl HP column, and the eluted fractions were loaded into a Superose 6 10/300GL size exclusion column (GE Healthcare) in 20mM sodium phosphate pH 8.0, 150mM sodium chloride. Wild type (BEI NR 52309) and mutant RBD, pre-fusion (fusion) S ectodomain (BEI NR 52394) and Fc receptor (FcRn and FcγRI) of mice and humans were purified using a HisTrap Ni-NTA column (GE Healthcare). In the case of S trimer, ni-NTA purification was followed by Superose 6, in the case of RBD and Fc receptor Superdex 200 interference size exclusion column (GE healthcare), in each case in 20mM phosphate pH 8.0, 150mM NaCl buffer.

Design, expression and purification of multabodies

All molecules referred to herein as multhabodies contain scFab and scFc fragments. Flexible linker [ (GGGGS) using 70 amino acids _x14 ]scFab and scFc polypeptide constructs are produced to produce heterodimer and homodimer fragments, respectively. Specifically, the C-terminus of the Fab light chain is fused to the N-terminus of the Fab heavy chain via a linker. In the case of scFc, two Fc single chains forming a functional homodimer Fc are fused in tandem. Each domain uses a 25 amino acid linker (GGGGS) _x5 Fusion with apoferritin monomer. Genes encoding scFab and scFc fragments linked to half apoferritin are produced by deleting residues 1 to 90 (C-ferritin) and 91 to 175 (N-ferritin) of the human apoferritin light chain. Transient transfection of multhady in HEK 293F cells was obtained by mixing 66 μg of plasmid scFab-human apoferritin scFc-human N-ferritin scFab-C-ferritin at a 2:1:1 ratio. scFab-human apoferritinThe addition of (2) allows for efficient Multabody assembly and increases the number of Fab compared to Fc in the final molecule, thus favoring Fab avidity over Fc avidity. In the case of multispecific Multabody, a scFab 1-human apoferritin to scFc human N-ferritin to scFab 2-C-ferritin to scFab 3-C-ferritin ratio of 4:2:1:1 was used. The DNA mixture was filtered and incubated with 66. Mu.l FectoPRO at Room Temperature (RT) before addition to the cell culture. Using HiTrap Protein A HP column (GE Healthcare) using 20mM Tris pH 8.0,3M MgCl ₂ And 10% glycerol elution buffer the protein containing fractions were concentrated by affinity chromatography purification partition (Split) multhady and further purified by gel filtration on a Superose 6/300 GL column (GE Healthcare).

Negative electron microscopy

Mu.l of Multabody at a concentration of about 0.02mg/mL was placed on the surface of a carbon coated copper grid that had been previously glow-discharged in air for 15 seconds, allowed to adsorb for 30 seconds, and stained with 3. Mu.l of 2% uranyl formate. Excess staining 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 a FEI Tecnai T20 electron microscope equipped with an Orius Charge Coupled Device (CCD) camera (Gatan Inc) operating at 200 kV.

Biological layer interference technique

Direct binding kinetics measurements were performed using the Octet RED96 BLI system (Sartorius ForteBio) in PBS pH7.4, 0.01% BSA and 0.002% tween at 25 ℃. His-tagged RBD, SARS-CoV-2Spike, was loaded onto Ni-NTA (NTA) biosensor (Sartorius ForteBio) to achieve a BLI signal response of 0.8 nm. The association rate was measured by transferring the loaded biosensor into wells containing a two-fold dilution series of 250 to 8nM (Fabs), 125 to 4nM (IgG) and 16 to 0.5nM (MB). The rate of dissociation is measured by immersing the biosensor in a well containing buffer. The duration of each step is 180 seconds. Fc characterization in the split multhady design was assessed by measuring binding to hfcyri and hFcRn loaded onto Ni-NTA (NTA) biosensors under the experimental conditions and concentration ranges described above. To probe the theoretical capacity of multhabody for endosomal recycling (endosomal recycling), binding to hfcrnβ2-microglobulin complex was measured at physiological (7.4) and endosomal (5.6) pH. Similarly, the Fc characterization of mice instead of MB was assessed by measuring binding to mfcyri and mFcRn pre-immobilized on Ni-NTA (NTA) biosensors. A two-fold dilution series of 100nM to 3nM (IgG) and 10nM to 0.3nM (MB) was used. The sensorgrams were analyzed using the Octet software with a 1:1 fitting model. Competition assays were performed in a two-step binding process. The Ni-NTA biosensor preloaded with His-tagged RBD was first immersed in a well containing 50 μg/mL primary antibody for 180 seconds. After a 30 second baseline period, the sensor was immersed in a well containing 50 μg/ml secondary antibody for 300 seconds. The incubation steps were all performed in PBS pH7.4, 0.01% BSA and 0.002% Tween at 25 ℃. ACE2-Fc was used to localize mAb binding to the receptor binding site.

Dynamic light scattering

The Rh of the Multabody was determined by Dynamic Light Scattering (DLS) using DynaPro Plate Reader III (Wyatt Technology). mu.L of Multabody at a concentration of 1mg/mL was added to 384 well black, clear bottom plates (Corning) and the measurements were performed at a fixed temperature of 25℃for a duration of 5 seconds per reading. Particle size determination and polydispersity were obtained from the accumulation of five readings using dynamic software (Wyatt Technology).

Aggregation temperature

Determination of aggregation temperatures (T) of Multabody and parent IgG using a UNit instrument (Unchained Labs) _agg ). The sample was concentrated to 1.0mg/mL and subjected to a thermal ramp from 25 ℃ to 95 ℃ in 1 ℃. T (T) _agg It was determined that a 50% increase in static light scattering (i.e., the maximum of the differential curve) was observed at 266nm wavelength from baseline. The mean and standard error of two independent measurements were calculated using the UNit analysis software.

Pharmacokinetics and immunogenicity

The present study used a surrogate Multabody consisting of scFab and scFc fragments of mouse HD37 (anti hCD 19) IgG2a fused to the N-terminus of the mouse apoferritin (mfrritin) light chain. HD37 scFab-mFerritin: fc-mFerritin: mFerritit were transfected and purified according to the procedure described above at a ratio of 2:1:1 in. L234A, L A and P329G (LALALAP) mutations were introduced into the mouse IgG2a Fc construct to silence the effector functions of Multabody ⁴⁸ . In vivo studies were performed using 12 week old male C57BL/6 mice (strain code: 027) purchased from Charles River, placed in independently ventilated cages, with 12h light/dark cycles (7 am/7 pm), at a temperature of 21-23℃and humidity of 40-55%. All procedures were approved by the local animal care committee of the university of Toronto, st.Carb. Single injections of 5mg/kg Multabody or control sample (HD 37 single-chain IgG-IgG1 or IgG2a subtype) and H.pylori ferritin (HpFERRITin) -PfCSP malaria peptide were subcutaneously injected in 200. Mu.L PBS (pH 7.5). Blood samples were collected at various time points and serum samples were assessed for circulating antibody and anti-drug antibody levels by ELISA. Briefly, 96-well Pierce Nickel coated plates (Thermo Fisher) were treated with 50. Mu.L of 0.5. Mu.g/ml His _6x The labelled antigen hCD19 was coated to determine the circulating HD37 specific concentration using the reagent specific standard curve of IgG and multhabody. HRP-protein A (Invitrogen) was used to detect the level of bound IgG/MB (dilution 1:10000). For the determination of anti-drug antibodies, nunc MaxiSorp plates (Biolegend) were coated with 12-mer HD37 scFab-mfrrin or hpferrrin-PfCSP malaria peptides. The 1:100 serum dilutions were incubated for 1 hour at room temperature and further developed using HRP-protein a (Invitrogen) as the second molecule (dilution 1:10000). Chemiluminescent signals at 450nm were quantified using a Synergy Neo2 multi-mode assay microplate analyzer (Biotek Instruments).

Biodistribution of living beings

8 week old male BALB/c mice were purchased from The Jackson Laboratory and placed in individually ventilated cages. The mice are kept under light for 14 hours/dark for 10 hours, the intensity from dawn to dusk is staged, the maximum value is reached in noon, the temperature is 20-21 ℃, and the humidity is 40-60%. All procedures were approved by the local animal care committee of the university of Toronto. The study used multhady consisting of scFab and scFc fragments of mouse HD37IgG2a fused to the N-terminus of the mouse apoferritin light chain. According to the manufacturer's instructions, alexa Fluor was used ^TM 647 antibody labelling kit (Invitrogen) HD37IgG2a Multabody or control samples (HD 37 single-chain IgG2 a) were fluorescent conjugated to Alexa-647. By Alexa Fluor ^TM 647 labeled 15nm gold nanoparticles were purchased from Creative Diagnostics (GFLV-15). Non-invasive biodistribution experiments were performed using PerkinElmer IVIS Spectrum (PerkinElmer). About 5mg/kg MB, HD37IgG2a, or gold nanoparticles in 200. Mu.L PBS (pH 7.5) were subcutaneously injected into the shoulder-relaxed skin of BALB/c mice and imaged at time 0, 1 hour, 6 hours, 24 hours, 2 days, 3 days, 4 days, 8 days, and 11 days post-injection. Prior to imaging, mice were placed in an anesthesia induction chamber containing a mixture of isoflurane and oxygen for 1 minute. The anesthetized mice were then placed in a prone position in the center of a built-in 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 seconds (excitation 640nm and emission 680 nm). The data were analyzed using IVIS software (in vivo image software for IVIS). After confirming the fluorescence signal from the 2D epi-illumination image, a 3D transmission fluorescence imaging tomography (FLIT) is performed on the region of interest using a built-in scan field of 3 x 3 or 3 x 4 transmission positions. A series of 2D fluorescent surface radiation images were taken at different transmission positions using excitation of 640 and 680nm emissions. A series of CT scans are also performed at the corresponding locations. A 3D profile of the fluorescence signal is reconstructed by combining the fluorescence signal and the CT scan. The resulting 3D fluoroscopic image is thresholded based on the 3D image of the PBS-injected mice taken at the corresponding body locations. The image was mapped to a rainbow LUT in IVIS software, and the upper end of the color patch was set to 50pmolM for mice injected with gold nanoparticles ^-1 cm ^-1 For mice injected with MB and IgG2a, the upper end of the color patch was set at 1000 pmole M ^-1 cm ^-1 To better show the biodistribution over time. The mouse organ registration function (organ registration feature) of the IVIS software is used as a general guideline for assessing the body position of a sample from 3D images.

RBD panning phage library against SARS-CoV-2

A commercial Superhuman 2.0 phage library (Distributed Bio/Charles River Laboratories) was used to identify monoclonal antibodies against SARS-CoV-2RBDAn antibody conjugate. For this purpose, the RBD-Fc-Avi tag construct of SARS-CoV-2 was expressed in the EXPi-293 mammalian expression system. The protein was then purified by the protein G Dynabeads, biotinylated and quality controlled for biotinylation and binding to ACE2 recombinant protein (Sino Biologics Inc). Superhuman 2.0 phage library (5X 10) ¹² ) Heating at 72℃for 10 min and targeting the protein GDynabeads ^TM (Invitrogen), M-280 streptavidin Dynabeads ^TM (Invitrogen), calf thymus histone (Sigma), human IgG (Sigma), and ssDNA-biotin NNK from Integrated DNA Technologies and DNA-biotin NNK from Integrated DNA Technologies were deselected (de-select). Next, the automation protocol on Kingfisher FLEX (Thermofisher) was used for the M-280 streptavidin Dynabeads ^TM Captured RBD panned the library. Selected phages were eluted from the beads and neutralized using Tris-HCl pH 7.9 (Teknova). By OD ₆₀₀ The neutralized phage library=0.5 infected ER2738 cells at a ratio of 1:10, after incubation at 37 ℃ and 100rpm for 40 min, the phage library was centrifuged and incubated on agar overnight at 30 ℃ with antibiotic selection. The rescued phages were precipitated by PEG and three additional rounds of automatic panning of the soluble phase were performed. PBST/1% BSA buffer and/or PBS/1% BSA was used for the deselection, washing and selection rounds.

Screening of bacterial PPE for anti-SARS-CoV-2 scFv Using SARS-CoV-2RBD

anti-SARS-CoV-2 RBD single chain antibodies selected from phage display were expressed and screened at 25℃using high throughput Surface Plasmon Resonance (SPR) on a Carterra LSA array SPR instrument (Carterra) equipped with HC200M sensor chip (Carterra). The V5 epitope tag was added to the scFv to enable capture by an immobilized anti-V5 antibody (Abcam, cambridge, MA) pre-immobilized on the chip surface by standard amine coupling. In short: the chip surface was first activated by 10 minutes of injection of a 1:1:1 (v/v/v) mixture of 0.4M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 0.1 MN-hydroxysuccinimide (sNHS) and 0.1M 2- (N-morpholino) ethanesulfonic acid (MES) pH 5.5. Then, 50. Mu.g/ml of anti-V5 tag antibody prepared in 10mM sodium acetate pH 4.3 was conjugated for 14min, and the excess of reactive ester was blocked during 10 min injection with 1M ethanolamine HCl pH 8.5. For screening, 384 ligand arrays containing crude bacterial periplasmic extracts (PPE) containing scFv (one spot per scFv) were prepared. Each extract was prepared as a two-fold dilution in running buffer (10mM HEPES pH 7.4, 150mM sodium chloride, 3mM EDTA and 0.01% (V/V) tween-20 (HBSTE)) and printed on anti-V5 surface for 15 min. SARS-CoV-2RBD Avi Tev His tags of 0, 3.7, 11.1, 33.3, 100, 37 and 300nM were then prepared in 10mM HEPES pH 7.4, 150mM NaCl and 0.01% (v/v) Tween-20 (HBST) supplemented with 0.5mg/ml BSA and injected as analyte for 5 minutes with a dissociation time of 15 minutes. The samples were injected at increasing concentrations without any regeneration step. Binding data from local reference spots was used to subtract the signal from active spots and the nearest buffer blank analyte response was subtracted to double-reference data. In the cartera kinetics detection tool (10 months edition 2019), double reference data were fitted to a simple 1:1langmuir binding model. 20 medium affinity binders from phage display screening were selected for this study.

Pseudovirus production and neutralization

SARS-CoV-2 pseudovirus (PsV) uses the HIV-based lentiviral system ⁴⁹ With minor modifications. Briefly, 293T cells were co-transfected with a lentiviral backbone encoding a luciferase reporter gene (BEI NR 52516), a plasmid expressing spike (BEI NR 52310) and a plasmid encoding HIV structural and regulatory proteins Tat (BEI NR 52518), gag-pol (BEI NR 52517) and Rev (BEI NR 52519) using a BioT transfection reagent (Bioland Scientific) according to the manufacturer's instructions. 24 hours after transfection, 5mM sodium butyrate was added to the medium at 37℃and the cells were incubated at 30℃for an additional 24 to 30 hours. SARS-CoV-2 spike protein mutant D614G is provided by D.R. Burton (The Scripps Research Institute), SARS-COV-2PsV variant B.1.351 is provided by D.D.Ho (university of Columbia), and the remaining PsV mutants were generated using the KOD Plus mutagenesis kit (Toyobo, osaka, japan) using the primers described in Table 1. The PsV granules were harvested and sterile passed through 0.45 μm wellsThe filters were finally concentrated using a 100K Amicon (Merck Millipore Amicon Ultra 2.0.0 centrifugal filtration device).

TABLE 1 primer sequences

Neutralization was determined in a single cycle neutralization assay using 293T-ACE2 cells (BEI NR 52511) and HeLa-ACE2 cells (supplied by D.R. Burton; the Scripps Research Institute). Cells were seeded at a density of 10000 cells/well in a volume of 100 μl one day prior to the experiment. In the case of 293T cells, the plates were pre-coated with poly-L-lysine (Sigma-Aldrich). On the day of the experiment, 50. Mu.l of serially diluted IgG and MB samples were incubated with 50. Mu.l of PsV for 1 hour at 37 ℃. After 1 hour of incubation, incubation volumes were added to the cells and incubated for 48 hours. Neutralization was monitored PsV by adding 50 μl briitelite plus reagent (PerkinElmer) to 50 μl cells, after 2 minutes of incubation, the volume was transferred to a 96-well white plate (Sigma-Aldrich) and luminescence in Relative Light Units (RLU) was measured using a Synergy Neo2 multi-mode assay microplate analyzer (Biotek Instruments). Two to three biological replicates were performed, two technical replicates each. IC (integrated circuit) ₅₀ Is calculated as:

IgG IC ₅₀ (μg/mL)/MB IC ₅₀ (μg/mL)

neutralization of true viruses

VeroE6 cells were seeded at 30000/well in DMEM supplemented with 100U penicillin, 100U streptomycin and 10% FBS in 96F plates. Cells were allowed to adhere to the plates and left to stand overnight. After 24 hours, 5-fold serial dilutions of IgG and MB samples were prepared in DMEM supplemented with 100U penicillin and 100U streptomycin, which were performed in quadruplicates (25 μl/well) in 96R plates. About 25. Mu.L SARS-CoV-2/SB2-P4-PB ⁵⁰ Clone 1 was added to each well at 100 TCID/well and incubated for 1 hour at 37 ℃ with shaking every 15 minutes. After CO-cultivation, the medium was removed from the VeroE6 plates and VeroE6 cells were inoculated in quadruplicate using 50 μl of antibody-virus samples at 37 ℃ for 1 hour, 5% CO ₂ Every 15 minutesZhong Yaodong once. 1 hour after inoculation, the inoculum was removed and 200. Mu.L of fresh DMEM supplemented with 100U penicillin, 100U streptomycin and 2% FBS was added to each well. The plates were further incubated for 5 days. Cytopathic effect (CPE) was monitored and IC was calculated using PRISM ₅₀ Values. Three biological replicates were performed, four technical replicates each time.

Crosslinking of spike proteins with Fab 80, fab 298 and Fab 324

Approximately 100 μg of spike trimer was mixed with a 2-fold molar excess of Fab 80, fab 298, or Fab 324 in 20mM HEPES pH 7.0 and 150mM sodium chloride. The protein was crosslinked by adding 0.075% (v/v) glutaraldehyde (Sigma-Aldrich) and incubating for 120 min at RT. The complex was purified by size exclusion chromatography (Superose 6 Increase 10/300GL,GE Healthcare), concentrated to 0.5mg/mL, and used directly in frozen EM grid preparation.

Crosslinking of Fab 46-RBD complexes

Approximately 100 μg Fab 46 was mixed with a 2-fold molar excess of RBD in 20mM HEPES pH 7.0 and 150mM sodium chloride. The complex was crosslinked by adding 0.05% (v/v) glutaraldehyde (Sigma-Aldrich) and incubating for 45 min at RT. The crosslinked complex was purified by size exclusion chromatography (Superdex 200 Increate 10/300GL,GE Healthcare), concentrated to 2.0mg/ml, and used directly in frozen EM grid preparation.

Frozen EM data collection and image processing

Internally prepared multi-well Jin Geshan with 3 microliter sample deposited ⁵¹ The grids were glow-discharged in air for 15 seconds with peloc easigow (Ted Pella) before use. Samples were spotted with modified FEI Mark III Vitrobot (maintained at 4 ℃ and 100% humidity) using-5 lithography (offset) for 6 seconds and then frozen by immersion in a mixture of liquid ethane and propane. Data were obtained at 300kV using a Thermo Fisher Titan Krios G electron microscope and a prototype Falcon 4 camera operating in electron counting mode of 250 frames/sec. About 5e using a 29 exposure score ^- Perpix/s camera exposure rate and aboutThe total exposure of the samples was taken as a 9.6 second film.No objective aperture is used. The pixel size is calibrated from gold diffraction standard to +. >A pixel. EPU software package for microscope realizes automation, and cryoSPARC Live for data collection ⁵² Monitoring is performed.

To overcome the preferential direction encountered by certain samples, oblique data collection was employed ⁵³ . For spike-Fab 80 complexes, 820 0 ° oblique movies and 2790 40 ° oblique movies were collected. For the spike-Fab 298 complex, 4259 0 ° oblique movies and 3513 40 ° oblique movies were collected. For the spike-Fab 324 complex, 1098 0 ° oblique movies and 3380 40 ° oblique movies were collected. For the RBD-Fab 46 complex, 4722 0℃oblique movies were collected. For 0 tilt movies, crySPARC patch motion correction is performed. For 40 inclined movies Relion MotionCorr is used ^54,55 . The micrograph is then imported into crySPARC and patch CTF estimation is performed. Templates generated from 2D classification during cryosprc Live were used for template selection of particles. Using 2D classification to remove the garbage pellet image, a dataset was generated consisting of a pellet image of 80951 spike-Fab 80 complexes, a pellet image of 203138 spike-Fab 298 complexes, a pellet image of 64365 spike-Fab 324 complexes, and a pellet image of 2143629 RBD-Fab 46 complexes. Particle image stacks were cleaned up using multi-round multi-class de novo refinement (multi-class ab initio refinement) and uniform refinement (homogeneous refinement) was used to obtain consistent structures. For tilted particles, particle polishing was performed in a Relion at this stage and cryospharc was reintroduced. For spike-Fab complexes, a wide range of flexibility was observed. 3D variability analysis was performed ⁵⁶ And together with non-uniform refinement is used to classify the different states that exist. The final set of particle images is then non-uniformly refined (Nonuniform refinement) ⁵⁷ . For RBD-Fab 46 complexes, three classes of crysparc were iteratively used to clean up the particle image stack from de novo refinement. Then, the stack of particle images with refined Euler angles (refined Euler angle) is brought into the cisTEM for processingReconstruction of ⁵⁸ To produceResolution map. The data transmission between the Relion and crySPARC is performed by using the pyem ⁵⁹ And (3) finishing.

Crystallization and Structure determination

200 μg RBD was mixed with a 2-fold molar excess of each Fab in 20mM Tris pH 8.0, 150mM NaCl to give a 52Fab-298 Fab-RBD ternary complex, which was subsequently purified by size exclusion chromatography (Superdex 200Incase 10/300GL,GE Healthcare). The fraction containing the complexes was concentrated to 7.3mg/ml and mixed with 20% (w/v) 2-propanol, 20% (w/v) PEG 4000 and 0.1M sodium citrate pH 5.6 in a 1:1 ratio. Crystals appeared after about 1 day and were cryoprotected in 10% (v/v) ethylene glycol, then flash frozen in liquid nitrogen.

Data was collected on the 23-ID-D beam line of Argonne National Laboratory Advanced Photon Source. Data set usage XDS ⁶⁰ And XPREP for processing. Phaser was used with CNTO88Fab as model 52Fab (PDB ID:4DN 3), 20358Fab as model 298Fab (PDSBID: 5 CZX) and PDB ID:6XDG as search model of RBD ⁶¹ The phases are determined by molecular replacement. Using phenylix ⁶² And Coot ⁶³ The manual construction iteration in (a) optimizes the structure. Structural analysis and graphics rendering with PyMOL ⁶⁴ . By SBgrid ⁶⁵ Access to all software is supported. Representative electron densities for the two Fab-RBD interfaces are shown in FIGS. 2e, f.

Availability of materials

Electron microscopy images have been deposited in Electron Microscopy Data Bank (EMDB) with 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 from Protein Data Bank under the accession number PDB ID 7K9Z. The sequences of the monoclonal antibodies used are provided herein (table 4). Additional PDB/EMDB entries were used throughout the manuscript to perform comparative analysis on different epitope bins targeted by mabs. The entries used in this analysis are: REGN10933 (PDB ID:6 XDG), CV30 (PDB ID:6XE 1), C105 (PDB ID:6 XCM), COVA2-04 (PDB ID:7 JMO), COVA2-39 (PDB ID:7 JMP), CC12.1 (PDB ID:6XC 2), BD23 (PDB ID:7 BYR), B38 (PDB ID:7BZ 5), P2C-1F11 (PDB ID:7 BWJ), 2-4 (PDB ID:6 XEY), CB6 (PDB ID:7C 01), REGN10987 (PDB ID:6 XDG), S309 (PDB ID:6WPS,6 WPT), EY6A (PDB ID:6 ZCZ), CR3022 (PDB ID:6 YLA), H014 (PDB ID:7 CAH), 4-8 (EMDB ID: 22159), 4A8 (PDB ID:7C 2L) and EMDB 2-22275 (EM43).

Table 2.Cryo-EM data collection and image processing

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Table 3.X ray crystallography data collection and refinement statistics

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TABLE 4 Table 4

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Materials and methods references

46.Kabsch,W.et al.XDS.Acta Crystallogr.Sect.D Biol.Crystallogr.66,125–132(2010).

47.McCoy,A.J.et al.Phaser crystallographic software.J.Appl.Crystallogr.40,658–674(2007).

48.Adams,P.D.et al.PHENIX:A comprehensive Python-based system for macromolecular structure solution.Acta Crystallogr.Sect.D Biol.Crystallogr.66,213–221(2010).

49.Emsley,P.,Lohkamp,B.,Scott,W.G.&Cowtan,K.Features and development of Coot.Acta Crystallogr.Sect.D Biol.Crystallogr.66,486–501(2010).

50.Morin,A.et al.Collaboration gets the most out of software.Elife 2,(2013).

51.Marr,C.R.,Benlekbir,S.&Rubinstein,J.L.Fabrication of carbon films with～500nm holes for cryo-EM with a direct detector device.J.Struct.Biol.185,42–47(2014).

52.Punjani,A.,Rubinstein,J.L.,Fleet,D.J.&Brubaker,M.A.CryoSPARC:Algorithms for rapid unsupervised cryo-EM structure determination.Nat.Methods 14,290–296(2017).

53.Zi Tan,Y.et al.Addressing preferred specimen orientation in single-particle cryo-EMthrough tilting.Nat.Methods 14,(2017).

54.Zivanov,J.et al.New tools for automated high-resolution cryo-EM structure determination in RELION-3.Elife 9,e42166(2018).

55.Scheres,S.H.W.RELION:Implementation of a Bayesian approach to cryo-EM structure determination.J.Struct.Biol.180,519–530(2012).

56.Punjani,A.&Fleet,D.3D Variability Analysis:Directly resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM images.bioRxiv(2020).

57.Punjani,A.,Zhang,H.&Fleet,D.Non-uniform refinement:Adaptive regularization improves single particle cryo-EM reconstruction.bioRxiv(2019).

58.Grant,T.,Rohou,A.&Grigorieff,N.CisTEM,user-friendly software for single-particle image processing.Elife 7,e35383(2018).

59.Asarnow,D.,Palovcak,E.&Cheng,Y.asarnow/pyem:UCSF pyem v0.5.(2019).

Results

Affinity enhancing neutralization potency

We used self-assembly of human apoferritin light chains to multimerize antigen-binding moieties targeting SARS-CoV-2S glycoprotein. Apoferritin multimers (protomers) self-assemble into octahedral symmetry structures with hydrodynamic radius (Rh) of about 6 nm consisting of 24 identical polypeptides ³³ . The N-terminus of each apoferritin subunit is directed towards the outside of the spherical nanocage and thus can be used for gene fusion of the protein of interest. After folding, the apoferritin multimer served as a building block driving multimerization of 24 proteins fused to its N-terminus (FIG. 1 a).

First, we have studiedEffect of multivalent on the ability of single chain variable domain VHH-72 to block viral infection. VHH-72 has been previously described as neutralizing SARS-CoV-2 upon fusion to the Fc domain, but not in its monovalent form ³¹ . Human apoferritin light chains displaying 24 copies of VHH-72 assembled into monodisperse, well formed spherical particles (fig. 1b, c) and showed an enhanced binding affinity with S glycoprotein compared to bivalent VHH-72-Fc (fig. 1 d). Remarkably, the neutralizing potency of VHH-72 displayed on the human apoferritin light chain against SARS-CoV-2 pseudovirus (PsV) was increased by about 10000-fold compared to conventional Fc fusion (FIG. 1 e), demonstrating the ability of the avidity to convert the binding moiety to a potent neutralizing agent.

Multhady has IgG-like properties

Fc imparts IgG in vivo half-life and effector function by interacting with neonatal Fc receptor (FcRn) and fcγreceptor (fcγr), respectively. To impart these IgG-like properties to our multimeric scaffold, we next tried to incorporate both the binding moiety and Fc domain. Because Fab is a heterodimer consisting of a light chain and a heavy chain, and Fc is a homodimer, we created single chain Fab (scFab) and single chain Fc (scFc) polypeptide constructs. scFab and scFc domains are fused directly to the N-terminus of the apoferritin multimer. For in vivo proof of principle experiments we generated a species-matched surrogate molecule consisting of a fusion of mouse light chain apoferritin with mouse scFab and mouse scFc (IgG 2a subtype). Binding kinetics showed that the MB molecules produced bound to mouse FcRn in a pH-dependent manner, bound at endosomal pH (5.6), and not at physiological pH (7.4), similar to the parent IgG (fig. 3 a). Not expected, binding to high affinity mouse fcγr1 is enhanced by the avidity effect compared to the parental IgG. Thus, we generated a modified mouse scFc version that included fcγr silent mutations LALAP to reduce Fc binding in multimers (fig. 3 a). The subcutaneous administration of MB in C57BL/6 or BALB/C mice was well tolerated with no weight loss or visible adverse events. MB showed a good IgG-like serum half-life (fig. 3 b), with lower detectable potency prolongation of fcγr binding to MB (LALAP-Fc sequence) in serum compared to WT MB, indicating that Fc plays a role in determining in vivo bioavailability. Real-time 2D and 3D imaging showed that fluorescently labeled MBs were systemically biodistributed like the corresponding IgG, not accumulated in any specific tissue (fig. 3c and 4). In contrast, 15 nm Gold Nanoparticles (GNPs) with similar Rh as MB diffuse rapidly from the injection site (fig. 3c and fig. 4). It is speculated that, since all sequences were derived from the host, replacement mouse MB did not induce an anti-drug antibody response in the mouse (fig. 3 d), further highlighting the IgG-like nature of the MB platform.

Realizing higher-priced protein engineering

In view of these advantageous results of mouse MB surrogate, we aimed at IgG BD23 targeting SARS-CoV-2 spike RBD and N-terminal domain (NTD), respectively, from previous reports ¹² And IgG4A8 ¹³ Producing an all-human MB. Addition of scFc to MB reduces the number of scFab that can be multimerized. To confer MB platform Fc without affecting Fab affinity and thus neutralization potency, we designed apoferritin protomers so that each particle contains more than 24 components. Human apoferritin multimers are split into two halves according to their four helix bundle folding: two N-terminal alpha helices (N-ferritin) and two C-terminal alpha helices. In this configuration, the scFc fragment of human IgG1 and the scFab of anti-SARS-CoV-2 IgG are genetically fused to the N-terminus of each apoferritin half-body, respectively. The split apoferritin complement (Split apoferritin complementation) results in heterodimerization of the two halves, resulting in a very efficient heterodimerization process of the fusion protein. Co-expression of scFab-C-ferritin and scFc-N-ferritin genes with excess scFab-ferritin genes resulted in complete apoferritin self-assembly, which showed high amounts of scFab and low amounts of scFc at the nanocage periphery (FIG. 5a and materials and methods). Conveniently, this design allows direct purification of MB using protein a, similar to IgG purification.

This split MB design forms 16nm Rh spherical particles with uninterrupted density loops and regularly spaced protruding scFab and scFc (fig. 5b, c). Thus, MB is found in native IgM ³⁴ In the lower size range of (2), but with a greater weight build-up on similar sizes to achieve high multivalent. Binding kinetics experiments showed that high binding affinity of MB to spike was preserved after addition of Fc fragment (fig. 5d and table 5). Binding to human fcyri and FcRn at pH 5.6 and 7.4 confirmed that scFc folded correctly in split MB design (tables 6 and 7). Furthermore, the LALAP mutation in scFc reduced the binding affinity to human fcyri (fig. 5 e), as previously observed in the replacement mouse MB (fig. 3 a). The neutralization assay of SARS-CoV-2PsV using split design MB showed that enhancement of spike binding affinity translates into an increase in neutralization potency, BD23 and 4A8 increased by approximately 1600-fold and, respectively, as compared to their IgG counterparts>2000 times (fig. 5 f). Taken together, these data support a further search for MB as an IgG-like platform that confers fine binding affinity and PsV neutralization between epitopes of different spike domains.

TABLE 5 kinetic constants and affinities of Multabody for SARS-CoV-2 spike antigen as determined by BLI

TABLE 6 human ferritin Multabody to human FcRn derived from BD23 antibody (IgG 1) targeting SARS-CoV-2 Kinetic constant and affinity

TABLE 7 Multabody versus human FcγRI derived from BD23 antibody (IgG 1) targeting SARS-CoV-2 Kinetic constant and affinity of (a)

The Fc mutations of the IgG1 scaffold evaluated in multhabody included: LALAP (L234A, L235A and P329G) and I235A, and combinations thereof that reduce binding of antibodies to FcgammaR. (numbering is according to the EU numbering scheme)

Tables 6-10 summarize k for multabodies _on 、k _off And the equilibrium dissociation constant (K) resulting therefrom _D ) Is measured by the above method. Binding kinetics showed that the resulting mouse MB molecule bound to the mouse FcRn in a pH-dependent manner-binding at endosomal pH (5.6) and not at physiological pH (7.4) -similar to the parent IgG (fig. 3A). Binding to high affinity mouse fcγr1 is enhanced by the avidity effect compared to the parental IgG. Binding of human MB to human fcyri and FcRn at endosomal pH confirmed that scFc folded correctly in the split MB design, and that LALAP and I253A mutations reduced binding affinity to fcyri and FcRn, respectively.

Table 8: mouse ferritin multhady pair derived from CD 19-targeting HD37 antibody (IgG 2 a) as determined by BLI Kinetic constants and affinities of murine FcRn

Table 9: mouse ferritin Multabody pair derived from CD 19-targeting HD37 antibody (IgG 2) as determined by BLI Kinetic constant and affinity of mouse fcyri

Table 10: human ferritin Multabody to human FcRn derived from BD23 antibody (IgG 1) targeting SARS-CoV-2 Kinetic constant and affinity of (a)

From antibody discovery to super-potent neutralizing agents

Next, we 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 (fig. 6 a). According to the standard biological screening protocol for SARS-CoV-2RBD, 20 were selected with 10 ^-6 To 10 ^-8 M medium affinity human mAb binders (Table 4; table 11). These mabs were generated as full-length IgG and MB and compared for their ability to block viral infection in a neutralization assay against SARS-CoV-2PsV (fig. 6b and 7 a). Notably, MB expression yield, uniformity, and thermostability were similar to the parental IgG (fig. 8 and table 12), and MB increased potency by up to four orders of magnitude for 18 (90%) of 20 IgG (table 13). The maximum increase in mAb 298 was observed, which was averaged from IC as IgG ₅₀ About 0.3 μg/mL (in IgG) to average IC as MB ₅₀ 0.0001. Mu.g/mL. Remarkably, 11 mabs were converted from non-neutralizing IgG to neutralizing MB over the concentration range tested. Using two different target cells (293T-ACE 2 and HeLa-ACE2 cells; FIGS. 6b and 7 b), 7 MB showed extraordinary potency, IC against SARS-CoV-2PsV ₅₀ The value is between 0.2 and 2 ng/mL. PsV neutralization assays using recombinant mabs REGN10933 and REGN10987 as a benchmark showed a comparison with that reported previously ⁸ Similar IC ₅₀ Values (0.0044 and 0.030. Mu.g/mL, respectively) confirm the remarkable efficacy of MB observed in our assay. For the mAb with the highest potency, MB enhanced neutralization potency was further demonstrated with the true SARS-CoV-2 virus (FIGS. 6c and 7 c), and baseline tests were also performed with the two recombinant REGN mAbs. The less sensitive neutralization phenotype against true viruses than PsV was observed and was also reported previously ^5,6,9,12 And consistent.

Table 11: human mAb binding agent for SARS-CoV-2RBD

nb = binding below detection limit

_agg Table 12: aggregation temperature (T) of multhady and related antibodies

Multabody/antibody	T _agg [℃]
		MB 298	73
MB 82	81
		MB 46	69
MB 324	71
		MB 236	71
MB 52	74
		MB 80	85
IgG 298	74
		IgG 82	75
IgG 46	75
		IgG 324	70
IgG 236	70
		IgG 52	73
IgG 80	81

Table 13: neutralization of SARS-CoV-2 by RBD-targeted Multabody

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Retrospectively, all IgG and MB were tested for their ability to bind to the spike glycoprotein and RBD of SARS-CoV-2 (fig. 9). The increase in avidity resulted in a higher apparent binding affinity without a detectable dissociation rate (off-rates) for the spike glycoprotein, likely due to the conversion to high neutralizing potency of the inter-spike cross-links (fig. 6b-d and fig. 9). Overall, the data indicate that the MB platform is compatible with rapid delivery of super-potent IgG-like molecules, even starting from mabs of neutral neutralizing character.

Epitope mapping

Based on their neutralizing potency, 7 mabs were selected for further characterization: 298 (IGHV 1-46/IGKV 4-1), 82 (IGHV 1-46/IGKV 1-39), 46 (IGHV 3-23/IGKV 1-39), 324 (IGHV 1-69/IGKV 1-39), 236 (IGHV 1-69/IGKV 2-28), 52 (IGHV 1-69/IGKV 1-39) and 80 (IGHV 1-69/IGKV 4-1) (FIG. 6b and Table 4). Epitope binding experiments showed that these mabs target RTwo major sites on BD, one of these bins overlapped with the ACE2 binding site (fig. 10a and 11). Global resolution of aboutThe cryo-electron microscopy structure of the Fab-SARS-CoV-2S complex of (c) confirmed that mabs 324, 298 and 80 bind to overlapping epitopes (fig. 10b, fig. 12a-c and table 2). To gain insight into the binding of mabs targeting another bin, we obtained a global resolution of +.>The Fab 46 and RBD complex cryo-electron microscope structure (FIG. 10c, FIG. 12d and Table 2), and resolution of +.>Ternary complex crystal structures of Fab 298 and 52 with RBD (fig. 10d, fig. 2 and table 3).

The crystal structure shows that Fab 298 binds almost exclusively to the ACE2 Receptor Binding Motif (RBM) of RBD (residues 438-506). In fact, of the 16 RBD residues involved in binding to Fab 298, 12 were also involved in binding to ACE2-RBD (FIGS. 2a-c and Table 14). RBM is stabilized by 11 hydrogen bonds from the heavy and light chain residues of Fab 298. In addition, RBM Phe486 contacts 11 Fab 298 residues, embedded (24% of the total RBD embedded surface area) and therefore is the core of antibody-antigen interactions (fig. 2a and table 14).

Detailed analysis of the RBD-52Fab interface showed that the epitope of mAb 52 drifts toward the core of the RBD, covering 20 residues of RBM and 7 residues in the core domain (fig. 10c, fig. 2b and table 14). Consistent with competition data, antibody 52 and antibody 46 share similar binding sites, although they approach RBD at slightly different angles (fig. 10c, d and fig. 2 d). Examination of the previously reported RBD-antibody complex structure showed that antibodies 46 and 52 target the fragile site of SARS-CoV-2 spike (site of vulnerability), which was not previously described (fig. 10 e). The epitopes targeted by these antibodies are partially blocked by NTD into an S "closed" conformation, suggesting that the mechanism of action of such antibodies may involve spike destabilization. Taken together, these data indicate that the enhanced neutralizing potency of the MB platform observed by avidity is related to mabs that can target different epitope bins on RBD.

/>

vdW: van der Waals interactionsCut-off value

HB: hydrogen bond [ ]Cut-off value

SB: salt bridge [ ]Cut-off value

Multhady overcomes spike sequence variability

To explore whether MB can potentially resist viral escape through its enhanced binding affinity, we tested four naturally occurring RBD mutations ³⁵ Has the highest efficacy on 7 speciesBinding and neutralization of human mabs: L452R-within the epitope of antibodies 46 and 52 (bin 1), A475V and V483A-within the ACE2 binding site (bin 2), and circulating RBD variant N439K ³⁶ (FIGS. 13 a-c). In addition, the effect of mutating Asn234 (an N-linked glycosylation site) to Gln was also evaluated, as the absence of glycosylation at this site has been previously reported to reduce sensitivity to neutralizing antibodies targeting RBD ³⁵ . More infectious PsV variant D614G ³⁷ Is also included in the group. As expected, mutation L452R significantly reduced the binding and efficacy of mabs 52 and 46, while antibody 298 was sensitive to mutation a475V (fig. 13b, c). The deletion of the N-linked glycans at Asn234 position increased the resistance of the virus to most antibodies, especially mabs 46, 80 and 324, underscores the importance of glycans in viral antigenicity (fig. 13 c). Remarkably, the following antibody specificities of MB forms are minimally affected by any S mutation in terms of their specific neutralizing potency: 298. 80, 324 and 236 (fig. 13 d). The mutation L452R reduced the sensitivity of 46-MB and 52-MB, but they remained neutralised for this PsV variant, in contrast to their parent IgG (FIG. 13 d). The more infectious SARS-CoV-2PsV variant D614G was neutralized by IgG and MB with similar potency as wild-type PsV (fig. 13c and 14 a).

The MB mixture consisting of three monospecific MBs resulted in the ubiquity of all PsV variants without significant loss of potency, thus 100-1000 fold higher potency compared to the corresponding IgG mixture (fig. 13e and 14c, d). To achieve breadth in a single molecule, we next generated trispecific MBs by combining multimerized subunits displaying three different fabs in the same MB assembly (fig. 14 b). Notably, the resulting trispecific MBs exhibited ubiquitination while retaining the excellent neutralizing potency of the monospecific versions, including against the b.1.351psv variant (fig. 13e, f and fig. 14c, d). The highest potency was observed for the 298-324-46 combination (fig. 14c, e), with trispecific MBs achieving superior potency beyond that reported so far and that we observed for some of the most potent IgG recombinantly produced from the available sequences (fig. 13 g). In addition, the MB format was able to increase the potency of these previously reported high potency IgG against PsV and live replicating SARS-CoV-2 virus by one to two more orders of magnitude (fig. 13 h), highlighting the plug-and-play (plug-and-play) nature of MB and the multivalent ability to enhance mAb neutralization capability in a range of potency.

Neutral median IC of assay ₅₀ The values are summarized in tables 13 and 15.

Table 15: neutralization of SARS-CoV-2 by Multabody

Discussion of the invention

In this study we disclose how binding avidity can be used as an effective mechanism to promote antibody neutralization efficacy and resistance to viral mutations. For this, we developed a plug-and-play antibody multimerization platform using protein engineering that increases the affinity of the mAb for targeting SARS-CoV-2. Seven of the most effective MBs were directed against SARS-CoV-2PsV IC ₅₀ Has a value of 0.2 to 2ng/mL (9X 10) ^-14 To 9X 10 ^-13 M), and thus to our knowledge it is the most effective antibody-like molecule against SARS-CoV-2 reported so far.

MB platform is designed to contain key advantageous properties from a developability perspective. First, the ability to enhance the efficacy of an antibody is independent of the antibody sequence, form, or epitope targeted. The modularity and flexibility of the platform is exemplified by enhanced VHH and efficacy of multiple Fab targeting non-overlapping regions on two SARS-CoV-2S subdomains (RBD and NTD). The use of MB to enhance the potency of VHH domains may provide particular value for such molecules, as their small size allows for efficient multimerization. Second, other methods of enhancing affinity by tandem fusion of single-stranded variable fragments ^38,39 In contrast, MB does not exhibit low stability and in fact self-assembles into highly stable particles with aggregation temperatures similar to those of the parent IgG. Third, alternative multimerization strategiesSlightly e.g. streptavidin ⁴⁰ Verotoxin B subunit scaffolds ⁴¹ Or virus-like nanoparticles ⁴² The challenge of immunogenicity and/or poor bioavailability is faced, as it lacks the Fc fragment and therefore cannot undergo FcRn-mediated recovery. The light chain of apoferritin is fully human, is biologically inactive, has been engineered to include an Fc domain, and although>Multimerization of 24 Fab/Fc fragments, but still with Rh similar to IgM. Thus, replacement mouse MB did not elicit anti-drug antibodies in mice and could be detected in one week serum similar to its parent IgG. However, the in vivo bioavailability of MB depends on its binding affinity to fcγr, suggesting that careful fine tuning of Fc affinity is required to effectively convert MB to clinic. In addition, further studies are needed to assess the distribution of MB at anatomical sites of interest, such as the lungs in the case of SARS-CoV-2 infection. The plug-and-play nature of Multabody also helps to explore alternative half-life extending moieties beyond Fc if bioavailability is the only desired feature of absence of effector function, such as human serum albumin ⁴³ Or binding moieties that bind human serum albumin ^44,45 。

Different mAb sequences against SARS-CoV-2 were tested on MB and a different increase in neutralization potency was observed. This suggests that the ability of MB to enhance potency may depend on the epitope location on the spike protein, or how Fab binds to antigen to achieve a neutralising geometry. The fact that neutralization of two of the 20 SARS-CoV-2RBD conjugates was not rescued by the MB platform suggests limitations based solely on mAb sequence and binding properties. Nevertheless, the ability of MB to convert avidity into neutralizing potency in a range of epitope specificities for SARS-CoV-2 spike protein highlights the potential for widespread use of this technology. Exploration of MB platform for low surface spike density viruses (e.g., HIV-1 ⁴⁶ ) Or other targets (e.g., tumor necrosis factor receptor superfamily), where the bivalent nature of conventional antibodies limits their effective activation ⁴⁷ 。

Viral escape may occur under selective pressure in response to therapy or during natural selection. Fight against escapeThe traditional approach for mutants is to use a mixture of antibodies targeting different epitopes. MB is less susceptible to S mutation than the parent IgG, probably because the loss of affinity is compensated by the enhancement of binding affinity. Thus, MB overcomes variability in viral sequences with exceptional potency when used in mixtures. Furthermore, split MB design allows combining multiple antibody specificities within a single multimerization molecule, resulting in similar potency and breadth as MB mixtures. Importantly, several mabs can escape ²¹ , ^22,23 The neutralized b.1.351-mer variant was efficiently neutralized by the trispecific multhady, further highlighting the ability of these molecules to resist viral escape. The multi-specificity within the same particle can provide additional advantages such as synergy of the S-avidity and the correct combination of mabs, which lays a foundation for further investigation of different combinations of mAb specificities on MB. Avidity and multispecific can also be exploited to deliver single molecules that are effective in neutralizing viruses such as beta coronaviruses.

Overall, the MB platform provides a tool to surpass the affinity limit of antibodies and produce broadly and effectively neutralizing molecules, while bypassing significant efforts for antibody discovery or engineering. This platform is a timeline that illustrates how binding affinity can be used to expedite the discovery of the most effective biological agents for infectious diseases that are of interest to global health.

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Example 2

Virus production and pseudovirus neutralization assay

SARS-CoV-2 pseudotype virus (PsV) is produced using the method described previously ⁴⁴ Is generated by the HIV-based lentiviral system with minor modifications. Briefly, 293T cells were co-transfected with lentiviral backbone encoding a luciferase reporter gene (BEI NR 52516), a spike protein-expressing plasmid (BEI NR 52310) and plasmids encoding HIV structural and regulatory proteins Tat (BEI NR 52518), gag-pol (BEI-NR 52517) and Rev (BEI-NR 52519). After 24 hours of transfection at 37 ℃, 5mM sodium butyrate was added to the medium and the cells were incubated at 30 ℃ for an additional 24 to 30 hours. PsV mutants were generated using KOD Plus mutagenesis kit (Toyobo, osaka, japan). David Ho (Columbia) is good at providing SARS-CoV-2 spike-related variants B.1.117, B.1.351, P.1 and B.1.617.2. Neutralization was determined in a single cycle neutralization assay using 293T-ACE2 cells (BEI NR 52511). Neutralization was monitored PsV by adding briitelite plus reagent (PerkinElmer) to the cells and measuring luminescence in Relative Light Units (RLU) using a Synergy Neo2 multi-mode assay microplate analyzer (Biotek Instruments). The IC is calculated as follows ₅₀ Increase the multiple: igG (immunoglobulin G) _IC50 (μg/mL)/MB _IC50 (μg/mL). Two to three biological replicates were performed, two technical replicates each.

Results

Identification of sequence liability (sequence liability) in mAb52

Computer analysis of the leader VH/VL sequence identified the deamidation site at position N92 in CDRL3 of mAb 52. Deamidation sites in monoclonal antibodies can lead to changes in binding kinetics and heterogeneity in pharmaceutical products. To avoid this potential effect, we generated a variant (N92T) in which the asparagine residue was mutated to threonine. Figure 15 shows that this mutation did not have any effect on potency as IgG or monospecific MB in WT pseudovirus neutralization assays. Subsequently, 298-80-52 trispecific MBs containing the N92T mutation in the VL of mAb52 were screened in the p.1psv neutralization assay, confirming that no potency loss was observed compared to the parental trispecific MBs (fig. 16).

Neutralization of trispecific MB in related variants

In the pseudovirus neutralization assay, the efficacy of trispecific MB 298-80-52 in the relevant Variant (VOC) was evaluated. As shown in Table 16 and FIG. 17, this MB retains activity in VOCs, average IC measured by WT, B.1.1.7, B.1.351 and P.1PsV ₅₀ About 0.2ng/ml [97fM]Average IC of the corresponding IgG mixture ₅₀ 91ng/ml [0.61nM](n=5 experiments). This result represents an increase in potency of about 200 to 1000 times (ng/ml) or about 3000 to 16000 times (molar concentration). These results highlight that the trispecific MB overcomes the viral escape of SARS-CoV-2 with excellent efficacy.

Table 16

Sequence listing

SEQ ID NO:1：hFerritinLC

MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLRHD

SEQ ID NO. 2: joint 1

GGGGSGGGGSGGGGSGGGGSGGGGSGG

SEQ ID NO:3：VHH-hFerr

(underlined indicates linker sequence; bold indicates hFerritinLC)

SEQ ID NO:4：VHH-Fc

(underlined indicates the linker sequence)

SEQ ID NO:5：N-hFerritinLC

MSSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWSEQ ID NO:6：C-hFerritinLC

SEQ ID NO. 7: signal sequence

MGILPSPGMPALLSLVSLLSVLLMGCVAE

SEQ ID NO. 8: joint 1

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

SEQ ID NO. 9: joint 2

GGGGSGGGGSGGGGSGGGGSGGGGSGG

SEQ ID NO:10：BD23-scFab-hFerritinLC

(underlined indicates linker sequence; bold indicates hFerritinLC)

SEQ ID NO. 11: v of BD23 _K

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPYTFGQGTKLEIK

SEQ ID NO. 12: v of BD23 _H

QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARPQGGSSWYRDYYYGMDVWGQGTTVTVSS

SEQ ID NO:13：BD23-scFab-C_hFerritinLC

(underlined indicates linker sequence; bold indicates C_hFerritinLC)

SEQ ID NO:14：scFc-N_hFerritinLC

(underlined indicates linker sequence; bold indicates hFerritinLC)

SEQ ID NO:15：scFc(LALAP)

(boxes are residues mutated relative to wild-type Fc)

DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:16：4A8-scFab-hFerritinLC

(underlined indicates linker sequence; bold indicates hFerritinLC)

SEQ ID NO. 17: v of 4A8 _K

EIVMTQSPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCTQATQFPYTFGQGTKVDIK

SEQ ID NO. 18: v of 4A8 _H

EVQLVESGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQAPGKGLEWMGGFDPEDGETMYAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATSTAVAGTPDLFDYYYGMDVWGQGTTVTVSS

SEQ ID NO:19：4A8-scFab C_hFerritinLC

(underlined indicates linker sequence; bold indicates C_hFerritinLC)

SEQ ID NO:20：mFerritin

MTSQIRQNYSTEVEAAVNRLVNLHLRASYTYLSLGFFFDRDDVALEGVGHFFRELAEEKREGAERLLEFQNDRGGRALFQDVQKPSQDEWGKTQEAMEAALAMEKNLNQALLDLHALGSARTDPHLCDFLESHYLDKEVKLIKKMGNHLTNLRRVAGPQPAQTGAPQGSLGEYLFERLTLKHD

SEQ ID NO:21：HD37-scIgG

(underlined indicates the linker sequence)

SEQ ID NO:22：IgG2a Fc_mFerr

(underlined indicates linker sequence; bold indicates mFerritin)

SEQ ID NO:23：scFc-N-hFerr LALAP I253A

( Underlined indicates linker sequences; bold indicates hfrrinnlc; boxes indicate residues mutated with respect to wild-type IgG1 Fc. )

/>

SEQ ID NO. 24: wild type human IgG1 Fc

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO. 25: antibody 56 light chain

DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO. 26: antibody 56 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWM

GWISAYNGNTNYAQKLQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA

RDIGPIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKKVEPKSC

SEQ ID NO. 27: antibody 349 light chain

DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDT

SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPWTFGQGT

RLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 28: antibody 349 heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWV

SGISSAGSITNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG

NHAGTTVTSEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 29: antibody 178 light chain

EIVMTQSPATLSVSPGERATLSCKASQSVSGTYLAWYQQKPGQAPRLLIY

GASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCLQTHSYPPTFGQG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

SEQ ID NO. 30: antibody 178 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYHMHWVRQAPGQGLEW

MGWINPNSGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC

ARDISSWYEITKFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 31: antibody 108 light chain

DIQMTQSPSSLSASVGDRVTITCRASQVITNNLAWYQQKPGKAPKLLIYD

ASTLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTFPYTFGQGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 32: antibody 108 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGYIFSRYAIHWVRQAPGQGLEWM

GWMNPISGNTDYAPNFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA

KDGSQLAYLVEYFQHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 33: antibody 128 light chain

DIQMTQSPSSLSASVGDRVTITCRASQNISRYLNWYQQKPGKAPKLLIYD

ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANGFPPTFGQGT

KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 34: antibody 128 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGYTFTHYYMHWVRQAPGQGLEW

MGIINPSSSSASYSQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR

DGRYGSGSYPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 35: antibody 160 light chain

DIQMTQSPSSLSASVGDRVTITCRASQSVSSWLAWYQQKPGKAPKLLIYA

ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTTPYTFGQGT

KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 36: antibody 160 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGHDMHWVRQAPGQGLEW

MGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA

RANSLRYYYGMDVWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 37: antibody 368 light chain

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQL

LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPA

TFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

SEQ ID NO. 38: antibody 368 heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYDINWVRQAPGQGLEWM

GAIMPMFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR

GSSGYYYGWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC

NVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 39: antibody 192 light chain

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQL

LIYAASSLQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPY

TFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

SEQ ID NO. 40: antibody 192 heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWM

GWINPNSGGANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCST

YYYDSSGYSTDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC

LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

QTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 41: antibody 158 light chain

DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYDA

SNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTK

VDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

SEQ ID NO. 42: antibody 158 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEW

MGWINPLNGGTNFAPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC

ARDPGGSYSNDAFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 43: antibody 180 light chain

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQL

LIYAASSLQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYSSPYT

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW

KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

SEQ ID NO. 44: antibody 180 heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYAMHWVRQAPGQGLEW

MGRISPRSGGTKYAQRFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAR

EAVAGTHPQAGDFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 45: antibody 254 light chain

DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYDA

SSLQIGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQSYSTPPWTFGQGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 46: antibody 254 heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSSAMHWVRQAPGKGLEWVS

AIGTGGDTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARE

GDGYNFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 47: antibody 120 light chain

EIVMTQSPATLSVSPGERATLSCRASQSVSSRYLAWYQQKPGQAPRLLIYG

ASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYYTTPRTFGQGT

RLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 48: antibody 120 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWM

GMIDPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAK

DFGGGTRYDYWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS

SLGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 49: antibody 64 light chain

DIQMTQSPSSLSASVGDRVTITCRASQGISSHLAWYQQKPGKAPKLLIYDA

SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYSTPWTFGQGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 50: antibody 64 heavy chain

EVQLLESGGGLVQPGGSLRLSCAASGFPFSQHGMHWVRQAPGKGLEWV

SAIDRSGSYIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

DTYGGKVTYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 51: antibody 298 light chain

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP

KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP

PTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

SEQ ID NO. 52: antibody 298 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGTFSTYGISWVRQAPGQGLEWM

GWISPNSGGTDLAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAS

DPRDDIAGGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 53: antibody 82 light chain

DIQMTQSPSSLSASVGDRVTITCRASQVISNYLAWYQQKPGKAPKLLIYD

ASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPPPTFGQGT

RLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 54: antibody 82 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGSFSTSAFYWVRQAPGQGLEWM

GWINPYTGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCA

RSRALYGSGSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 55: antibody 46 light chain

DIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDA

SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGPGTK

VDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

SEQ ID NO. 56: antibody 46 heavy chain

EVQLLESGGGLVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS

TIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGD

SRDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKKVEPKSC

SEQ ID NO. 57: antibody 324 light chain

DIQMTQSPSSLSASVGDRVTITCRASQSITTYLNWYQQKPGKAPKLLIYDA

SNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

SEQ ID NO. 58: antibody 324 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGTFNNYGISWVRQAPGQGLEWM

GWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC

ARVGDYGDYIVSPFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS

SLGTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO 59: antibody 236 light chain

DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQL

LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPP

TFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

SEQ ID NO. 60: antibody 236 heavy chain

QVQLVQSGAEVKKPGASVKVSCKASGGTFTSYGINWVRQAPGQGLEWM

GWMNPNSGNTGYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYC

ASRGIQLLPRGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSC

SEQ ID NO. 61: antibody 52 light chain

DIQMTQSPSSLSASVGDRVTITCRASQGISNNLNWYQQKPGKAPKLLIYA

ASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNGFPLTFGPGT

KVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 62: antibody 52 light chain N92T

DIQMTQSPSSLSASVGDRVTITCRASQGISNNLNWYQQKPGKAPKLLIYA

ASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGTGFPLTFGPGT

KVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

SEQ ID NO. 63: antibody 52 heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYGISWVRQAPGQGLEWM

GGIIPMFGTTNYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARD

RGDTIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKKVEPKSC

SEQ ID NO. 64: antibody 80 light chain

DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP

KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSAP

LTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

SEQ ID NO. 65: antibody 80 heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGGTFNRYAFSWVRQAPGQGLEWM

GGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARST

RELPEVVDWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKKVEPKSC

Equivalent/other embodiments

While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and, in general, of covering any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth.

Claims

1. A fusion protein comprising a nanocage monomer linked to a SARS-CoV-2 binding moiety, wherein a plurality of said fusion proteins self-assemble to form a nanocage.

2. The fusion protein of claim 1, wherein the SARS-CoV-2 binding moiety targets a SARS-CoV-2S glycoprotein.

3. Fusion protein according to claim 1 or 2, wherein the SARS-CoV-2 binding moiety decorates the inner and/or outer surface, preferably the outer surface, of the assembled nanocages.

4. The fusion protein of any one of claims 1-3, wherein the SARS-CoV-2 binding moiety comprises an antibody or fragment thereof.

5. The fusion protein of claim 4, wherein the antibody or fragment thereof comprises a Fab fragment.

6. The fusion protein according to claim 4, wherein the antibody or fragment thereof comprises a scFab fragment, scFv fragment, sdAb fragment, VHH domain, or a combination thereof.

7. The fusion protein of claim 4, wherein the antibody or fragment thereof comprises a heavy chain and/or a light chain of a Fab fragment.

8. The fusion protein according to any one of claims 4 to 7, wherein the SARS-CoV-2 binding moiety comprises the single chain variable domain VHH-72, BD23 and/or 4A8.

9. The fusion protein of any one of claims 4-8, wherein the SARS-CoV-2 binding moiety comprises the mabs listed in table 4.

10. The fusion protein of claim 9, wherein the SARS-CoV-2 binding moiety comprises mAb 298, 324, 46, 80, 52, 82, or 236 from table 4, or a variant thereof.

The fusion protein of claim 10, wherein the SARS-CoV-2 binding moiety comprises mabs 298, 80 and 52 from table 4, or variants thereof.

11. The fusion protein of any one of claims 1-10, wherein the SARS-CoV-2 binding moiety is linked at the N-or C-terminus of the nanocage monomer, or wherein there is a first SARS-CoV-2 binding moiety linked at the N-terminus of the nanocage monomer and a second SARS-CoV-2 binding moiety linked at the C-terminus of the nanocage monomer, wherein the first and second SARS-CoV-2 binding moieties are the same or different.

12. The fusion protein of any one of claims 1-11, wherein the nanocage monomer comprises a first nanocage monomer subunit linked to the SARS-CoV-2 binding moiety; wherein the first nanocage monomer subunit self-assembles with the second nanocage monomer subunit to form the nanocage monomer.

13. The fusion protein of claim 12, wherein the SARS-CoV-2 binding moiety is linked to the N-or C-terminus of the first nanocage monomer, or wherein there is a first SARS-CoV-2 binding moiety linked to the N-terminus of the first nanocage monomer subunit 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.

14. The fusion protein of claim 12 or 13, in combination with the second nanocage monomer subunit.

15. The fusion protein of any one of claims 12-14, wherein the second nanocage monomer subunit is linked to a biologically active moiety.

16. The fusion protein of claim 15, wherein the biologically active moiety comprises an Fc fragment.

17. The fusion protein of claim 16, wherein the Fc fragment is an IgG1-Fc fragment.

18. The fusion protein according to claim 15 or 16, wherein the Fc fragment comprises one or more mutations, such as LS, YTE, LALA, I253A and/or LALAP, which modulate the half-life of the fusion protein from, for example, minutes or hours to days, weeks or months.

19. The fusion protein of any one of claims 15-18, wherein the Fc fragment is a scFc fragment.

20. The fusion protein of any one of claims 1-19, wherein about 3 to about 100 nanocage monomers, e.g., 24, 32, or 60 monomers, or about 4 to about 200 nanocage monomer subunits, e.g., 4, 6, 8, 10, 12, 14, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or more subunits, optionally in combination with one or more full nanocage monomers, self-assemble to form a nanocage.

21. The fusion protein of any one of claims 1-20, wherein the nanocage monomers are selected from the group consisting of ferritin, apoferritin, encapsulin, SOR, tetrahydropteridine dioxygenase (lumazine synthase), pyruvate dehydrogenase, carboxylase, vault protein, groEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

22. The fusion protein of claim 21, wherein the nanocage monomer is apoferritin, optionally human apoferritin.

23. The fusion protein of claim 22, wherein the first and second nanocage monomer subunits interchangeably comprise the "N" and "C" regions of apoferritin.

24. The fusion protein of claim 23, wherein the "N" region of the 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:

25. the fusion protein of claim 22 or 23, wherein the "C" region of the 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 a sequence that is:

Or (b)

26. The fusion protein of any one of claims 1-25, further comprising a linker between the nanocage monomer subunit and the biologically active moiety.

27. The fusion protein of claim 26, wherein the linker is flexible or rigid and comprises about 1 to about 30 amino acid residues, such as about 8 to about 16 amino acid residues.

28. The fusion protein of claim 26 or 27, wherein the linker comprises a GGS repeat, such as 1, 2, 3, 4 or more GGS repeats.

29. The fusion protein of claim 28, wherein 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 a sequence that is:

GGGGSGGGGSGGGGSGGGGSGGGGSGG。

30. the fusion protein of any one of claims 1-29, further comprising a C-terminal linker.

31. The fusion protein of claim 30, wherein 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 a sequence that is:

GGSGGSGGSGGSGGGSGGSGGSGGSG。

32. A nanocage comprising at least one fusion protein according to any one of claims 1 to 31 and at least one second nanocage monomer subunit that self-assembles with the fusion protein to form a nanocage monomer.

33. The nanocage of claim 32, wherein each nanocage monomer comprises the fusion protein of any one of claims 1-31.

34. The nanocage of claim 32 or 33, wherein about 20% to about 80% of the nanocage monomers comprise the fusion protein of any one of claims 1 to 27.

35. The nanocage of any of claims 32-34, comprising at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different SARS-CoV-2 binding moieties, such as 3 different SARS-CoV-2 binding moieties.

36. The nanocage of any one of claims 32-35, wherein said nanocage is multivalent and/or multispecific.

37. The nanocage of any one of claims 32-36, comprising one or more mabs from table 4.

38. The nanocage of claim 37, comprising 3 mabs from table 4.

39. The nanocage of claim 37 or 38, comprising mabs 298, 324, 46, 52, 80, 82, and/or 236 from table 4.

40. The nanocapsule of any one of claims 32-39 comprising scFab1 human apoferritin scFc human N-ferritin scFab 2-C-ferritin scFab 3-C-ferritin in a ratio of 4:2:1:1.

41. Nanocage according to any of claims 32 to 40, which carries cargo molecules, such as pharmaceutical agents, diagnostic agents and/or imaging agents.

42. The nanocapsule of claim 41 wherein the cargo molecules are not fused to the fusion protein and are contained internally within the nanocapsule.

43. The nanocapsule of claim 42 wherein the cargo molecule is a protein and is fused to the fusion protein such that the cargo molecule is contained internally within the nanocapsule.

44. Nanocage according to any of claims 41 to 43, wherein the cargo molecules are fluorescent proteins, such as GFP, EGFP, ametrine, and/or flavin-based fluorescent proteins, such as LOV proteins, such as iLOV.

45. A trispecific antibody construct that targets SARS-CoV-2.

A sars-CoV-2 therapeutic or prophylactic composition comprising the nanocage of any one of claims 32 to 44 or the antibody of claim 45.

47. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 31.

48. A vector comprising the nucleic acid molecule of claim 47.

49. A host cell comprising the vector of claim 48 and producing the fusion protein of any one of claims 1 to 31.

50. A method of treating and/or preventing SARS-CoV-2, the method comprising administering a nanocage according to any one of claims 32 to 33 or an antibody according to claim 45 or a composition according to claim 46.

51. Use of a nanocage according to any one of claims 32 to 33 or an antibody according to claim 45 or a composition according to claim 46 for the treatment and/or prophylaxis of SARS-CoV-2.

52. The nanocage of any one of claims 32 to 33 or the antibody of claim 45 or the composition of claim 46 for use in the treatment and/or prevention of SARS-CoV-2.

53. A polypeptide comprising an amino acid sequence having at least 70% identity to any of the sequences listed in the following table, or a functional fragment thereof:

54. the polypeptide of claim 53, comprising a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the listed sequence.

55. The polypeptide of claim 54, which consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the listed sequence.

56. An antibody or fragment thereof comprising the polypeptide of any one of claims 53 to 55.

57. A fusion polypeptide comprising (1) a fragment crystallizable (Fc) region linked to (2) a nanocage monomer or subunit thereof, wherein the Fc region comprises an I253A mutation, wherein numbering is according to the EU index.

58. The fusion polypeptide of claim 57, wherein the Fc region further comprises a LALAP (L234A/L235A/P329G) mutation, wherein numbering is according to the EU index.

59. The fusion polypeptide of claim 57 or 58, wherein the Fc region is an IgG1 Fc region.

60. The fusion polypeptide of any one of claims 57-59, wherein the nanocage monomers are ferritin monomers.

61. The fusion polypeptide of claim 60, wherein the ferritin monomer is a ferritin light chain.

62. The fusion polypeptide of claim 61, wherein the ferritin light chain is a human ferritin light chain.

63. The fusion polypeptide of any one of claims 57-62, wherein the Fc region is linked to the nanocage monomers or subunits thereof via an amino acid linker.

64. The fusion polypeptide of any one of claims 57-63, wherein the Fc region is linked to the N-terminus of the nanocage monomer or subunit thereof.

65. The fusion polypeptide of any one of claims 57-64, wherein the Fc region is a single chain Fc (scFc).

66. The fusion polypeptide of any one of claims 57-65, wherein the Fc region is an Fc monomer.

67. A self-assembled polypeptide complex comprising:

68. The self-assembled polypeptide complex according to claim 67, wherein the nanocage monomers are ferritin monomers.

69. The self-assembled polypeptide complex according to claim 68, wherein the nanocage monomers are ferritin light chains.

70. The self-assembled polypeptide complex according to claim 69, which does not comprise any ferritin heavy chain or subunits of ferritin heavy chain.

71. The self-assembled polypeptide complex according to claim 68 or 69, wherein the nanocage monomers are human ferritin light chains.

72. The self-assembling polypeptide complex of any one of claims 67-71, wherein the SARS-CoV-2 binding antibody fragment binds to a receptor binding domain or spike protein of SARS-CoV-2.

73. The self-assembled polypeptide complex of any one of claims 67-72, wherein the SARS-CoV-2 binding antibody fragment comprises a light chain variable domain and a heavy chain variable domain.

74. The self-assembling polypeptide complex of any one of claims 67-73, wherein the SARS-CoV-2 binding antibody fragment comprises a Fab of antibodies capable of binding to SARS-CoV-2.

75. The self-assembled polypeptide complex of claim 73 or 74, wherein the SARS-CoV-2 binding antibody fragment comprises V _K Domain and V _H A domain.

76. The self-assembling polypeptide complex of any one of claims 67 to 75, wherein the self-assembling polypeptide complex is characterized by a ratio of 1:1 of first fusion polypeptide to second fusion polypeptide.

77. The self-assembled polypeptide complex according to any one of claims 67 to 76, wherein the Fc region is an IgG1 Fc region.

78. The self-assembled polypeptide complex of any one of claims 67-77, wherein the Fc region is linked to the nanocage monomer or subunit thereof by an amino acid linker.

79. The self-assembled polypeptide complex of any one of claims 67-78, wherein the Fc region is linked to the N-terminus of the nanocage monomer or subunit thereof.

80. The self-assembling polypeptide complex of any one of claims 67-79, comprising a total of at least 24 fusion polypeptides.

81. The self-assembling polypeptide complex of claim 80, comprising a total of at least 32 fusion polypeptides.

82. The self-assembling polypeptide complex of claim 81, having a total of about 32 fusion polypeptides.

83. A self-assembled polypeptide complex comprising:

(a) A plurality of first fusion polypeptides, each first fusion polypeptide comprising (1) an IgG1-Fc region linked to (2) a human ferritin monomer or subunit thereof, wherein said IgG1 Fc region comprises LALAP (L234A/L235A/P329G) and an I253A mutation, wherein numbering is according to the EU index, and

(b) A plurality of second fusion polypeptides, each comprising (1) a Fab fragment of an antibody capable of binding to a SARS-CoV-2 protein, said Fab fragment being linked to (2) a human ferritin monomer or subunit thereof.

84. The self-assembling polypeptide complex of claim 83, wherein:

(1) Each first fusion polypeptide comprises a ferritin monomer subunit that is C-half-ferritin, and each second fusion polypeptide comprises a ferritin monomer subunit that is N-half-ferritin; or (b)

85. The self-assembling polypeptide complex of claim 83 or 84, wherein the self-assembling polypeptide complex is characterized by a ratio of 1:1 of the first fusion polypeptide to the second fusion polypeptide.

86. The self-assembled polypeptide complex according to any one of claims 83 to 85, wherein each first fusion polypeptide comprises a ferritin monomer subunit that is C-half-ferritin.

87. The self-assembled polypeptide complex according to claim 86, wherein the IgG1 Fc region is linked to the C-half-ferritin by an amino acid linker.

88. The self-assembled polypeptide complex according to claim 86 or 87, wherein the IgG1 Fc region is linked to the C-half-ferritin via the N-terminus of the C-half-ferritin.

89. The self-assembled polypeptide complex according to any one of claims 83 to 88, wherein each second fusion polypeptide comprises a ferritin monomer subunit that is N-half-ferritin.

90. The self-assembled polypeptide complex of claim 89 wherein the Fab fragment is linked to the N-half-ferritin via an amino acid linker.

91. The self-assembled polypeptide complex of claim 89 or 90 wherein the Fab fragment is linked to the N-half-ferritin via the N-terminus of the N-half-ferritin.

92. The self-assembling polypeptide complex of any one of claims 83-91, further comprising a plurality of third fusion polypeptides, each third fusion polypeptide comprising (1) a human ferritin monomer linked to (2) a Fab fragment of an antibody capable of binding to a SARS-CoV-2 protein.

93. The self-assembling polypeptide complex of claim 92, wherein the self-assembling polypeptide complex is characterized by a ratio of 1:1:2 of first fusion polypeptide to second fusion polypeptide to third fusion polypeptide.

94. The self-assembling polypeptide complex of any one of claims 83-93, comprising a total of at least 24 fusion polypeptides.

95. The self-assembling polypeptide complex of claim 94, comprising a total of at least 32 fusion polypeptides.

96. The self-assembling polypeptide complex of claim 95, having a total of 32 fusion polypeptides.

97. The self-assembled polypeptide complex of any one of claims 74 or 83-93, wherein the Fab fragment comprises V _K Domain and V _H Domain, wherein:

(1)V _K the domain has the amino acid sequence shown in SEQ ID NO. 11 and V _H The structural domain has an amino acid sequence shown in SEQ ID NO. 12;

(2)V _K the domain has the amino acid sequence shown in SEQ ID NO. 17 and V _H The structural domain has an amino acid sequence shown as SEQ ID NO. 18;

(3)V _K the structural domain has SEQ ID NO:25 internal V _K And V _H The structural domain has SEQ ID NO. 26 internal V _H Amino acid sequence of (a);

(4)V _K the structural domain has SEQ ID NO:27 internal V _K And V _H The domain has SEQ ID NO. 28 internal V _H Amino acid sequence of (a);

(5)V _K the domain has the sequence of SEQ ID NO. 29 internal V _K And V _H The structural domain has SEQ ID NO:30 internal V _H Amino acid sequence of (a);

(6)V _K the structural domain has SEQ ID NO. 31 internal V _K And V _H The structural domain has SEQ ID NO:32 internal V _H Amino acid sequence of (a);

(7)V _K the domain has the sequence of SEQ ID NO 33 internal V _K And V _H The domain has SEQ ID NO:34 internal V _H Amino acid sequence of (a);

(8)V _K the structural domain has SEQ ID NO:35 internal V _K And V _H The structural domain has SEQ ID NO:36 internal V _H Amino acid sequence of (a);

(9)V _K the domain has SEQ ID NO. 37 internal V _K And V _H The domain has SEQ ID NO:38 internal V _H Amino acid sequence of (a);

(10)V _K the domain has the sequence shown in SEQ ID NO 39 _K And V _H The structural domain has SEQ ID NO:40 internal V _H Amino acid sequence of (a);

(11)V _K the domain has SEQ ID NO. 41 internal V _K And V _H The domain has SEQ ID NO:42 internal V _H Amino acid sequence of (a);

(12)V _K the domain has SEQ ID NO:43 internal V _K And V _H The domain has SEQ ID NO:44 internal V _H Amino acid sequence of (a);

(13)V _K the domain has SEQ ID NO:45 internal V _K And V _H The domain has the sequence of SEQ ID NO:46 internal V _H Amino acid sequence of (a);

(14)V _K the domain has the sequence shown in SEQ ID NO. 47 _K And V _H The domain has SEQ ID NO:48 internal V _H Amino acid sequence of (a);

(15)V _K the domain has the sequence of SEQ ID NO:49 in V _K And V _H The structural domain has SEQ ID NO:50 internal V _H Amino acid sequence of (a);

(16)V _K the structural domain has the sequence shown in SEQ ID NO. 51 _K And V _H The domain has SEQ ID NO:52 internal V _H Amino acid sequence of (2)；

(17)V _K The structural domain has SEQ ID NO:53 internal V _K And V _H The domain has the sequence shown in SEQ ID NO. 54 _H Amino acid sequence of (a);

(18)V _K the domain has the sequence of SEQ ID NO:55 in V _K And V _H The domain has the sequence shown in SEQ ID NO. 56 _H Amino acid sequence of (a);

(19)V _K the domain has the sequence of SEQ ID NO:57 internal V _K And V _H The domain has SEQ ID NO:58 internal V _H Amino acid sequence of (a);

(20)V _K the domain has the sequence of SEQ ID NO:59 internal V _K And V _H The structural domain has SEQ ID NO:60 internal V _H Amino acid sequence of (a);

(21)V _K the domain has the sequence of SEQ ID NO 61 or V in SEQ ID NO 62 _K And V _H The domain has the sequence of SEQ ID NO:63 internal V _H Amino acid sequence of (a); or (b)

(22)V _K The structural domain has SEQ ID NO:64 internal V _K And V _H The structural domain has SEQ ID NO:65 internal V _H Is a sequence of amino acids of (a).

98. The self-assembled polypeptide complex according to any one of claims 83 to 97, wherein the human ferritin monomer is a human ferritin light chain.

99. The self-assembled polypeptide complex of claim 98 which does not comprise any ferritin heavy chain or subunits of ferritin heavy chain.

100. A method of treating, ameliorating or preventing a SARS-CoV-2 associated disorder, the method comprising administering to a subject a composition comprising the self-assembled polypeptide complex of any one of claims 67-99.

101. The method of claim 100, wherein the subject is a mammal.

102. The method of claim 101, wherein the subject is a human.